Fab arm exchange prevention type Fc variants capable of eliminating effector function

Abstract

The present invention relates to a Fab-arm (Fab-arm) exchange prevention Fc variant, the effector function of which is reduced due to elimination of binding force with Fc [gamma] Rs and C1q, and the human antibody Fc domain variant of the present invention is a novel variant different from the conventional Fab-arm exchange prevention variant, which can overcome the Fab arm exchange phenomenon, which is the disadvantage of IgG4, and which does not bind to all human Fc [gamma] Rs and C1q, and which can be used as a novel human antibody Fc domain variant having a reduced effector function due to elimination of binding force with Fc [gamma] Rs and C1q. The compound does not bind to mouse and monkey Fc [gamma] Rs, has excellent blood half-life and thermal stability, and thus can be used for preventing immune cell / normal cell death (toxicity) caused by a therapeutic antibody or an antibody Fc region of an Fc-fusion protein drug.

Description

Technical Field

[0001] This invention relates to a Fab arm exchange-blocking Fc variant whose effector function is reduced due to the elimination of binding forces with FcγRs and C1q. Background Technology

[0002] Because protein therapeutics are highly specific to disease targets and have lower side effects and toxicity, they are rapidly replacing non-specific small molecule compounds and are widely used in clinical practice. Currently, antibody therapeutics and Fc-fusion proteins incorporating the Fc region of antibodies dominate the clinical application of protein therapeutics. Therapeutic antibodies are considered one of the most effective cancer treatments because they have higher targeting specificity, lower biotoxicity and side effects than existing small molecule drugs, and a long blood half-life of approximately three weeks. In fact, major pharmaceutical companies and research institutes worldwide are accelerating the development of therapeutic antibodies that can specifically bind to and effectively eliminate cancer cells (including carcinogens). Companies developing therapeutic antibody drugs mainly include Roche, Amgen, Johnson & Johnson, Abbott, and Bristol-Myers Squibb. Roche, in particular, achieved global sales of approximately $19.5 billion in 2012 with its representative anti-cancer antibody products Herceptin, Avastin, and Rituximab, generating substantial profits and leading the global antibody drug market. Johnson & Johnson, the company that developed Remicade, has also rapidly expanded in the global antibody market thanks to its continuously growing sales. Pharmaceutical companies such as Abbott and Bristol-Myers Squibb also have multiple therapeutic antibodies in the final stages of development. As a result, biopharmaceutical products, including therapeutic antibodies with disease-targeting specificity and fewer side effects, are rapidly replacing small molecule drugs in the global pharmaceutical market. Antibodies provide a bridge between the humoral and cellular immune systems. The Fab region of an antibody recognizes antigens, while the Fc domain binds to cell surface antibody (immunoglobulin) receptors (Fc receptors or FcRs). These receptors are differentially expressed on all immune-active cells, and their binding mechanisms differ depending on the type of FcγR expressed on the surface of the immune cells they bind to. When the Fc receptor binding site of the antibody Fc region binds to the Fc receptor (FcR) on the cell, the antibody binds to the Fc receptor on the cell surface through the Fc region, triggering a variety of important biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killing cells and lysing antibody-coated target cells (antibody-dependent cell-mediated cytotoxicity (ADCC)), release of inflammatory mediators, control of placental transport, and production of immunoglobulins (Deo, YM et al., Immunol. Today 18(3): 127-135 (1997)).As mentioned above, the Fc domain plays a crucial role in immune cell recruitment and in ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis). In particular, as effector functions of antibodies, ADCC and ADCP function depend on their interaction with Fc receptors present on various cell surfaces. Human Fc receptors are classified into five types, and the type of immune cells recruited by an antibody depends on the type of Fc receptor it binds to. For example, the antibody's Fc domain induces effector function in ADCC by binding to FcγRⅢa, effector function in ADCP by binding to FcγRI or FcγRⅡa, and effector function in CDC (complement-dependent cytotoxicity) by binding to C1q, thereby exerting toxicity on target antigens bound to the Fab region and thus being responsible for the main therapeutic effect of therapeutic antibodies.

[0003] However, from a therapeutic perspective, the effector function of antibodies is often not optimal; they can activate the host's immune defenses, leading to safety risks and adverse side effects. For example, some therapeutic antibodies, such as immune checkpoint inhibitors and bispecific immune cell engagers, have the following side effect: they damage immune cells due to the immune mechanisms that target them. Immune checkpoint inhibitors that target immune checkpoint proteins expressed on the surface of immune cells (such as T cells) have the following side effect: they destroy immune cells that should be eliminating cancer cells by activating the immune response, thereby reducing the original efficacy of the antibody. Furthermore, in FcγR, the immunorepressive receptor (FcγRⅡb) is expressed on T cells, and it has been reported that it can reduce the efficacy of immune checkpoint inhibitor antibodies by interacting with antibody Fc (Bennion et al., Sci Transl Med., 2023). Therefore, it is known that the interaction between the immune cell surface and the antibody Fc on FcγR can not only lead to the side effects of immune checkpoint antibodies but also reduce their efficacy. Moreover, if bispecific immune cell linker antibodies coexist with Fc-mediated immune mechanisms, they may cause immune cell destruction, failing to effectively clear cancer cells and thus causing side effects. These bispecific immune cell linker antibodies bind to antigens on the surface of cancer cells on one side and to immune cells on the other, thereby attracting immune cells to attack cancer cells and more effectively clearing them. Additionally, agonist antibodies that bind to target cells and induce cell activation or antagonist antibodies that block the interaction between target antigens and ligands also have the problem of toxicity to target cells and antigens due to Fc-mediated immune mechanisms, thereby reducing the original efficacy of the antibody. Meanwhile, when an Fc-fusion protein with an Fc region is developed to increase the half-life of active substances such as proteins or compounds for therapeutic, diagnostic, or research purposes, there is a risk of toxicity due to the Fc-mediated immune mechanism.

[0004] Therefore, to prevent off-target toxicity caused by the antibody's immune mechanism and ensure effective therapeutic efficacy, eliminating the Fc-mediated immune mechanism is crucial. For this reason, when developing antibodies, IgG2 antibodies, which have the lowest binding affinity to FcγR among human IgG subclasses and thus the lowest immune mechanism, are considered. However, due to disulfide bond exchange in the hinge region, IgG2 antibodies exhibit multiple isotypes and suffer from stability issues leading to aggregation. Therefore, IgG4 antibodies, with the next lowest binding affinity, are considered, and such antibodies are currently prepared for clinical development. In this regard, anti-PD-1 antibodies targeting the T-cell-expressed immune checkpoint protein PD-1 (programmed cell death-1) (Merck & Co.'s pembrolizumab (Keytruda), Bristol-Myers Squibb) are being developed. Squibb's nivolumab (Opdivo) and Regeneron's cimiprimab (Libtayo) are both FDA-approved human IgG4 antibodies and widely used in clinical practice due to various needs. Pembrolizumab has been approved for the treatment of multiple cancers and ranked second in global pharmaceutical sales in 2020, reaching $14.3 billion, while nivolumab ranked eighth, with sales of $7.9 billion. However, IgG4 antibodies also bind to all FcγRs, especially FcγRIs, exhibiting strong binding affinity of several nM, which can activate multiple immune mechanisms. Therefore, to prevent the immune mechanisms of antibodies from causing target cell destruction, research is actively underway to develop an Fc antibody that eliminates the binding affinity to all FcγRs. Furthermore, wild-type human IgG4 differs from human IgG1 at amino acid position 228 in the CH2 region, presenting as serine and mediated through a flexible core hinge. The hinge forms an intrachain disulfide bond, leading to the production of a half antibody through a non-covalent bond.In this way, IgG4 existing in hapten form can undergo Fab-arm exchange (FAE), where two IgG4 haptens bind to different antigens. To prevent this, replacing amino acid 228 of IgG4 with proline (amino acid 228 of human IgG1) stabilizes the hinge region and prevents Fab-arm exchange. Therefore, the S228P variant is under development for routine clinical use of IgG4 antibodies. However, this traditional S228P variant has the same FcγR binding affinity as wild-type IgG4, thus still presenting the problem of causing immune cell death side effects / off-target toxicity to normal cells. Summary of the Invention

[0005] Technical issues

[0006] The purpose of this invention is to provide a novel variant of the Fc domain of a human antibody.

[0007] Furthermore, the object of the present invention is to provide an antibody or a fragment thereof with reduced effector function or an immunologically active fragment thereof.

[0008] Furthermore, the purpose of this invention is to provide an Fc-fusion protein.

[0009] Furthermore, the object of the present invention is to provide an antibody therapeutic agent.

[0010] Furthermore, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer.

[0011] Furthermore, the purpose of this invention is to provide a method for preparing a human antibody Fc domain variant.

[0012] Furthermore, the purpose of this invention is to provide a method for preparing an antibody or fragment thereof with reduced effector function.

[0013] Furthermore, the object of the present invention is to provide a use in the preparation of antibody therapeutic agents.

[0014] Furthermore, the object of the present invention is to provide a use in the prevention or treatment of cancer.

[0015] Meanwhile, the purpose of this invention is to provide a cancer treatment method.

[0016] Technical solution

[0017] To address the above problems, this invention provides a novel human antibody Fc domain variant with reduced effector function.

[0018] Furthermore, the present invention provides an antibody comprising the novel human antibody Fc domain variant or an immunomodulatory fragment thereof.

[0019] Furthermore, the present invention provides an Fc-fusion protein formed by fusing the human antibody Fc domain variant and a protein therapeutic agent.

[0020] Furthermore, the present invention provides an antibody therapeutic agent comprising the antibody or its immunologically active fragment and a pharmaceutical portion.

[0021] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the human antibody Fc domain variant, the antibody or its immunologically active fragment or the antibody therapeutic agent as an active ingredient.

[0022] Furthermore, the present invention provides a method for preparing the human antibody Fc domain variant.

[0023] Furthermore, the present invention provides a method for preparing an antibody or fragment thereof with reduced effector function.

[0024] Furthermore, the present invention provides the use of an antibody comprising a variant of the Fc domain of the present invention or an immunologically active fragment thereof in the preparation of an antibody therapeutic agent.

[0025] Furthermore, the present invention provides the use of the human antibody Fc domain variant of the present invention, the antibody of the present invention or its immunomodulatory fragment, or the antibody therapeutic agent of the present invention in the prevention or treatment of cancer.

[0026] Meanwhile, the present invention provides a cancer treatment method comprising the step of administering a pharmaceutically effective amount of the human antibody Fc domain variant of the present invention, the antibody of the present invention or its immunologically active fragment, or the antibody therapeutic agent of the present invention to a subject suffering from cancer.

[0027] The effects of the invention

[0028] The human antibody Fc domain variant of the present invention is a novel variant that differs from traditional Fab arm exchange-blocking variants. It overcomes the Fab arm exchange phenomenon, a drawback of IgG4, and does not bind to human FcγRs and C1q, nor to mouse and monkey FcγRs. It has excellent blood half-life and thermal stability, and therefore can be used to block immune cell / normal cell death (toxicity) caused by the antibody Fc region of therapeutic antibodies or Fc-fusion protein drugs. Attached Figure Description

[0029] Figure 1The figure shows the results of SDS-PAGE (sodium dodecyl sulfonate-polyacrylamide gel electrophoresis) analysis of glycosylated IgG4 Fc variants (Stapled Fc-1, Stapled Fc-2, Stapled Fc-3, Stapled Fc-4, Stapled Fc-5) after purification to prevent Fab arm exchange and eliminate FcγRs and C1q binding.

[0030] Figure 2 The figure shows the expression and SDS-PAGE analysis results of purified FcγRI-GST, FcγRIIIa-131H-GST, FcγRIIIa-131R-GST, FcγRIIIb-GST, FcγRIIIIa-158V-GST and FcγRIIIIa-158F-GST.

[0031] Figure 3 A graph showing the binding affinity of the glycosylated pembrolizumab IgG4Fc variant of the present invention to FcγRI, FcγRIIIa-131H, FcγRIIIa-131R, FcγRIIIb, FcγRIIIIa-158V and FcγRIIIIa-158F by ELISA (enzyme-linked immunosorbent assay).

[0032] Figure 4 The figure shows the expression of purified mouse FcγRI-GST, mouse FcγRIIIb-GST, mouse FcγRIIII-GST, and mouse FcγRIIV-GST, as well as the results of SDS-PAGE analysis after purification.

[0033] Figure 5 This is a graph showing the binding affinity of the glycosylated pembrolizumab IgG4 Fc variant of the present invention to mouse FcγRI, mouse FcγRIIIb, mouse FcγRIIII, and mouse FcγRIIV by ELISA analysis.

[0034] Figure 6 The figure shows the expression and SDS-PAGE analysis results of purified cynomolgus monkey FcγRI-GST, cynomolgus monkey FcγRI1a-GST, cynomolgus monkey FcγRI1b-GST and cynomolgus monkey FcγRI3-GST.

[0035] Figure 7 This is a graph showing the binding affinity of the glycosylated pembrolizumab IgG4 Fc variant of the present invention to cynomolgus monkey FcγRI, cynomolgus monkey FcγRIIa, cynomolgus monkey FcγRIIb and cynomolgus monkey FcγRIIII by ELISA.

[0036] Figure 8 This is a graph showing the C1q binding affinity of the glycosylated pembrolizumab IgG4 Fc variant of the present invention analyzed by ELISA.

[0037] Figure 9 The figure shows the expression of purified FcRn-GST and the results of SDS-PAGE analysis after purification.

[0038] Figure 10 This is a graph showing the pH-dependent FcRn binding affinity of the glycosylated pembrolizumab IgG4 Fc variant of the present invention analyzed by ELISA.

[0039] Figure 11 This is an in vivo analysis of the blood half-life of the glycosylated pembrolizumab IgG4 Fc variant of the present invention in mice expressing human FcRn.

[0040] Figure 12 This is a DSF (differential scanning fluorescence) analysis chromatogram of the thermal stability of the glycosylated pembrolizumab IgG4 Fc variant of the present invention.

[0041] Best practice

[0042] The present invention will now be described in detail with reference to the accompanying drawings and through examples. However, the following examples are provided as illustrations of the invention, and detailed descriptions of techniques or structures well-known to those skilled in the art may be omitted when it is deemed that such descriptions would unnecessarily obscure the spirit of the invention; the invention is not limited thereto. The invention can be modified and applied in various ways within the scope of the claims and their equivalents.

[0043] Furthermore, the terminology used in this specification is intended to properly express preferred embodiments of the invention and may vary depending on the intent of the user, operator, or convention in the art to which this invention pertains. Therefore, the definitions of these terms should be based on the entire specification. Throughout this specification, when a part is stated to "comprise" a structure, it means, unless specifically stated otherwise, that other structural elements may be included, rather than excluded.

[0044] In this invention, unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, while preferred methods or embodiments are described in this specification, similar or equivalent methods or embodiments are also included within the scope of this invention. The contents of all publications described in this specification as referenced are incorporated herein by reference.

[0045] Throughout this specification, in addition to the commonly used 1-letter and 3-letter codes for natural amino acids, the generally permissible 3-letter codes for other amino acids such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine) are also used. Furthermore, the amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB (International Union of Biochemistry and Biotechnology) nomenclature as follows.

[0046] 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.

[0047] In one embodiment, the present invention relates to a variant of the Fc domain of a human antibody, wherein, in the Fc domain of a wild-type human antibody, one or more amino acids selected from the group consisting of amino acids 231, 232, 234, and 235 numbered according to the Kabat numbering system are replaced by a sequence different from the wild-type amino acid sequence.

[0048] In one example, the human antibody Fc domain variant of the present invention may include one or more amino acid substitutions selected from the group consisting of A231C, P232C, F234C and L235C.

[0049] In one example, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-1 containing an amino acid substitution of A231C, the variant may contain the amino acid sequence of SEQ ID NO: 1, and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 2.

[0050] In one example, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-2 containing an amino acid substitution of P232C, the variant may contain the amino acid sequence of SEQ ID NO: 3, and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 4.

[0051] In one example, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-4 containing an amino acid substituted for F234C, the variant may contain the amino acid sequence of SEQ ID NO: 5, and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 6.

[0052] In one example, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-5 containing an amino acid substitution of L235C, the variant may contain the amino acid sequence of SEQ ID NO: 7, and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 8.

[0053] In one instance, the human antibody (immunoglobulin) may be IgA, IgM, IgE, IgD, or IgG or variants thereof, more preferably IgG4.

[0054] In one instance, the human antibody (immunoglobulin) may be IgG4 or a variant thereof, may be a humanized antibody, and may be trastuzumab, pembrolizumab, or atezolizumab.

[0055] In one instance, the human antibody (immunoglobulin) may be IgG4 or a variant thereof, the Fc domain of wild-type IgG4 containing hinge, CH2 and CH3 may contain the amino acid sequence of SEQ ID NO: 9, and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 10.

[0056] In this invention, the human antibody IgG4 of SEQ ID NO: 9 comprises the Fc domain of the entire sequence of wild-type IgG4, namely 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). The variant is a variant in which the amino acid CH2 of wild-type IgG4 is substituted. For example, in the amino acids of SEQ ID NO: 9, amino acid L at position 17 is numbered as amino acid 235 according to the Kabat numbering system, which is substituted by C in the Stapled Fc-5 (L235C) variant.

[0057] In one example, compared to the wild-type human antibody Fc domain, the binding affinity of the human antibody Fc domain variant of the present invention to Fcγ receptors (FcγRs) can be reduced. The Fcγ receptors can be human, mouse, or monkey Fcγ receptors (FcγRs), and can be FcγRI, FcγRI1a, FcγRI1b, FcγRI3, FcγRI3a, or FcγRIV.

[0058] In one instance, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb, or FcγRⅢa, wherein FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and 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.

[0059] In one instance, the binding affinity of the human antibody Fc domain variant of the present invention to C1q can be reduced compared to the wild-type human antibody Fc domain.

[0060] In one instance, the effector function of the human antibody Fc domain variant of the present invention can be reduced compared to the wild-type human antibody Fc domain.

[0061] In one instance, 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-γ receptor binding, protein A binding, protein G binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, endocytosis of Fc-containing peptides, target downregulation, antibody-drug conjugate (ADC) uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof, preferably ADCC or CDC.

[0062] In one instance, the thermal stability of the human antibody Fc domain variant of the present invention can be improved compared to the wild-type human antibody Fc domain.

[0063] In one instance, the in vivo half-life of the human antibody Fc domain variant of the present invention can be extended compared to the wild-type human antibody Fc domain.

[0064] In one example, the half-life of the human antibody Fc domain variant of the present invention can be extended by more than 3%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the wild-type human antibody Fc domain, or by more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to the wild-type Fc domain.

[0065] In one instance, the human antibody Fc domain variant of the present invention can have a high binding affinity for FcRn at pH 5.6 to 6.5 compared to the wild-type human antibody Fc domain, and the endosome may be in a weakly acidic environment with a pH of 5.8 to 6.0. Compared to the wild-type Fc domain, the pH-sensitive Fc variant of the present invention exhibits a binding affinity to FcRn that is increased by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% within the stated pH range, or by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to the wild-type Fc domain. In this example, compared to the wild-type immunoglobulin Fc region, the Fc variant of the present invention can exhibit a low binding affinity to FcRn at pH 7.0 to 7.8, where the blood pH can be within the normal range, specifically 7.2 to 7.6. Within the pH range, the degree of dissociation between the Fc variants of the present invention and FcRn can be the same or substantially unchanged compared to the wild-type Fc domain.

[0066] In one instance, the Fc domain variant of the present invention can be used to prevent the death of the bound cells.

[0067] In one instance, the Fc domain variant of the present invention can be applied to antibodies that target immune cells or normal cells.

[0068] In one instance, the Fc domain variant of the present invention can be used for immune checkpoint inhibitor antibodies or bispecific immune cell engaging bispecific antibodies.

[0069] In this invention, variants containing amino acid variations in the Fc region of the human antibody of this invention are defined according to the amino acid modifications constituting the Fc region of the parent antibody, and the conventional antibody numbering follows the European Union (EU) index developed by Kabat (Kabat et al., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991).

[0070] The term "Fc domain variant" used in this invention can be used interchangeably with "Fc variant".

[0071] As used in this invention, the term "wild-type polypeptide" refers to an unmodified polypeptide that is subsequently modified to generate a derivative. A wild-type polypeptide can be a naturally occurring polypeptide, a derivative of a natural polypeptide, or an engineered form. A wild-type polypeptide can refer to the polypeptide itself, a composition containing the wild-type polypeptide, or the amino acid sequence encoding the wild-type polypeptide. Therefore, as used in this invention, the term "wild-type antibody" refers to a derivative antibody polypeptide generated by modifying amino acid residues. Interchangeably with this terminology, "parent antibody" can be used to refer to an unmodified antibody polypeptide that is derived by introducing amino acid modifications.

[0072] The term "amino acid modification / variation" as used in this invention refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence, preferably substitution. The term "amino acid substitution" or "replacement" as used in this invention refers to replacing an amino acid at a specific position in the polypeptide sequence of the Fc domain of a wild-type human antibody with another amino acid. For example, an Fc variant containing an A231C substitution refers to replacing alanine, the amino acid residue at position 231 of the Fc domain of a wild-type antibody, with cysteine.

[0073] As used in this specification, the term "Fc variant" refers to a modification that includes one or more amino acid residues compared to the Fc domain of the wild-type antibody.

[0074] Compared to the wild-type antibody Fc domain (region or fragment), the Fc variants of the present invention contain one or more amino acid modifications, resulting in differences in the amino acid sequence. The amino acid sequence of the Fc variants of 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 of the present invention has about 80% or more, preferably about 90% or more, and most preferably about 95% or more homology to the amino acid sequence of the wild-type antibody Fc domain. Amino acid modifications can be performed through genetic engineering using molecular biological methods, or through enzymatic or chemical methods.

[0075] The Fc variants of the present invention can be prepared by any method known in the art. In one embodiment, the Fc variant of the human antibody of the present invention, after encoding a polypeptide sequence containing specific amino acid modifications, is cloned into a host cell as needed and used to form nucleic acids for expression and identification. Various methods for achieving this purpose 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).

[0076] The nucleic acid encoding the Fc variant of the present invention can be inserted into an expression vector for protein expression. It typically contains a single protein, i.e., a functionally related protein operatively linked to a regulatory sequence, selection marker, any fusion chaperone, and / or additional elements. Under suitable conditions, the Fc variant of the present invention can be produced by using a host cell transformed with nucleic acid, preferably by culturing an expression vector containing the nucleic acid encoding the Fc variant of the present invention and inducing protein expression. A variety of 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 depend on the host cell used. Preferably, *Escherichia coli*, which has low production costs and high industrial value, is used as the host cell for producing the Fc variant of the present invention.

[0077] Therefore, the scope of the present invention covers methods for preparing Fc variants, the methods comprising the steps of: culturing host cells into which nucleic acids encoding Fc variants have been introduced under conditions suitable for protein expression; and purifying or isolating the Fc variants expressed from the host cells.

[0078] As used in this invention, the term "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody, at least a portion of which is encoded by an FcRn gene. The FcRn can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, functional FcRn proteins comprise two polypeptides commonly referred to as a light chain and a heavy chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated in this specification, FcRn or an FcRn protein refers to a complex of the FcRn heavy chain and β-2-microglobulin.

[0079] In one embodiment, the present invention relates to an antibody comprising a variant of the Fc domain of the present invention or an immunologically active fragment thereof.

[0080] In one instance, the antibody or its immunologically active fragment can reduce its binding affinity to Fcγ receptors (FcγRs) or C1q compared to wild-type human antibodies by eliminating its binding affinity to FcγRs.

[0081] In one instance, the Fcγ receptor can be a human, mouse, or monkey Fcγ receptor (FcγRs), and can be FcγRI, FcγRIIIa, FcγRIIIb, FcγRIIII, FcγRIIIIa, or FcγRIV.

[0082] In one instance, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb, or FcγRⅢa, wherein FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and 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.

[0083] In one instance, the effector function of the antibody or its immunologically active fragment may be reduced compared to a wild-type human antibody.

[0084] In one instance, the antibody may be a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, IgG-like bispecific antibody, bispecific immune cell engager, antibody mimic, chimeric antibody, antibody conjugate, human antibody, humanized antibody, bivalent antibody, or bispecific molecule. The immunologically active fragment may be Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity-determining region fragment, or diabody of the antibody.

[0085] Antibodies can be isolated or purified using various methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassay, precipitation, dialysis, filtration, concentration, and chromatofocusing. As known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies and can be used for purification. Typically, specific fusion partners can be used for purification.

[0086] The antibody is not only in its whole form but also includes functional fragments of the antibody molecule. A whole antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond. The functional fragment of the antibody molecule refers to the fragment carrying the antigen-binding function. Examples of antibody fragments include: (i) the Fab fragment, consisting of the variable region (VL) of the light chain, the variable region (VH) of the heavy chain, the constant region (CL) of the light chain, and the first constant region (CH1) of the heavy chain; (ii) the Fd fragment, consisting of the VH and CH1 domains; (iii) the Fv fragment, consisting of the VL and VH domains of a single antibody; and (iv) the dAb fragment, consisting of the VH domain (Ward ES et al., Nature 341:544-546 (1989)). (1989)); (v) a separated CDR region; (vi) an F(ab')2 fragment as a bivalent fragment containing two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) bound by a peptide linker, which binds a VH domain and a VL domain to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer (PCT / US92 / 09965); and (ix) a diabody (WO94 / 13804), which is a multivalent or multispecific fragment made by gene fusion, etc.

[0087] The antibodies or their immunologically active fragments of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and their immunologically active fragments. The antibodies may be produced by recombinant or synthetic methods.

[0088] The antibody or its immunologically active fragment can be isolated from an organism (not present in the organism) or produced non-naturally, for example, by synthetic or recombinant methods.

[0089] In this invention, "antibody" refers to a substance produced within the immune system in response to antigen stimulation. Its type is not particularly limited and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies exhibit high stability both in vitro and in vivo, and have a long half-life, thus facilitating large-scale expression and production. Furthermore, due to their inherently dimer structure, antibodies possess extremely high affinity. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain via disulfide bonds. The constant regions of an antibody are divided into heavy chain constant regions and light chain constant regions. The heavy chain constant regions have γ, μ, α, δ, and ε types, and subclasses γ1, γ2, γ3, γ4, α1, and α2. The light chain constant regions have κ and λ types.

[0090] In this invention, the term "heavy chain" is interpreted to include both full-length heavy chains and their segments, wherein the full-length heavy chains and their segments include variable region structural domains V. H (Contains an amino acid sequence with a variable region sequence sufficient to confer antigen specificity), 3 constant region domains C H 1. C H 2 and C H 3. And hinges. Furthermore, the term "lightchain" is interpreted to include the entire length of the lightchain and its segments, which includes the variable region structural domain V. L (Contains an amino acid sequence with a variable region sequence sufficient to confer antigen specificity) and a constant region structural domain C L .

[0091] In this invention, the terms "Fc domain," "Fc fragment," or "Fc region" together with the Fab domain / fragment constitute the antibody, the Fab domain / fragment being composed of a variable region (V) of the light chain. L ) and the variable region (V) of the heavy chain H ), the constant region of the light chain (C) L ) and the first constant region of the heavy chain (C H 1) Composition: The Fc structural domain / segment consists of the second constant region (C) of the heavy chain. H 2) and the third constant region (C) H 3) Composition.

[0092] In one embodiment, the present invention relates to a nucleic acid molecule encoding a variant of the Fc domain of the present invention or an antibody or an immunologically active fragment thereof.

[0093] In one embodiment, the present invention relates to a vector containing the nucleic acid molecule and a host cell containing the vector.

[0094] The nucleic acid molecules of this invention can be isolated or recombinant, including single-stranded and double-stranded DNA and RNA, and corresponding complementary sequences. When the isolated nucleic acid is isolated from a natural source, it is a nucleic acid isolated from the surrounding genetic sequence present in the genome of the isolated organism. For nucleic acids synthesized from a template by enzymatic or chemical methods, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids produced through these stages can be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules refer to nucleic acid molecules as individual fragments or components of larger nucleic acid constructs. Nucleic acids are operatively linked when establishing functional relationships with other nucleic acid sequences. For example, when a polypeptide is expressed in a pre-secretory form, i.e., as a preprotein, the DNA of the leader sequence or secretory leader sequence is operatively linked to the DNA of the polypeptide; a promoter or enhancer is operatively linked to the coding sequence when influencing the transcription of the polypeptide sequence; or a ribosome binding site is operatively linked to the coding sequence when set in a manner that promotes translation. Ooperative linking generally refers to the adjacent DNA sequences to be linked, and for secretory leader sequences, it refers to adjacent sequences located within the same reading frame. However, enhancers do not need to be adjacent. Ligation is achieved by joining at a suitable restriction endonuclease site. If such a site is unavailable, synthetic oligonucleotide adapters or linkers are used according to standard methods.

[0095] Due to codon degeneracy or considering the codon preferences of the organism to which it is to be expressed, various modifications can be made to the coding region of isolated nucleic acid molecules encoding Fc domain variants of the present invention or antibodies or immunologically active fragments thereof, without altering the amino acid sequence of the Fc domain variant or the antibody or immunologically active fragment containing it. Various modifications or alterations can also be made to portions other than the coding region without affecting gene expression. Those skilled in the art will understand that such modified genes are also included within the scope of the present invention. That is, more than one nucleic acid base can be mutated by substitution, deletion, insertion, or combinations thereof, as long as the nucleic acid molecule of the present invention encodes a protein with equivalent activity. The sequence of such nucleic acid molecule can be single-stranded or double-stranded, and can be a DNA molecule or an RNA (mRNA) molecule.

[0096] Isolated nucleic acid molecules encoding variants of the Fc domain of the present invention, or antibodies containing the Fc domain, or immunologically active fragments thereof, can be inserted into expression vectors for protein expression. Expression vectors typically contain proteins, i.e., proteins with functional relationships, operatively linked to regulatory sequences, selection markers, any fusion chaperones, and / or additional elements. Under suitable conditions, variants of the Fc domain of the present invention, or antibodies containing the Fc domain, or immunologically active fragments thereof, can be produced by using nucleic acid-converted host cells, preferably culturing expression vectors containing nucleic acid molecules encoding variants of the Fc domain of the present invention, or antibodies containing the Fc domain, or immunologically active fragments thereof, and inducing protein expression. A variety of 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 depend on the host cell used. Preferably, *E. coli*, which has low production costs and high industrial value, is used as the host cell for production.

[0097] The vectors of this invention include, but are not limited to, plasmid vectors, granular vectors, phage vectors, and viral vectors. Suitable vectors, in addition to expression control elements such as promoters, operons, start codons, stop codons, polyadenylation signals, and enhancers, may also contain signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector can be constitutive or inducible. Regarding the signal sequences, when the host is *Escherichia sp.*, PhoA signal sequences, OmpA signal sequences, etc., can be used; when the host is *Bacillus sp.*, α-amylase signal sequences, subtilisin signal sequences, etc., can be used; when the host is yeast, MFα signal sequences, SUC2 signal sequences, etc., can be used; and when the host is animal cells, insulin signal sequences, α-FcγIV signal sequences, antibody molecule signal sequences, etc., can be used, but are not limited to these. Furthermore, the vector may include selection markers for selecting host cells containing the vector, and if the expression vector is reproducible, it may include an origin of replication.

[0098] In this invention, the term "vector" refers to a carrier into which a nucleic acid sequence can be inserted to introduce it into a cell capable of replicating that nucleic acid sequence. The nucleic acid sequence can be exogenous or heterologous. Examples of vectors include plasmids, granules, and viruses (e.g., bacteriophages), but are not limited thereto. Those skilled in the art can construct vectors using standard recombination techniques (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).

[0099] In one instance, when preparing the vector, depending on the host cell type for which the Fc domain variant or antibody containing it or its immunologically active fragment is to be produced, expression regulatory sequences such as promoters, terminators, and enhancers, membrane-targeting or secretory sequences may be appropriately selected and combined in various ways according to the purpose.

[0100] In this invention, the term "expression vector" refers to a vector containing at least a portion of the nucleic acid sequence encoding a transcribed gene product. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. Expression vectors may contain a variety of regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.

[0101] In this invention, the term "host cell" includes eukaryotes and prokaryotes, referring to any transformable organism capable of replicating a vector or expressing the gene encoded by the vector. Host cells can be transfected or transformed by the vector, which refers to the process of transferring or introducing exogenous nucleic acid molecules into the host cell.

[0102] In one instance, the host cell may be a bacterial cell or an animal cell; the animal cell line may be a CHO cell, HEK cell, or NSO cell; and the bacteria may be Escherichia coli.

[0103] In one embodiment, the present invention relates to a fusion protein in which a human Fc domain variant or antibody or its immunologically active fragment is linked to a carrier molecule.

[0104] In one instance, the carrier molecule may be a detection agent, therapeutic agent, drug, peptide, growth factor, cytokine, receptor trap, compound, carbohydrate moiety, enzyme, antibody or fragment thereof, DNA molecule, viral vector or cytotoxic agent; or one or more liposomes or nanocarriers loaded with a detection agent, therapeutic agent, drug, peptide, enzyme, antibody or fragment thereof, DNA molecule, viral vector or cytotoxic agent; or one or more nanoparticles, nanowires, nanotubes or quantum dots.

[0105] In one instance, the fusion protein may be an agonist antibody, an antagonist antibody, or an antibody therapeutic agent.

[0106] In one embodiment, the present invention relates to an Fc-fusion protein formed by fusing a human antibody Fc domain variant of the present invention with a protein therapeutic agent.

[0107] In one instance, the protein therapeutic agent may be an immune checkpoint protein or immune effector cell-specific targeting molecule that is a T-cell modulatory polypeptide (TMP), an immune checkpoint inhibitor antibody, a bispecific immune cell engaging bispecific antibody, an agonist antibody, or an antagonist antibody.

[0108] In one instance, the immunotherapeutic cells may be effector T cells, regulatory T cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, or B cells.

[0109] In one instance, the immune checkpoint protein may be CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, 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.

[0110] In one instance, the immune checkpoint inhibitor antibody may be atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents.

[0111] In one embodiment, the present invention relates to an antibody therapeutic agent comprising the antibody of the present invention or an immunologically active fragment thereof and a pharmaceutical portion.

[0112] In one example, the drug component could be an immunomodulatory drug (IMiD), an immunogenic cell death inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a chimeric antigen receptor (CAR) cell therapy agent, or an oncolytic drug. Drugs, immunotherapeutic agents, cytotoxic agents, angiogenesis inhibitors, kinase inhibitors, co-stimulatory molecule blockers, adhesion molecule blockers, anti-cytokine agents, anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, TNF-α cross-linking agents, TRAIL cross-linking agents, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agents, tigretin, FcγIV-α, clobetasol, polyethylene glycol FcγIV, prednisone, romidixin, bexarotine, methotrexate, triamcinolone acetonide cream, anti-chemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable biomarkers or reporter molecules, TNF antagonists, antirheumatic agents, muscle relaxants, anesthetics, non-steroidal anti-inflammatory drugs (NSAIDs). Drugs, analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blocking agents, antibacterial agents, psoriasis treatment agents, corticosteroids, anabolic steroids, erythropoietin, immunizations, immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, psychotropic drugs, stimulants, asthma medications, beta-agonists, inhaled steroids, adrenaline 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.

[0113] In one instance, the antibody therapeutic agent may have reduced effector function.

[0114] In one instance, the antibody therapeutic agent could be an immune checkpoint inhibitor or a bispecific immune cell engager.

[0115] In one embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising, as an active ingredient, a human antibody Fc domain variant of the present invention, an antibody containing the present invention or an immunologically active fragment thereof, an Fc-fusion protein or an antibody therapeutic agent.

[0116] In one instance, cancer can be any of the following: 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 and neck cancer, cervical cancer, ovarian cancer, colon cancer, small bowel cancer, rectal cancer, fallopian tube cancer, anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, 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 ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem gliomas, and pituitary adenomas.

[0117] In one example, the composition of the present invention may further comprise an immunogenic cell death inducer, which may be selected from anthracycline anticancer agents, taxane anticancer agents, anti-EGFR antibodies, BK channel agonists, bortezomib, and cardiac glycosides. The group consists of one or more of the following: glycosides, cyclophosphamide anticancer agents, GADD34 / PP1 inhibitors, LV-tSMAC, measles virus, bleomycin, mitoxantrone, or oxaliplatin. Anthracycline anticancer agents may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin. Taxane anticancer agents may be paclitaxel or docetaxel.

[0118] The pharmaceutical compositions of the present invention can be used as a standalone therapy or in combination with other conventional biological therapies, chemotherapy, or radiotherapy. When used in such combination therapy, cancer can be treated more effectively.

[0119] The pharmaceutical composition of the present invention for the prevention or treatment of cancer can enhance the cancer treatment efficacy of conventional anticancer agents by enhancing the death effect on cancer cells when administered co-administered with chemopreventive drugs (anticancer agents). Co-administration can be performed simultaneously or sequentially with the said anticancer agent. Examples of the anticancer agents include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anticancer antibiotics such as actinomycin D, plicamycin, and mitomycin C; and plant alkaloids. Alkaloids, such as vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan, but not limited to these.

[0120] In this invention, the term "prevention" refers to all actions that inhibit or delay the occurrence, spread, and recurrence of cancer by applying the pharmaceutical composition of this invention.

[0121] The term "treatment" as used in this invention refers to all actions that kill cancer cells or improve or beneficially alter cancer symptoms by applying the compositions of this invention. Those skilled in the art can refer to materials provided by the Korean Medical Association, etc., to understand the specific criteria for the diseases in which the compositions of this invention are effective, and can determine the degree of improvement, enhancement, and treatment.

[0122] In this invention, the "therapeutic effective amount" used with the active ingredient refers to a pharmaceutically acceptable amount of salt of the composition capable of effectively preventing or treating the target disease. The therapeutic effective amount of the compositions of this invention may vary due to various factors, such as route of administration, target site, and patient condition. Therefore, when used in humans, the dosage should be appropriately determined based on safety and efficacy. The human dosage can also be estimated based on the effective dosage determined in animal studies. Such factors to be considered when determining the effective amount are, for example, found in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW. It is described in "Remington's Pharmaceutical Sciences", 18th ed. (1990), Mack Publishing Co., edited by Martin.

[0123] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. As used herein, the term "pharmaceutically effective amount" refers to a dose sufficient to treat a disease without causing side effects, given a reasonable benefit / risk ratio. Effective dose levels can be determined based on a variety of factors, including the patient's health status, cancer type and severity, drug activity and sensitivity, method of administration, time of administration, route of administration and excretion rate, duration of treatment, combination or simultaneous use of drugs, and other factors known in the medical field. The compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or administered once or multiple times. Considering all of the aforementioned factors, it is important to achieve maximum efficacy with the minimum amount and without causing side effects; this amount can be readily determined by those skilled in the art.

[0124] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable additives. In this case, pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silica, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, omeprazole, sodium carboxymethyl starch, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, glucose, sorbitol, and talc. The content of pharmaceutically acceptable additives in the present invention is preferably from 0.1 parts by weight to 90 parts by weight relative to the composition, but is not limited thereto.

[0125] The compositions of the present invention may also contain carriers, diluents, excipients, or combinations thereof commonly used in biological agents. There are no particular limitations on pharmaceutically acceptable carriers, provided they are suitable for in vivo delivery of the composition. For example, compounds described in the Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered salt solutions, glucose solutions, maltodextrin solutions, glycerol, ethanol, and one or more of these components may be used. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added as needed. Furthermore, various primary dosage forms, such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, can be formulated by adding diluents, dispersants, surfactants, binders, and lubricants. Furthermore, formulations may be preferably made according to appropriate methods in the art or those disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990), depending on the disease or ingredient.

[0126] The compositions of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally) or orally according to the intended method, with dosage ranges varying depending on the patient's weight, age, sex, health status, diet, time of administration, route of administration, excretion rate, and disease severity. The daily dose of the compositions of the present invention is 0.0001–10 mg / ml, preferably 0.0001–5 mg / ml, and preferably administered once or multiple times daily.

[0127] The oral liquid formulations of the compositions of the present invention include suspensions, solutions, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.

[0128] In one embodiment, the present invention relates to a method for preparing a human antibody Fc domain variant, the method comprising: step a), culturing a host cell containing a vector, the vector containing a nucleic acid molecule encoding the human antibody Fc domain variant of the present invention; and step b), recovering a polypeptide expressed by the host cell.

[0129] In one embodiment, the present invention relates to a method for preparing an antibody or a fragment thereof with reduced effector function, the method comprising: step a) culturing a host cell containing a vector, the vector containing a nucleic acid molecule encoding the antibody or an immunologically active fragment thereof of the present invention; and step b) purifying the antibody expressed from the host cell.

[0130] In one instance, antibody purification may include filtration, high-performance liquid chromatography (HPLC), anion exchange or cation exchange, affinity chromatography, or a combination thereof, preferably using affinity chromatography with protein A.

[0131] In one embodiment, the present invention relates to the use of an antibody comprising a variant of the Fc domain of the present invention or an immunologically active fragment thereof in the preparation of an antibody therapeutic agent.

[0132] In one embodiment, 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 an immunologically active fragment thereof, or an antibody therapeutic agent of the present invention in the prevention or treatment of cancer.

[0133] In one embodiment, the present invention relates to a cancer treatment method 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 an immunologically active fragment thereof, or an antibody therapeutic agent of the present invention. Detailed Implementation

[0134] The present invention will be described in more detail through the following embodiments. However, the following embodiments are intended to specifically describe the content of the present invention, and not to limit the present invention.

[0135] Example 1. Preparation of IgG4 Fc variants that prevent Fab arm exchange and eliminate FcγRs binding.

[0136] In order to prepare Fc variants that prevent the binding of wild-type human IgG4 in a half-antibody form and eliminate the binding force of FcγRs to C1q to avoid the toxicity of conventional S228P Fc variants, various Fab arm exchange blocking variants were prepared by introducing cysteine ​​into the lower hinge of the glycosylated Fc variant SL001 (E233C) that eliminates the binding force of FcγRs, which was previously studied by the inventors (patent application number 10-2023-0077425). (Table 1)

[0137] Table 1

[0138]

[0139] Example 2. Expression and purification of the Fc variant of glycosylated pembrolizumab IgG4.

[0140] The glycosylated Fc variant prepared in Example 1 was cloned into the pembrolizumab heavy chain gene (model antibody) without the Fab arm exchange variant to prepare an expression vector. Then, in 3 ml of Freestyle 293 expression medium (Gibco, 12338-018), the heavy chain gene and light chain gene of the variant were first mixed at a 1:1 ratio, then mixed at a PEI:variant gene ratio of 4:1, and incubated at room temperature for 20 minutes. This was then combined with the mixture prepared the previous day at a 2×10⁻⁶ ratio. 6Expi293F cells were passaged at a density of cells / ml and cultured for 7 days in a CO2 shaker at 37°C, 125 rpm, and 8% CO2. The mixture was then centrifuged, and only the supernatant was collected. The supernatant was equilibrated with 25X PBS and filtered through a 0.2 μm syringe filter for later use. Protein A resin was then added to the medium containing the pembrolizumab Fc variant and stirred at 4°C for 16 hours. The resin was recovered by spin-down, washed with 2 ml of PBS, and eluted with 600 μl of 100 mM glycine (pH 2.7) buffer. The eluent was neutralized with 200 μl of 1 M Tris-HCl (pH 8.0) and the buffer was exchanged using Amicon Ultra-4 30K ultrafiltration centrifugal filter units (Merck Millipore, UFC503096). SDS-PAGE gel analysis confirmed that the glycosylated pembrolizumab Fc variant was purified to high purity. Specifically, the wild-type IgG4 antibody produced a hapten (75 kDa), while the glycosylated pembrolizumab Fc variant of this invention did not produce a hapten, confirming that Fab arm exchange was prevented. Figure 1 ).

[0141] Example 3. Binding affinity analysis of glycosylated pembrolizumab IgG4 Fc variant to human FcγRs

[0142] 3-1. Expression and purification of human FcγRs

[0143] To analyze the binding affinity of the glycosylated Fc variants of this invention to FcγRs using ELISA, FcγRI-GST, FcγRIIIa-131H-GST, FcγRIIIa-131R-GST, FcγRIIIb-GST, FcγRIIIIa-158V-GST, and FcγRIIIIa-158F-GST were cloned into animal cell expression vectors, transfected into Expi293F cells with PEI, and cultured for 7 days at 37°C, 125 rpm, and 8% CO2. After culture, the supernatant was recovered and equilibrated with 25X PBS, and each receptor protein was purified by anti-GST affinity chromatography. Then, SDS-PAGE analysis was performed to confirm that FcγRI-GST, FcγRIIIa-131H-GST, FcγRIIIa-131R-GST, FcγRIIIb-GST, FcγRIIIIa-158V-GST, and FcγRIIIIa-158F-GST were purified to high purity. Figure 2 ).

[0144] 3-2. Analysis of the binding force with FcγRs

[0145] To confirm the FcγRs binding affinity of the purified glycosylated pembrolizumab Fc variant in Example 2, an ELISA analysis was performed. Specifically, the purified FcγRs-GST (FcγRI-GST, FcγRⅡa-131H-GST, FcγRⅡa-131R-GST, FcγRⅡb-GST, FcγRⅢa-158V-GST and FcγRⅢa-158F-GST) from Example 3-1 was diluted to 4 μg / ml with 0.05M Na2CO3 (pH 9.6), and then added to a flat bottom polystyrene high bind 96 well microplate (Costar, 3590), 50 μl per well, and fixed at 4°C for 16 hours. Then, it was 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 the glycosylated pembrolizumab Fc variant, serially diluted with 1% skim milk powder, was aliquoted into each well and reacted at room temperature for 1 hour. Following washing, the antibody was reacted with 50 μl of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and the reaction was terminated by adding 50 μl of 2M H2SO4 to each well. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0146] The results showed that, compared with the wild-type IgG4 antibody, the Stapled Fc-1 (A231C) variant had reduced binding affinity to all FcγRs except FcγRI, the Stapled Fc-2 (P232C) variant had similar binding affinity to FcγRs as the wild-type IgG4 antibody, and the Stapled Fc-4 (F234C) variant had eliminated binding affinity to all FcγRs except FcγRI. Figure 3 In particular, the binding affinity of the Stapled Fc-5 (L235C) variant to all FcγRs was eliminated, exhibiting significantly reduced FcγR binding affinity compared to the existing S228P / L235E variant (SPLE), which previously inhibited Fab arm exchange and reduced FcγR binding affinity in clinically used variants. Figure 3 ).

[0147] Example 4. Binding affinity analysis of glycosylated pembrolizumab IgG4 Fc variant to mouse FcγRs

[0148] 4-1. Expression and purification of mouse FcγRs

[0149] To analyze the binding affinity of the glycosylated Fc variants of this invention to mouse FcγRs, which are widely used as preclinical animal models, using ELISA, mouse FcγRI-GST, mouse FcγRIIIb-GST, mouse FcγRIIII-GST, and mouse FcγRIIV-GST were cloned into animal cell expression vectors, transfected into Expi293F cells with PEI, and cultured for 7 days at 37°C, 125 rpm, and 8% CO2. After culture, the supernatant was recovered and equilibrated with 1X PBS, and each receptor protein was purified by anti-GST affinity chromatography. Then, SDS-PAGE analysis confirmed that mouse FcγRI-GST, mouse FcγRIIIb-GST, mouse FcγRIIII-GST, and mouse FcγRIIV-GST were purified to high purity. Figure 4 ).

[0150] 4-2. Binding affinity analysis of mice to FcγRs

[0151] To confirm the binding affinity of the purified glycosylated pembrolizumab Fc variant from Example 2 to mouse FcγRs, an ELISA analysis was performed. Specifically, the mouse FcγRI-GST, mouse FcγRIIIb-GST, mouse FcγRIIII-GST, and mouse FcγRIIV-GST purified in Example 4-1 were diluted to 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), and then added to a flat-bottomed polystyrene high-binding-aft 96-well microplate (Costar, 3590), 50 μl per well. The plate was fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk powder (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of the glycosylated pembrolizumab Fc variant, serially diluted with 1% skim milk powder, was aliquoted into each well and reacted at room temperature for 1 hour. Following washing, the antibody was reacted with 50 μl of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and the reaction was terminated by adding 50 μl of 2M H2SO4 to each well. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0152] The results showed that the Stapled Fc-5 (L235C) variant had significantly lower FcγR binding affinity than the wild-type IgG4 antibody and the conventional existing S228P / L235E variant (SPLE), and in particular, binding affinity with all mouse FcγRs was completely eliminated. Figure 5 ).

[0153] Example 5. Binding affinity analysis of glycosylated pembrolizumab Fc variant to monkey FcγRs

[0154] 5-1. Expression and purification of FcγRs in cynomolgus monkeys

[0155] To analyze the binding affinity of the glycosylated Fc variants of this invention to cynomolgus monkey FcγRs, which are widely used as preclinical animal models, using ELISA, cynomolgus monkey FcγRI-GST, FcγRI1a-GST, FcγRI1b-GST, and FcγRI3-GST were cloned into animal cell expression vectors, transfected into Expi293F cells with PEI, and cultured for 7 days at 37°C, 125 rpm, and 8% CO2. After culture, the supernatant was recovered and equilibrated with 25X PBS, and each receptor protein was purified by anti-GST affinity chromatography. Then, SDS-PAGE analysis confirmed that cynomolgus monkey FcγRI-GST, FcγRI1a-GST, FcγRI1b-GST, and FcγRI3-GST were purified to high purity. Figure 6 ).

[0156] 5-2. Analysis of the binding force between cynomolgus monkeys and FcγRs

[0157] To confirm the binding affinity of the purified glycosylated pembrolizumab Fc variant from Example 2 to cynomolgus FcγRs, an ELISA analysis was performed. Specifically, the cynomolgus FcγRI-GST, FcγRIIIa-GST, FcγRIIIb-GST, and FcγRIIII-GST purified in Example 5-1 were diluted to 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), and then added to a flat-bottomed polystyrene high-binding-aft 96-well microplate (Costar, 3590), 50 μl per well. The plate was fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk powder (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of the glycosylated pembrolizumab Fc variant, serially diluted with 1% skim milk powder, was aliquoted into each well and reacted at room temperature for 1 hour. Following washing, the antibody was reacted with 50 μl of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and the reaction was terminated by adding 50 μl of 2MH2SO4 to each well. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0158] The results showed that the Stapled Fc-5 (L235C) variant had significantly lower FcγR binding affinity than the wild-type IgG4 antibody and the conventional existing S228P / L235E variant (SPLE), and in particular, binding affinity with all cynomolgus monkey FcγRs was completely eliminated. Figure 7 ).

[0159] Example 6. Binding affinity analysis of glycosylated pembrolizumab Fc variant to human C1q

[0160] To confirm the binding affinity of the purified glycosylated pembrolizumab Fc variant to C1q in Example 2, an ELISA analysis was performed. The glycosylated pembrolizumab Fc variant, diluted to 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), was added to each well of a flat-bottom polystyrene high bind 96-well microplate (Costar, 3590), and fixed at 4°C for 16 hours. Then, it was blocked with 100 μl of 4% skim milk powder (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of C1q (Quidel, A400) protein, serially diluted with 1% skim milk powder, was aliquoted into each well and reacted at room temperature for 1 hour. After washing, the antibody was reacted with 50 μl of anti-C1q-HRP (Invitrogen, PA1-84324) at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and then 50 μl of 2M H2SO4 was added to each well to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0161] The results showed that none of the Fc variants of the glycosylated pembrolizumab of the present invention had binding affinity to C1q. Figure 8 ).

[0162] Example 7. In vivo half-life analysis of glycosylated pembrolizumab Fc variant

[0163] 7-1. Analysis of the binding force between human FcRn

[0164] To confirm the binding affinity of the glycosylated Fc variant of this invention to human FcRn, which participates in the in vivo half-life, ELISA analysis was performed based on pH. First, FcRn-GST was cloned into an animal cell expression vector and transfected into Expi293F cells with PEI. The cells were then cultured for 7 days at 37°C, 125 rpm, and 8% CO2. After culture, the supernatant was recovered and equilibrated with 25X PBS. FcRn-GST was purified by anti-GST affinity chromatography, and its high purity was confirmed by SDS-PAGE analysis. Figure 9Furthermore, the purified glycosylated pembrolizumab Fc variant from Example 2 was diluted to 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), and then added to a flat-bottomed polystyrene high-binding 96-well microplate (Costar, 3590), 50 μl per well. The plate was fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk powder (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 with 1% skim milk powder (pH 6.0 / pH 7.4), 50 μl was dispensed into each well, and the plate was reacted at room temperature for 1 hour. After washing, each well was treated with 50 μl of anti-GST-HRP conjugate (Conjugate) (GE Healthcare, RPN1236V) and the antibody reaction was carried out at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and then 50 μl of 2M H2SO4 was added to each well to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0165] The results showed that all Fc variants of the glycosylated pembrolizumab of the present invention retained the FcRn characteristics of the wild-type IgG4 antibody. In particular, the Stapled Fc-5 (L235C) variant showed significantly enhanced binding affinity to FcRn at pH 6.0 compared to the wild-type IgG4 antibody. Figure 10 It was confirmed that its half-life is longer than that of the wild type.

[0166] 7-2. In vivo half-life analysis in mice expressing human FcRn

[0167] To compare the in vivo half-life of the Stapled Fc-5 (L235C) variant, which exhibits enhanced binding affinity to pH-dependent human FcRn, with the conventional variant S228P, pharmacokinetic analyses were performed using Tg276 female mice carrying human FcRn. Specifically, two or three mice of each variant were selected and intravenously (iv) injected with 5 mg / kg of pembrolizumab containing each variant. Blood samples were then collected from each mouse at 0.5, 24, 168, 336, 504, and 696 hours, and serum was collected by centrifugation at 1000 x g for 15 minutes. To measure serum antibody concentration using ELISA, HER2-His, diluted to 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), was added to each well of a flat-bottomed polystyrene high-binding-strength 96-well microplate (Costar, 3590), at a concentration of 50 μl. The plate was fixed at 4 °C for 16 hours, then blocked with 100 μl of 4% skim milk powder (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, pembrolizumab containing each variant was serially diluted with 1% skim milk powder starting from a concentration of 1 μg / ml to establish a standard curve. 50 μl of serum was aliquoted into each well, and the plate was reacted at room temperature for 1 hour. After washing, 50 μl of goat anti-human IgG H+L (Jackson Immunoresearch, 109-036-003) was used for antibody reaction at room temperature for 1 hour, followed by washing. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well for color development, and then 50 μl of 2M H2SO4 was added to each well to stop the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek). Antibody concentration was analyzed using a standard curve.

[0168] The results showed that the Stapled Fc-5 (L235C) variant had a longer blood half-life than the previously used Fab arm exchange-blocking variant S228P in clinical practice. Figure 11 ).

[0169] Example 8. Thermal stability analysis of glycosylated pembrolizumab Fc variant

[0170] To confirm the thermostability of the purified glycosylated pembrolizumab Fc variant in Example 2 at different temperatures, DSF (differential scanning fluorimetry) analysis was performed. Specifically, 45 μl of the glycosylated pembrolizumab Fc variant diluted to 5 μM with 1X PBS was mixed with 5 μl of SYPROOrange (Invitrogen, S6651) dye diluted to 200X and aliquoted into PCR plates (ThermoScientific, AB0900W) as a control group. 1X PBS was prepared in the same manner for use (all samples were tested in triplicate). An optically clear sealing film (ThermoScientific, AB1170) was attached to a plate containing the sample. Fluorescence intensity was measured using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, A28567) at a temperature increase of 0.03℃ / s within a temperature range of 25℃ to 99.9℃. The fluorescence values ​​at each temperature were fitted to a Boltzmann model using OriginPro software to determine the midpoint of the sigmoidal transition curve.

[0171] The results showed that the degradation temperature of the glycosylated pembrolizumab Fc variant of the present invention was higher than that of the wild-type IgG4 antibody, the conventional Fab arm exchange-blocking variant (S228P), and the existing variant SPLE (S228P / L235E). In particular, the StapledFc-5 (L235C) variant had the highest degradation temperature. Figure 12 It was confirmed that it has excellent thermal stability.

Claims

1. A variant of the Fc domain of a human antibody, characterized in that, In the Fc domain of the wild-type human antibody, one or more amino acids selected from the group consisting of amino acids 231, 232, 234 and 235 numbered according to the Kabat numbering system are replaced by a sequence different from the wild-type amino acid sequence.

2. The human antibody Fc domain variant according to claim 1, characterized in that, It includes substitutions of one or more amino acids selected from the group consisting of A231C, P232C, F234C, and L235C.

3. The human antibody Fc domain variant according to claim 1, characterized in that, The human antibody is IgG4.

4. The human antibody Fc domain variant according to claim 1, characterized in that, Compared to the Fc domain of wild-type human antibodies, its binding affinity to the Fcγ receptor is reduced.

5. The human antibody Fc domain variant according to claim 4, characterized in that, It is a human, mouse, or monkey Fcγ receptor.

6. The human antibody Fc domain variant according to claim 1, characterized in that, Compared to the Fc domain of wild-type human antibodies, its binding affinity to C1q is reduced.

7. The human antibody Fc domain variant according to claim 1, characterized in that, Compared to the Fc domain of wild-type human antibodies, its effector function is reduced.

8. The human antibody Fc domain variant according to claim 7, characterized in that, The effector function is an Fc-mediated effector function, selected from C1q binding, complement activation, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, Fc receptor binding including Fc-γ receptor binding, protein A binding, protein G binding, antibody-dependent cell-mediated phagocytosis, complement-dependent cell-mediated cytotoxicity, complement-enhanced cytotoxicity, opsonization, endocytosis of Fc-containing peptides, target downregulation, antibody-drug conjugate uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

9. The human antibody Fc domain variant according to claim 1, characterized in that, Compared with the Fc domain of wild-type human antibodies, its thermal stability is improved.

10. The human antibody Fc domain variant according to claim 1, characterized in that, Compared with the Fc domain of wild-type human antibodies, its in vivo half-life is prolonged.

11. An antibody or an immunologically active fragment thereof, characterized in that, It includes a variant of the human antibody Fc domain as described in claim 1.

12. The antibody or its immunologically active fragment according to claim 11, characterized in that, Compared with wild-type human antibodies, its binding affinity to the Fcγ receptor is reduced.

13. The antibody or its immunologically active fragment according to claim 11, characterized in that, Compared with wild-type human antibodies, its binding affinity to C1q is reduced.

14. The antibody or its immunologically active fragment according to claim 11, characterized in that, Compared to wild-type human antibodies, their effector function is reduced.

15. The antibody or its immunologically active fragment according to claim 11, characterized in that, Antibodies can be polyclonal antibodies, monoclonal antibodies, mini antibodies, domain antibodies, bispecific antibodies, IgG-like bispecific antibodies, bispecific immune cell linker antibodies, antibody mimics, chimeric antibodies, antibody conjugates, human antibodies, humanized antibodies, bivalent antibodies, or bispecific molecules.

16. A nucleic acid molecule, characterized in that, Encoding the human antibody Fc domain variant according to claim 1, the antibody according to claim 11, or an immunologically active fragment thereof.

17. An Fc-fusion protein, characterized in that, It is formed by fusing the human antibody Fc domain variant according to claim 1 with a protein therapeutic agent.

18. The Fc-fusion protein according to claim 17, characterized in that, Protein therapeutic agents include immune checkpoint proteins or immune effector cell-specific targeting molecules that are T-cell regulatory peptides, immune checkpoint inhibitor antibodies, immune cell-binding bispecific antibodies, agonist antibodies, or antagonist antibodies.

19. The Fc-fusion protein according to claim 18, characterized in that, Immune checkpoint proteins include CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, 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.

20. The Fc-fusion protein according to claim 18, characterized in that, Immune checkpoint inhibitor antibodies include atezolizumab, avelumumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monozumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents.

21. An antibody therapeutic agent, characterized in that, It comprises the antibody or its immunologically active fragment as described in claim 11 and the pharmaceutical portion.

22. The antibody therapeutic agent according to claim 21, characterized in that, The drug component includes immunomodulatory drugs, immunogenic cell death inducers, microtubule formation inhibitors, meiosis inhibitors, topoisomerase inhibitors, DNA intercalation agents, toxins, chimeric antigen receptor cell therapy agents, oncolytic drugs, immunotherapeutic agents, cytotoxic agents, angiogenesis inhibitors, kinase inhibitors, co-stimulatory molecule blockers, adhesion molecule blockers, anti-cytokine agents, anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, TNF-α cross-linking agents, TRAIL cross-linking agents, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agents, tigretin, interferon-α, clobetasol, pegylated interferon, prednisone, romidesin, bexarotine, and methotrexate. Triamcinolone acetonide cream, antichemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable biomarkers or reporter molecules, TNF antagonists, antirheumatic agents, muscle relaxants, anesthetics, nonsteroidal anti-inflammatory agents, analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blocking agents, antibacterial agents, psoriasis treatment agents, corticosteroids, anabolic steroids, erythropoietin, immunizations, immunoglobulins, immunosuppressants, growth hormone, hormone replacement drugs, radiopharmaceuticals, antidepressants, psychotropic drugs, stimulants, asthma medications, beta-agonists, inhaled steroids, adrenaline 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.

23. The antibody therapeutic agent according to claim 21, characterized in that, It has reduced effector functionality.

24. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, The active ingredient comprises a human antibody Fc domain variant according to claim 1, an antibody or its immunologically active fragment according to claim 11, an Fc-fusion protein according to claim 17, or an antibody therapeutic agent according to claim 21.

25. A method for preparing a human antibody Fc domain variant, characterized in that, include: Step a) Culturing host cells containing a vector, said vector containing a nucleic acid molecule encoding a variant of the human antibody Fc domain according to claim 1; as well as Step b), recover the peptides expressed by the host cells.

26. A method for preparing an antibody or fragment thereof with reduced effector function, characterized in that, include: Step a) Culturing host cells containing a vector, said vector containing a nucleic acid molecule encoding the antibody or an immunologically active fragment thereof according to claim 11; as well as Step b), purify the antibody expressed from the host cell.

27. Use of an antibody comprising a variant of the Fc domain according to claim 1 or an immunologically active fragment thereof in the preparation of an antibody therapeutic agent.

28. Use of a human antibody Fc domain variant according to claim 1, an antibody according to claim 11 or an immunologically active fragment thereof, or an antibody therapeutic agent according to claim 21 in the prevention or treatment of cancer.

29. A cancer treatment method, characterized in that, The procedure includes administering a pharmaceutically effective amount of the human antibody Fc domain variant of claim 1, the antibody of claim 11 or its immunomodulatory fragment thereof, or the antibody therapeutic agent of claim 21 to a subject suffering from cancer.

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