Antibody variants and uses thereof

Abstract

To provide polypeptides and related antibodies comprising a variant Fc domain.SOLUTION: The present invention provides a method of increasing an effector function of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, the method comprising introducing a mutation to the parent polypeptide in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W; a variant and composition thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to polypeptides and related antibodies comprising variant Fc domains. More particularly, the present invention relates to antibodies or polypeptides containing an Fc domain that have altered effector function due to one or more amino acid modifications in the Fc domain. [Background technology]

[0002] Background of the Invention Effector functions mediated by the Fc region of an antibody enable the destruction of foreign entities, such as killing pathogens and eliminating and degrading antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by binding of the Fc region to cells bearing Fc receptors (FcR), while complement-dependent cytotoxicity (CDC) is initiated by binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

[0003] Each IgG antibody contains two binding sites for C1q, one in each heavy chain constant (Fc) region. However, single IgG molecules in solution lack the affinity of monomeric IgG for C1q (K d Almost 10 -4 M) and does not activate complement (Sledge et al., 1973 J. Biol. Chem. 248,2818-13 (Non-Patent Document 1); Hughes-Jones et al., 1979 Mol. Immunol. 16,697-701 (Non-Patent Document 2)). Antigen-triggered IgG association is mediated by the much tighter binding of multivalent C1q molecules (K d Almost 10 -8IgM) and complement activation (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM naturally exists as a covalently linked pentamer or hexamer, and upon binding to expressed or immobilized cellular antigens, IgM pentamers and hexamers can efficiently induce CDC. Antigen binding is a prerequisite for inducing a conformational change in IgM that exposes the C1q binding site (Feinstein et al., 1986, Immunology Today, 169-174).

[0004] It has also been suggested that IgG can activate complement by forming a hexamer ring structure through interaction between the CH2 / CH3 domains of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69 (Non-Patent Document 5)). Evidence supporting the existence of such hexameric IgG structures has been found in two-dimensional crystals (Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, D. M. ed.), pp. 17.1-17.5. Blackwell, Edinburgh (Non-Patent Document 6); Pinteric et al., 1971 Immunochem. 8, 1041-5 (Non-Patent Document 7)) and three-dimensional crystals, as well as for IgG1, IgG2a and IgG4 and human Fc in solution (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115 (Non-Patent Document 8)). Hexamer ring formation was also observed in the crystal structure of the b12 human IgG1κ antibody (1HZH in the PDB) directed against HIV-1 gp120 (Saphire et al., Science 2001 Aug 10;293(5532),1155-9). In the b12 hexamer ring, six accessible C1q binding sites were displayed on the hexamer surface, one from each of the six antibodies, and the remaining six binding sites faced downward.

[0005] C1q has a shape resembling a bunch of tulips, with six globular heads containing antibody-binding domains tethered to six collagen stalks [Perkins et al., 1985 Biochem J. 228, 13-26 (Non-Patent Document 10); Poon et al., 1983 J Mol Biol. 168, 563-77 (Non-Patent Document 11); Reid et al., 1983 Biochem Soc Trans 11, 1-12 (Non-Patent Document 12); Weiss et al., 1986 J. Mol. Biol. 189, 573-81 (Non-Patent Document 13)]. C1q has been found to fit from above into the b12 hexamer assembly in the 1HZH crystal structure, resulting in each of the six globular heads contacting one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., July 5-10, 2010; "Crystal Structure of an Intact Human IgG: Implications for HIV-1 Neutralization and Effector Function," Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, California, November 2000). Mutations of selected amino acids in the Fc interface observed between symmetrically related b12 antibodies in the crystal structure have been observed to reduce C1q binding avidity, indicating the contribution of these amino acids to intermolecular Fc:Fc interactions.

[0006] US 2011 / 0123440 (Patent Document 1) describes modified antibody Fc regions and uses thereof. The modified Fc regions have one or more amino acid substitutions.

[0007] US 2008 / 0089892 (Patent Document 2) describes polypeptide Fc region variants and compositions comprising these Fc region variants.

[0008] US 2010 / 0184959 (Patent Document 3) describes a method for providing Fc polypeptide variants with altered Fc ligand recognition and / or effector function.

[0009] US 2010 / 015133 (Patent Document 4) describes a method for producing polypeptides by modulating the association of polypeptides.

[0010] US 2010 / 105873 describes an integrated approach to generating multidomain protein therapeutics.

[0011] US Pat. No. 6,737,056 describes polypeptide variants with altered effector functions.

[0012] Previous efforts have been made to identify antibody Fc variants with enhanced effector function or other altered properties. Such studies have aimed, for example, at exchanging segments between IgG isotypes to create chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72 (Non-Patent Document 16)), or at generating amino acid substitutions within the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138 (Non-Patent Document 17)) or within or near the C1q binding site in the CH2 domain centered on residues D270, K322, P329 and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575 (Non-Patent Document 18); Michaelsen et al., 2009 Scand J Immunol 70, 553-564 (Non-Patent Document 19) and WO 99 / 51642 (Patent Document 7)). For example, Moore et al. (2010 mAbs 2(2), 181-189) describe the investigation of various combinations of S267E, H268F, S324T, S239D, I332E, G236A, and I332E for enhancing effector function via CDC or ADCC. Other Fc mutations that affect binding to Fc receptors (WO 2006 / 105062, WO 00 / 42072, U.S. Pat. No. 6,737,056, and U.S. Pat. No. 7,083,784) or the physical properties of the antibody (WO 2007 / 005612 A1) have also been suggested.

[0013] However, despite these and other advances in the art, there remains a need for new and improved antibody-based therapeutics. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] US 2011 / 0123440

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Non-licensed literature

[0015] [Non-licensed document 1] Sledge et al., 1973 J. Biol. Chem. 248,2818-13 [Non-licensed document 2] Hughes-Jones et al., 1979 Mol. Immunol. 16,697-701 [Non-licensed document 3] Burton et al., 1990 Mol. Immunol. 22, 161-206

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Summary of the Invention

[0016] The present invention provides polypeptide and antibody variants with enhanced effector function compared to their parent polypeptides / antibodies. Without being limited by theory, it is believed that these variants provide a more avid surface, allowing for more stable binding interactions between the Fc regions of two polypeptide / antibody molecules, thereby leading to enhanced effector functions, such as enhanced or more specific CDC responses. Certain variants are also characterized by improved ADCC responses, ADCP responses, and / or other enhanced effector functions. This sophisticated procedure of polypeptide / antibody engineering can be applied, for example, to enhance the activity or specificity of antibody-based therapeutics, as described herein.

[0017] Thus, in one aspect, the present invention relates to variants of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, which variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0018] The present invention also provides for the use of at least one such mutation to enhance an effector function mediated by a polypeptide or antibody upon binding to an antigen, for example, on the surface of a cell, cell membrane, or virion expressing the antigen.

[0019] In one aspect, when referred to herein as a "single variant," the variant has enhanced effector function compared to the parent polypeptide or antibody.

[0020] In one aspect, when referred to herein as a "double mutant," the mutant comprises at least two mutations in the segment and has improved effector function compared to a mutant, parent polypeptide or antibody, or both, comprising only one of the two mutations.

[0021] In one aspect, when referred to herein as a "mixed variant," the variant, when used in combination with a second variant of the same or a different polypeptide or antibody containing mutations at different amino acid residues in said segment, results in enhanced effector function compared to one or more of the variant, the second variant, and the parent polypeptide or antibody alone.

[0022] Typically, the mutations are amino acid substitutions, such as mutations that replace the parent amino acid residue with one of different size and / or physicochemical properties that facilitate the formation of new intermolecular Fc:Fc bonds or enhance the strength of an existing pairwise interaction. Exemplary amino acid residues for mutations of the present invention are shown in Table 1 and Tables 2A and 2B, along with exemplary amino acid substitutions. A non-limiting illustration of various aspects of the present invention is provided in Figure 1.

[0023] [The present invention 1001] A method for enhancing the effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the step of introducing a mutation into the parent polypeptide at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W. [The present invention 1002] 1001. A method for enhancing the effector function of a parent antibody, comprising the step of introducing a mutation into said parent antibody at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W. [The present invention 1003] A method for reducing an effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising introducing into the parent polypeptide a mutation at one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W, e.g., the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R. [The present invention 1004] A method of reducing an effector function of a parent antibody, comprising a step of introducing into the parent antibody a mutation at one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W, for example, the mutation at the position corresponding to K439 in the Fc region of human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of human IgG1 heavy chain is S440K / H / R. [The present invention 1005] 1005. The method of any of claims 1002 or 1004, wherein said parent antibody is a monospecific, bispecific or multispecific antibody. [The present invention 1006] The parent antibody is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or different antigens, and the first CH2-CH3 region binds to K409, T366, L368, K370, D399, F4 in the Fc region of a human IgG1 heavy chain. 1005. The method of claim 1005, wherein said first CH2-CH3 region comprises a further amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain; and wherein said further amino acid substitution in said first CH2-CH3 region is different from said further amino acid substitution in said second CH2-CH3 region. [The present invention 1007] The parent antibody is a bispecific antibody and comprises a first polypeptide and a second polypeptide, the first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, the second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, the first and second antigen-binding regions binding to different epitopes on the same antigen or different antigens, and the method further comprises: The method of claim 1005 or 1006, comprising the step of introducing a mutation into at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, for each of the H3 regions, with the proviso that the mutation at S440 is S440Y or S440W. [The present invention 1008] introducing a mutation at one or more positions other than S440 and K447; and (i) a mutation at each of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; (ii) mutations in the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, respectively, such as K447K / R / H and 448E / D in the Fc region of the human IgG1 heavy chain, preferably K447K and 448E in the Fc region of the human IgG1 heavy chain; or (iii) mutations at amino acid residues corresponding to K447, 448, and 449 in the Fc region of the human IgG1 heavy chain, for example, K447D / E, 448K / R / H, and 449P in the Fc region of the human IgG1 heavy chain, preferably K447E, 448K, and 449P in the Fc region of the human IgG1 heavy chain Any of the methods 1001 to 1002 and 1005 to 1007 of the present invention, further comprising the step of introducing: [The present invention 1009] 1008. The method of claim 10, further comprising introducing a mutation at one or more positions other than S440, and further introducing a mutation at each of the amino acid residues corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W. [The present invention 1010] The method of the present invention 1009, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R. [The present invention 1011] 1. A method of enhancing effector function of a combination of at least a first and a second parent polypeptide, wherein the at least a first and a second parent polypeptide each comprise an Fc domain and a binding region of an immunoglobulin, the method comprising: (i) to said at least first and / or second parent polypeptides, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain A method comprising the step of introducing mutations into one or more amino acid residues selected from the group consisting of: [The present invention 1012] 1. A method for enhancing the effector function of a combination of at least a first and a second parent antibody, wherein the at least a first and a second parent antibody each comprise an Fc domain and an antigen-binding region of an immunoglobulin, the method comprising: (i) to said at least first and / or second parent antibody, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain 1011. The method of claim 10, comprising the step of introducing mutations into one or more amino acid residues selected from the group consisting of: [The present invention 1013] 1012. The method of claim 1012, wherein the mutation in said at least first and / or second parent antibody is at at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. [The present invention 1014] The method of claim 1012 or 1013, comprising introducing mutations into both said first and second parent antibodies. [The present invention 1015] (i) introducing a mutation into said first parent antibody at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W; (ii) providing the second parent antibody, which does not contain mutations in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain; The method of the present invention 1012, comprising: [The present invention 1016] Any of the methods of claims 1012 to 1015, comprising a step of introducing a mutation at one or more positions other than S440, and further comprising the step of: (i) introducing a second mutation into said first parent antibody at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing into said second parent antibody a second mutation at an amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W; wherein steps (ii) and (iii) may alternatively be: (i) introducing into said first parent antibody a second mutation at an amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W; and (ii) introducing a second mutation into said second parent antibody at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain. [The present invention 1017] The method of the present invention 1016, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R. [The present invention 1018] 1018. The method of any of claims 1012 to 1017, wherein said at least first and second parent antibodies may each be selected from the group of monospecific, bispecific and multispecific antibodies. [The present invention 1019] 1018. The method of claim 1018, wherein said at least first and second parent antibodies are bispecific or multispecific antibodies binding to different epitopes on the same antigen or on different antigens. [The present invention 1020] The at least first and second parent antibodies are bispecific antibodies each comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or different antigens, and the first CH2-CH3 region binds to K409, T366, L368, K370, D39 in the Fc region of a human IgG1 heavy chain. 9, F405 and Y407; and wherein said second CH2-CH3 region comprises further amino acid substitutions at positions selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and wherein said further amino acid substitutions in said first CH2-CH3 region are different from said further amino acid substitutions in said second CH2-CH3 region. [The present invention 1021] Any of the methods of present inventions 1001 to 1002, 1005 to 1007, 1011 to 1015 and 1018 to 1020, wherein the mutation is in at least one amino acid residue selected from those corresponding to E345, E430, S440 to Y or W, and Q386 in the Fc region of human IgG1 heavy chain, and preferably the mutation is at least one amino acid substitution selected from the group consisting of substitution of E345 with R, Q, N or K, substitution of E430 with T, S or G, substitution of S440 with Y or W, and substitution of Q386 with K. [The present invention 1022] Any of the methods of the present inventions 1001 to 1002, 1005 to 1007, 1011 to 1015 and 1018 to 1020, comprising a step of introducing mutations into the parent antibody at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. [The present invention 1023] The method of claim 1022, comprising a step of introducing mutations into the parent antibody at least two amino acid residues selected from those corresponding to E345, E430, Q386 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W, for example, the mutations at the at least two amino acids being selected from mutation of E345 to R, Q, N or K, mutation of E430 to T, S or G, mutation of S440 to Y or W, or mutation of Q386 to K. [The present invention 1024] 1024. The method of any of claims 1001 to 1023, wherein said effector function is an Fc-mediated effector function selected from C1q binding, complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-γ receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof. [The present invention 1025] The method of any one of claims 1001 to 1002 and 1005 to 1024, wherein the effector function is enhanced when the antibody binds to its antigen. [The present invention 1026] 1026. The method of claim 1025, wherein said antigen is on the surface of an antigen-expressing cell, cell membrane, or virion. [The present invention 1027] 1. A method for inducing an effector response against a cell, cell membrane, or virion expressing a target to which a parent polypeptide comprising an immunoglobulin Fc domain and a binding region binds, comprising the steps of: (i) providing a parent polypeptide, or a combination of at least a first parent polypeptide and a second parent polypeptide, mutated according to any one of claims 1001 to 1024; and (ii) contacting a preparation of the mutated parent polypeptide of step (i) or a combination of at least a first mutated parent polypeptide and a second mutated parent polypeptide of step (i) with a cell, cell membrane or virion expressing the antigen in the presence of human complement or effector cells. [The present invention 1028] 1027. A method of inducing an effector response against a cell, cell membrane or virion expressing an antigen to which the parent antibody binds, comprising the steps of: (i) providing a parent antibody, or a combination of at least a first parent antibody and a second parent antibody, mutated according to any of inventions 1001 to 1024; and (ii) contacting a preparation of the mutated parent antibody of step (i) or a combination of at least a first mutated parent antibody and a second mutated parent antibody of step (i) with a cell, cell membrane or virion expressing the antigen in the presence of human complement or effector cells. [The present invention 1029] 1029. The method of claim 1027 or 1028, wherein said effector response is an Fc-mediated effector response selected from complement dependent cytotoxicity (CDC), C1q binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof. [The present invention 1030] 1. A method for increasing the specificity of a combination of at least first and second parent polypeptides comprising an Fc domain and a binding region of an immunoglobulin, comprising the steps of: A) (i) introducing a mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing a mutation into said second parent polypeptide at an amino acid residue at a position corresponding to S440 in the Fc region of human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; B) (i) introducing a mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and (ii) introducing mutations into the second parent polypeptide at amino acid residues at positions corresponding to K447K / R / H and 448P in the Fc region of human IgG1 heavy chain; or C) (i) introducing a mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and (ii) introducing mutations into the second parent polypeptide at amino acid residues at positions corresponding to K447K / R / H, 448K / R / H and 449P in the Fc region of human IgG1 heavy chain. [The present invention 1031] 1030. A method of increasing the specificity of a combination of at least a first and a second parent antibody, comprising the steps of: (i) introducing a mutation into the first parent antibody at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing a mutation into the second parent antibody at an amino acid residue at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W. [The present invention 1032] The method of the present invention 1031, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R. [The present invention 1033] The method of any of claims 1001 to 1032, wherein said parent antibody or said combination of a first parent antibody and a second parent antibody is human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE, and optionally a human full-length antibody, for example a human full-length IgG1 antibody. [The present invention 1034] A variant of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. [This invention 1035] A variant of a parent antibody comprising an antigen-binding region and an Fc domain, comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W. [The present invention 1036] 1035. The variant of claim 1035, wherein said mutation in at least one amino acid residue is selected from those corresponding to E345X, E430X, S440W / Y, Q386K, P247G, I253V, S254L, Q311L / W, D / E356R, E382V and Y436I in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid. [This invention 1037] The variant of 1036 of the present invention, wherein said mutation in at least one amino acid residue is selected from those corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid. [The present invention 1038] The mutation in at least one amino acid residue in the Fc region of a human IgG1 heavy chain E345R / Q / N / K / A / C / D / F / G / H / I / L / M / P / S / T / V / W / Y, E430T / S / G / A / C / D / F / H / I / L / K / M / N / P / Q / R / V / W / Y, S440W / Y, and Q386K A variant of 1037 of the present invention selected from those corresponding to: [This invention 1039] The variant of 1038 of the present invention, wherein said mutation in at least one amino acid residue is selected from those corresponding to E345R / Q / N / K, E430T / S / G, S440Y / W, and Q386K in the Fc region of human IgG1 heavy chain. [The present invention 1040] A variant of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain wherein the at least two amino acid mutations are different. [The present invention 1041] A variant of a parent antibody comprising an antigen-binding region and an Fc domain, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain A variant of 1040 of the present invention, comprising mutations in at least two amino acid residues selected from the group consisting of: [The present invention 1042] A variant of 1041 of the present invention, comprising mutations in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. [This invention 1043] A variant of 1042 of the invention, comprising mutations in at least two amino acid residues selected from those corresponding to E345X, E430X, S440W / Y, Q386K, P247G, I253V, S254L, Q311L / W, D / E356R, E382V and Y436I in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid. [This invention 1044] A variant of 1043 of the invention, comprising mutations in at least two amino acid residues selected from the group corresponding to those of E345X, E430X, S440W / Y and Q386K in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid. [This invention 1045] A variant of 1044 of the present invention, comprising mutations in at least two amino acid residues selected from those corresponding to E345R / Q / N / K / A / C / D / F / G / H / I / L / M / P / S / T / V / W / Y, E430T / S / G / A / C / D / F / H / I / L / K / M / N / P / Q / R / V / W / Y, S440W / Y, and Q386K in the Fc region of a human IgG1 heavy chain. [The present invention 1046] A variant of 1045 of the present invention, wherein said mutations in said at least two amino acid residues are selected from those corresponding to E345R / Q / N / K, E430T / S / G, S440Y / W, and Q386K in the Fc region of a human IgG1 heavy chain. [This invention 1047] the mutation is at a position other than S440 and K447, and the mutant is (i) a mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is not S440W or S440Y; (ii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain; The variant of any one of 1034 to 1046 of the present invention further comprises: [This invention 1048] A variant of 1047 of the present invention, wherein said mutation is at a position other than S440, and said variant further comprises a mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440W nor S440Y. [This invention 1049] A variant of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that it does not contain a mutation corresponding to S440 in the Fc region of the human IgG1 heavy chain, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and comprising a mutation in at least one amino acid residue selected from the group (i) an additional mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is not S440W or S440Y; (ii) an additional mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) additional mutations in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain; Including, variants. [The present invention 1050] A variant of a parent antibody comprising an antigen-binding region and an immunoglobulin Fc domain, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that it does not contain a mutation corresponding to S440 in the Fc region of the human IgG1 heavy chain, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and comprising a mutation in at least one amino acid residue selected from the group (i) an additional mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is not S440W or S440Y; (ii) an additional mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) additional mutations in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain; A variant of 1049 of the present invention, comprising: [This invention 1051] A variant of 1049 or 1050 of the present invention, comprising amino acid mutations at both positions corresponding to K439 and S440 in the Fc region of the IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W. [This invention 1052] Any of the mutants 1047 to 1051 of the present invention, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R. [This invention 1053] A variant of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, (i) a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, with the proviso that the mutation in S440 is neither S440Y nor S440W, e.g., the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R; (ii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain; Including, variants. [This invention 1054] A variant of a parent antibody comprising an antigen-binding region and an immunoglobulin Fc domain, comprising a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is neither S440Y nor S440W, e.g., the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R. [This invention 1055] A variant of any of 1034 to 1054 of the present invention, selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody. [This invention 1056] A bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or on different antigens; A variant of 1055 of the present invention, wherein the first and second CH2-CH3 regions each comprise a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. [This invention 1057] the first polypeptide comprises additional mutations at amino acid residues selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; and 1056. The variant of claim 10, wherein said second polypeptide comprises a further mutation at an amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and wherein said further mutation in said first polypeptide is different from said further mutation in said second polypeptide. [This invention 1058] The variant of any of 1034 to 1057, which is conjugated to a drug, toxin or radiolabel, for example conjugated to a toxin via a linker. [This invention 1059] A variant of any of 1034 to 1057 of the present invention, which is part of a fusion protein. [The present invention 1060] The variant of any of claims 1034 to 1059, which is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally a human full-length antibody, for example a human full-length IgG1 antibody. [The present invention 1061] A composition comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide, wherein the first variant comprises a first Fc domain and binding region of an immunoglobulin and the second variant comprises a second Fc domain and binding region of an immunoglobulin; and (i) the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; or (ii) the first variant contains a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant contains a mutation at a position corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises mutations at positions corresponding to K447K / R / H, 448K / R / H, and 449P in the Fc region of a human IgG1 heavy chain. composition. [This invention 1062] A composition comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin; and (i) the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; or (ii) the first variant contains a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant contains a mutation at a position corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises mutations at positions corresponding to K447K / R / H, 448K / R / H, and 449P in the Fc region of a human IgG1 heavy chain. The composition of the present invention 1061. [This invention 1063] 1062. A composition of matter comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, wherein the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprises a mutation at a position corresponding to S440, with the proviso that the mutation at S440 is neither S440Y nor S440W. [The present invention 1064] The first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the first variant is (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; The composition of the present invention 1063, comprising: [This invention 1065] The first mutant (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and the second mutant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, and the mutation is neither S440Y nor S440W; Composition of the present invention 1063. [The present invention 1066] the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; The composition of the present invention 1063, comprising: [This invention 1067] Any of the compositions of the present inventions 1061 to 1066, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R. [The present invention 1068] 1. A composition comprising a first variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, and a second variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the first variant comprises a first Fc domain and a first antigen-binding region of an immunoglobulin, and wherein the first variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a first mutation in at least one amino acid residue selected from the group The second mutant is (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain does not contain a mutation in an amino acid residue selected from the group composition. [The present invention 1069] 1. A composition comprising a first variant of an antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and a first antigen-binding region of an immunoglobulin, and wherein the first variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a first mutation in at least one amino acid residue selected from the group the second variant comprises a second Fc domain and a second antigen-binding region of an immunoglobulin, and the second variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain does not contain a mutation in an amino acid residue selected from the group Composition of the present invention 1068. [The present invention 1070] Any of the compositions of claims 1061 to 1067, wherein the first and second mutants comprise a mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain. [This invention 1071] The composition of claim 1070, wherein the mutations in said first and second variants are different. [This invention 1072] 1068 or 1069, wherein said first variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W. [This invention 1073] The composition of any of claims 1061 to 1072, wherein the first variant and the second variant are each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, and optionally each are a human full-length antibody, for example each are a human full-length IgG1 antibody. [This invention 1074] The composition of any of claims 1061 to 1073, wherein said first mutant and said second mutant are selected from a monospecific antibody, a bispecific antibody or a multispecific antibody. [This invention 1075] The composition of any of claims 1061 to 1074, wherein said first and second variants bind to different epitopes on the same antigen or on different antigens. [This invention 1076] Any of the compositions of claims 1061 to 1075, wherein one or both of the first mutant and the second mutant are conjugated to a drug, a toxin or a radioactive label, for example, one or both of the first mutant and the second mutant are conjugated to a toxin via a linker. [This invention 1077] 1076. The composition of any of claims 1061 to 1075, wherein one or both of said first variant and second variant are part of a fusion protein. [This invention 1078] A composition comprising any one of the mutants of the present invention 1034 to 1060 or any one of the compositions of the present invention 1061 to 1077 and a pharmaceutically acceptable carrier. [This invention 1079] A kit-of-parts comprising a first variant and a second variant as defined in any of claims 1034 to 1060 for simultaneous, separate or sequential use in therapy. [The present invention 1080] A variant, composition or kit-of-parts of any of 1034 to 1079 for the treatment of a disease such as cancer. [This invention 1081] A method for the treatment of a human comprising administering a variant, composition or kit-of-parts of any of claims 1034 to 1079. [This invention 1082] A method for the treatment of cancer in a human comprising administering a variant, composition or kit-of-parts of any of 1034-1079 of the present invention. These and other aspects of the invention, particularly the various uses and therapeutic applications of the antibody variants, are described in further detail below. [Brief explanation of the drawings]

[0024] [Figure 1](A) Schematic representation of IgG molecules in hexamer formation. The dotted circles illustrate two adjacent Fc:Fc interaction pairs of two adjacent IgG molecules. The arrows in the boxes illustrate the viewing orientation of the illustrations in B, C, and D: two adjacent Fc molecules are rotated 90° (in the plane of the drawing) and looking from the Fab arms towards the CH3 domains. (B) Observed effect of oligomerization-enhancing mutations on CDC. Schematic representation illustrating Fc:Fc interaction pairs whose activity is enhanced by single and double mutant aspects of the invention. (C) Observed effect of oligomerization-inhibiting mutations on CDC. Schematic representation illustrating how at least two compensating oligomerization-inhibiting mutations, either combined in one molecule (double mutant aspect) or separated into two molecules (mixed mutant aspect), restore or enhance Fc:Fc interactions by double and mixed mutant aspects of the invention. The mixed mutant achieves specific effector function activation upon binding of both antibodies, which can recognize multiple different targets. (D) Theoretical effect of C1q binding-inhibitory mutations on CDC. Schematic representation of Fc:C1q interactions illustrating that if mutations inhibit C1q binding, they cannot be combined or mixed to restore CDC activity, because C1q cannot compensate for the defect introduced into the antibody. [Figure 2]Sequence alignment of the Fc segments of human IgG1, IgG1f, IgG2, IgG3, and IgG4, numbered according to the EU index as described in Kabat, using Clustal 2.1 software. The sequences shown represent residues 130-330 of the human IgG1 heavy chain constant region (SEQ ID NO:1; UniProt Accession No. P01857) and allotypic variant IgG1m(f) (SEQ ID NO:5), residues 126-326 of the IgG2 heavy chain constant region (SEQ ID NO:2; UniProt Accession No. P01859), residues 177-377 of the IgG3 heavy chain constant region (SEQ ID NO:3; UniProt Accession No. P01860), and residues 127-327 of the IgG4 heavy chain constant region (SEQ ID NO:4; UniProt Accession No. P01861). [Figure 3] Figures 3A and B: Sequence alignment of anti-EGFr antibody 2F8 in IgG1 (SEQ ID NO:6), IgG4 (SEQ ID NO:8), and (partial) IgG3 (SEQ ID NO:7) scaffolds, showing amino acid numbering according to the Kabat and EU index (both as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). [Figure 4] Detailed view of the K439 / S440 interaction between Fc of adjacent molecules (Fc and Fc', respectively) in a multimeric (e.g., hexamer) configuration, illustrating the interaction between wild-type, unmodified Fc and Fc' molecules. [Figure 5] Detailed view of the K439 / S440 interaction between Fc of adjacent molecules (Fc and Fc', respectively) in a multimeric (e.g., hexamer) arrangement, illustrating the interaction between mutant Fc and Fc' molecules containing K439E and S440K mutations. [Figure 6]C1q binding ELISA using 7D8 Fc:Fc variants. Microtiter plate wells were coated with a concentration series of the indicated antibodies and incubated with a fixed concentration of C1q. C1q binding efficiency was comparable to wild-type 7D8 for all coated variants except I253D. A representative of at least three experiments is shown. [Figure 7] CDC mediated by 7D8 mutants on CD20-positive Raji cells. To test CDC activity by measuring cell lysis, Raji cells were incubated with a concentration series of 7D8 mutants (K439E, S440K, K439E / S440K double mutant, and K439E+S440K mixed) and C1q. A representative graph of repeated experiments is shown. [Figure 8] CDC mediated by 7D8 mutants (7D8-WT, K439E, S440K, K439E / S440K double mutant, and K439E+S440K mixed mutant) on CD20+ Daudi cells. A concentration series of 7D8 mutants was examined for their ability to induce CDC. [Figure 9] CDC mediated by mutants of the CD38 antibody HuMAb 005 on CD38-positive cells. (A) CDC activity on Daudi cells by a concentration series of 005 mutants. (B) CDC activity on Raji cells by a concentration series of HuMAb 005 mutants. (C) CDC activity on Wien133 cells by the E345R mutant of HuMAb 005 with either 20% or 50% NHS. (D) CDC activity on Raji cells by the E345R mutants of HuMAb 005 and 7D8 with either 20% or 50% NHS. Non-purified antibody samples isolated from transient transfections were tested. Supernatant from mock-transfected cells was used as a negative control. [Figure 10]CDC by wild-type and E345R mutants of the CD38 antibody HuMAb 005 (A) and the CD20 antibody HuMAb 7D8 (B) in a competition experiment with Fc-binding peptides. Cytolysis was measured after CDC on antibody-opsonized Daudi cells incubated with a concentration series of the Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO:9). Unpurified antibody samples isolated from transient transfections were used. Supernatant from mock-transfected cells served as a negative control. [Figure 11] ADCC of wild-type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R against CD38-expressing Daudi cells. ADCC of PBMCs from one donor is shown and is expressed as percent lysis. [Figure 12] Binding of wild-type IgG1-7D8 and mutant IgG1-7D8-E345R to human, cynomolgus monkey, and mouse FcRn determined by ELISA at pH 6. [Figure 13] Plasma concentrations of wild-type IgG1-7D8, and the IgG1-7D8-E354R, IgG1-7D8-S440K and IgG1-7D8-K322A mutants after intravenous injection in SCID mice. [Figure 14] Figure 14A, B, C and D: CDC on CD20-positive and CD38-positive Wien133 cells. [Figure 15] Figure 15A and B: Evaluation of the in vivo activity of IgG1-7D8-E345R in a subcutaneous xenograft model using Raji-luc #2D1 cells. [Figure 16] Figures 16A and B: Evaluation of the in vivo activity of IgG1-005-E345R in a subcutaneous xenograft model using Raji-luc #2D1 cells. [Figure 17] CDC with a CD38 / EGFR bispecific antibody carrying the E345R mutation on CD38-positive, EGFR-negative Wien133 cells. [Figure 18]Figure 18A and B: CDC on CD20-positive, CD38-negative Wienl33 or Raji cells by CD20 / CD38 bispecific antibodies with and without the E345R mutation. [Figure 19] CDC on EGFR-positive A431 cells by the EGFR antibody 2F8 carrying the E345R mutation. [Figure 20] Figure 20A and B: CDC mediated by the E345R mutant antibody. [Figure 21] Colocalization analysis of TF antibody (FITC) with the lysosomal marker LAMP1 (APC). [Figure 22] Figures 22A-D: In a study of various B cell lines, introduction of E345R resulted in enhanced CDC-mediated killing compared to wild-type rituximab. [Figure 22E] In various B cell lines with comparable CD20 expression levels, introduction of E345R resulted in enhanced maximal CDC-mediated killing compared to wild-type rituximab, regardless of the expression levels of the complement regulatory proteins CD46 (A), CD55 (B), or CD59 (C). [Figure 23] CDC Kinetics. The E345R antibody results in more rapid and more substantial target cell lysis by CDC compared to the wild-type antibody. [Figure 24] CDC Kinetics. Introduction of the E345R mutation into a bispecific CD38xCD20 antibody results in faster and more substantial CDC-mediated target cell lysis. [Figure 25] Figures 25A-B: CDC kinetics. Introduction of the E345R mutation into the bispecific antibodies CD38xEGFR (A) and CD20xEGFR (B), which bind monovalently to EGFR-negative Raji cells, results in faster and more substantial CDC-mediated target cell lysis. [Figure 26] Figure 26A-F: CDC on Wien133 cells by combinations of wild-type antibody and mutant antibodies containing (A-C) E345R and Q386K, or (D-F) E345R, E430G, and Q386K. The IgG1-b12 mutant did not bind to Wien133 cells and was therefore used as a negative control antibody. [Figure 27] CDC activity of IgG1, IgG2, IgG3, and IgG4 isotype antibodies containing the E345R mutation. [Figure 28] Introduction of the Fc-Fc stabilizing E345R mutation into wild-type CD38 antibody 005 results in enhanced killing of primary CLL cells in an ex vivo CDC assay (mean ± standard error of the mean). DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention As described herein, surprisingly, mutations in amino acids not directly involved in Fc:C1q binding can nevertheless enhance antibody CDC and further improve other Fc-mediated effector functions of the antibody. This supports the hypothesis that antibody molecules, such as IgG1 antibodies, can form oligomeric structures that subsequently undergo C1q binding. Furthermore, although certain mutations have been found to reduce CDC induction, certain combinations of such mutations in the same or different antibody molecules restored CDC induction and demonstrated additional specificity for antibody oligomerization, thereby promoting more specific CDC induction. Specific mutations that enhance CDC responses were also characterized by improved ADCC responses, enhanced avidity, enhanced internalization, and in vivo activity in mouse tumor model systems, as shown in the Examples. These findings enable novel antibody-based therapeutics with enhanced CDC induction capabilities, more selective CDC induction, and / or other improved effector functions.

[0026] All antibody mutants of the present invention comprise an antigen-binding region and a full-length or partial Fc region containing at least one mutation in the segment corresponding to amino acid residues P247 to K447 in IgG1. Without being limited by theory, the identified mutations are believed to confer more effective and / or more specific CDC induction based on three different principles, referred to herein as "single mutants," "double mutants," and "mixed mutants," which are schematically represented in Figure 1.

[0027] The improved C1q and / or CDC effects of the variants of the present invention are primarily detectable only in assays that allow antibody oligomerization, such as cell-based assays in which the antigen is not immobilized but present on a flow membrane. Furthermore, it can be verified that these effects are due to more stable antibody oligomers and not to alterations in the direct C1q binding site, according to the principles shown in Figure 1C.

[0028] definition The term "single variant" is intended to be understood as a variant of the invention which has an enhanced effector function compared to the parent polypeptide or antibody.

[0029] The term "double mutant" shall be understood as a mutant comprising at least two mutations in said segment and having improved effector function compared to a mutant comprising only one of the two mutations, the parent polypeptide or antibody, or both.

[0030] The term "mixed variant" shall be understood as a variant that, when used in combination with a second variant of the same or different polypeptide or antibody containing mutations in different amino acid residues in said segment, results in enhanced effector function compared to one or more of the variant, the second variant, and the parent polypeptide or antibody alone.

[0031] The term "polypeptide comprising an immunoglobulin Fc domain and a binding region" in the context of the present invention refers to a polypeptide comprising an immunoglobulin Fc domain and a binding region capable of binding to any molecule, such as a polypeptide present on the surface of a cell, bacterium, or virion. The immunoglobulin Fc domain is defined as a fragment of an antibody that includes the two CH2-CH3 regions of the immunoglobulin and a linking region (e.g., hinge region) and is typically thought to result after digestion of an antibody with papain (known to those skilled in the art). The constant domain of the antibody heavy chain defines the antibody isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE). The Fc domain, together with cell surface receptors called Fc receptors and proteins of the complement system, mediates the effector functions of the antibody. The binding region may be a polypeptide sequence such as a protein, protein ligand, receptor, antigen-binding region, or ligand-binding region capable of binding to a cell, bacterium, or virion. When the binding region is, for example, a receptor, a "polypeptide comprising an immunoglobulin Fc domain and a binding region" can be prepared as a fusion protein of the immunoglobulin Fc domain and the binding region. When the binding region is an antigen-binding region, the "polypeptide comprising an immunoglobulin Fc domain and a binding region" may be a human antibody, a heavy chain-only antibody, or an antibody such as an ScFv-Fc fusion. A polypeptide comprising an immunoglobulin Fc domain and a binding region typically comprises a linking region (e.g., a hinge region) and two CH2-CH3 regions of the immunoglobulin heavy chain; therefore, a "polypeptide comprising an immunoglobulin Fc domain and a binding region" may also be a "polypeptide comprising at least one immunoglobulin Fc domain and a binding region." By "immunoglobulin Fc domain" in the context of the present invention is meant the presence of joining regions, such as the hinge, and the CH2 and CH3 regions of the immunoglobulin depending on the antibody subtype, for example human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2 or IgE.

[0032] The term "CH2 region" or "CH2 domain," as used herein, is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region may be that of any of the other subtypes described herein.

[0033] The term "CH3 region" or "CH3 domain," as used herein, is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system. However, the CH2 region may be that of any of the other subtypes described herein.

[0034] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low-molecular-weight chains and one pair of heavy (H) chains, all four of which may be interconnected by disulfide bonds. The structure of immunoglobulins has been extensively characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chains are interconnected via disulfide bonds in the so-called "hinge region." Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable in the sequence and / or configuration of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged in the following order from amino to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, the amino acids of constant region sequences are numbered herein according to the EU index (as described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)).

[0035] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, that has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of a meaningful period of time, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days or more, or any other reasonable, functionally defined period of time (e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a time sufficient for the antibody to elicit effector activity). The antibody of the present invention comprises an immunoglobulin Fc domain and an antigen-binding region. Antibodies generally comprise two CH2-CH3 regions and a connecting region (e.g., hinge region), e.g., comprise at least one Fc domain. Thus, an antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with antigen. The constant region, or "Fc" region, of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system (e.g., C1q, the first component in the classical pathway of complement activation). An antibody may also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. If the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. As noted above, unless otherwise stated or clearly contradicted by context, the term antibody, as used herein, includes fragments of antibodies that contain at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be prepared by any known method, for example, enzymatic cleavage,They can be obtained by techniques such as peptide synthesis and recombinant expression. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the scope of the term "Ab" or "antibody" include monovalent antibodies (described in WO 007059782 by Genmab); heavy chain antibodies consisting of only two heavy chains and occurring naturally, for example in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); Thiomab (Roche, WO2011069104), strand-exchange engineered domain (SEED or Seed-body) asymmetric bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); dual domain diabody (Unilever; Sanofi Aventis, WO20100226923), di-diabody (ImClone / Eli Lilly), knobs-into-holes antibody format (Genentech, WO9850431); Duobody (Genmab, WO 2011 / 131746); electrostatic steering antibody format (Amgen, EP1870459 and WO No. 2009089004; Chugai, US201000155133; Oncomed, WO 2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, No. WO11143545), CrossMAb (Roche, No. WO2011117329), LUZ-Y (Genentech),Biclonic (Merus), dual-targeting domain antibody (GSK / Domantis), two-in-one antibody recognizing two targets (Genentech, NovImmune), cross-linked Mab (Karmanos Cancer Center), CovX-body (CovX / Pfizer), IgG-like bispecific (ImClone / Eli Lilly, Shen, X, et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), as well as DIG-body and PIG-body (Pharmabcine), and dual affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), Zybody (Zyngenia), approaches using common light chains (Crucell / Merus, US7262028) or common heavy chains (κλBody by NovImmune), as well as fusion proteins such as scFv fusions, including those in which a polypeptide sequence is fused to an antibody fragment containing an Fc domain, such as BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPION by Emergent BioSolutions / Trubion, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb by f-Star, 2(WO2008 / 003116) and double scFv fusions are included, but are not limited to. The term antibody should also be understood to include, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals), such as those produced by technologies developed by Symphogen and Merus (Oligoclonics), as well as antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. The antibodies produced may potentially have any isotype.

[0036] The term "full-length antibody," as used herein, refers to an antibody (e.g., a parent antibody or a variant antibody) that includes all of the heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that isotype.

[0037] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0038] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of Ab molecules of monomolecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an Ab exhibiting a single binding specificity whose variable and constant regions are derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas comprising immortalized B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, whose genomes contain rearranged human heavy and light chain transgene repertoires to produce functional human antibodies.

[0039] As used herein, "isotype" refers to the class of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotypes thereof, such as IgG1m(za) and IgG1m(f)) encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0040] The term "monovalent antibody" in the context of the present invention means that an antibody molecule can bind to an antigen with only one of the antibody binding domains, e.g., has a single antigen-antibody interaction, and is therefore incapable of antigen cross-linking.

[0041] As used herein, the term "target" should be understood in the context of the present invention as any antigen-containing molecule against which an antibody is raised, when the binding region of a polypeptide comprising an Fc domain and a binding region is used in the context of antibody binding. The terms "antigen" and "target" can be used interchangeably in relation to an antibody and have the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0042] As used herein, the term "binding" in the context of binding of an antibody to a given antigen typically refers to a K, as determined by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using, for example, the antigen as the ligand and the antibody as the analyte. D is about 10 -6 M or less, e.g., 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less, and has a K that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, such as at least 100,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The amount at which the affinity is lower is the antibody's K D Therefore, the K D If K is very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000 times lower than the affinity for a nonspecific antigen. D The term "" (M), as used herein, refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.

[0043] A "variant" or "antibody variant" or "variant of a parent antibody" of the present invention comprises one or more mutations compared to an antibody molecule or "parent antibody." Similarly, a "variant" or "variant of a polypeptide comprising an immunoglobulin Fc domain and a binding region" or "variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region" of the present invention is a "polypeptide comprising an immunoglobulin Fc domain and a binding region" that comprises one or more mutations compared to a "parent polypeptide comprising an immunoglobulin Fc domain and a binding region." These different terms can be used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, or any combination thereof. Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may replace a naturally occurring amino acid with another naturally occurring amino acid or a non-natural amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within classes of amino acids represented in one or more of the following three tables: Classes of amino acid residues for conservative substitutions TIFF2024116396000001.tif50131 Another class of conservative amino acid residue substitutions TIFF2024116396000002.tif38131 Alternative physical and functional classifications of amino acid residues TIFF2024116396000003.tif67135

[0044] In the context of the present invention, substitutions in the variants are designated as follows: Original amino acid - position - substituted amino acid; Three-letter or one-letter codes are used to designate amino acid residues, including the codes Xaa and X. Thus, the designation "E345R" or "Glu345Arg" means that the variant contains a substitution of glutamic acid with arginine at the variant amino acid position corresponding to the amino acid at position 345 in the parent antibody, when the two are aligned as indicated below.

[0045] If such a position does not exist in the antibody, the variant may include an amino acid insertion, for example: Position-substituted amino acid; for example, use designation such as "448E".

[0046] Such designation is particularly applicable with respect to variations in a series of homologous polypeptides or antibodies.

[0047] Similarly, if the identity of the substituted amino acid residue is not important: Original amino acid position; or "E345".

[0048] For modifications in which the original and / or substituted amino acids include multiple, but not all, amino acids, for substitution of glutamic acid at position 345 with arginine, lysine, or tryptophan: "Glu345Arg,Lys,Trp" or "E345R,K,W" or "E345R / K / W" or "E345 to R, K or W" can be used interchangeably in the context of the present invention.

[0049] Moreover, the term "substitution" also encompasses substitution with any one of the other 19 naturally occurring amino acids, or with other amino acids, such as unnatural amino acids. For example, substitution of the amino acid E at position 345 includes each of the following substitutions: TIFF2024116396000004.tif10128. Incidentally, this is equivalent to the notation 345X, where X represents any amino acid. These substitutions may also be designated as E345A, E345C, etc., or E345A, C, etc., or E345A / C / etc. The same is equally true for each and every position referred to herein, and any such substitutions are specifically included herein.

[0050] An amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that (i) aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW, or the like, typically with default settings, and (ii) has at least 50%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. For example, the sequence alignments shown in Figures 2 and 3 can be used to identify any amino acid in an IgG2, IgG3, or IgG4 Fc sequence that corresponds to a particular amino acid in an IgG1 Fc sequence.

[0051] The present invention refers to variants, i.e., parent and / or mutant antibodies, that have a degree of identity to amino acids P247 to K447 of SEQ ID No: 1, 2, 3, 4 and 5, and such parent and / or mutant antibodies are hereinafter referred to as "homologous antibodies".

[0052] For purposes of the present invention, the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, is determined by the program "align," which is a Needleman-Wunsch alignment (i.e., global alignment). This program is used for aligning polypeptide and nucleotide sequences. The default scoring matrix, BLOSUM50, is used for polypeptide alignments, and the default identity matrix is ​​used for nucleotide alignments, with a penalty of -12 for the first residue of a gap for polypeptides and -16 for nucleotides. The penalty for additional residues in a gap is -2 for polypeptides and -4 for nucleotides.

[0053] "align" is part of the FASTA package, version v20u6 (see W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W.R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology 183:63-98). FASTA protein alignment uses the Smith-Waterman algorithm with no restrictions on gap size.

[0054] As used herein, the term "effector cell" refers to an immune cell involved in the effector stage of an immune response, rather than the recognition and activation stages. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Certain effector cells express Fc receptors (FcRs) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce ADCC. For example, FcR-expressing monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells are involved in specifically killing target cells and presenting antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, ADCC can further enhance antibody-triggered classical complement activation, which results in the deposition of activated C3 fragments on the surface of target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote Fc receptor-mediated ADCC enhancement. In some embodiments, antibody-triggered classical complement activation results in C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression enhances the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-triggered classical complement activation results in C3 fragments on target cells.These C3 cleavage products either promote direct phagocytosis by effector cells or indirectly by enhancing antibody-mediated phagocytosis.

[0055] The term "vector," as used herein, refers to a nucleic acid molecule capable of directing transcription of a nucleic acid segment ligated therein. One type of vector is a "plasmid," which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0056] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, as well as prokaryotic cells, such as Escherichia coli (E. coli), and other eukaryotic hosts, such as plant cells and fungi.

[0057] The term "transfectoma," as used herein, includes recombinant eukaryotic host cells, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, including yeast cells, that express an Ab or target antigen.

[0058] The term "preparation" refers to a preparation of antibody variants and mixtures of multiple different antibody variants that can enhance their ability to form oligomers when interacting with cell-bound antigens (e.g., antigens expressed on the surface of cells), cell membranes, virions, or other structures, thereby enhancing C1q binding, complement activation, CDC, ADCC, ADCP, other Fc-mediated effector functions, internalization, downmodulation, apoptosis, antibody-drug conjugate (ADC) uptake, avidity, or any combination thereof. Exemplary assays are provided in the Examples, such as for C1q binding (Example 4), CDC (Examples 5, 6, and 10, 16, 19, 22, 23, 24, 25); ADCC (Example 12), and in vivo activity (Examples 20, 21). The variants of various aspects herein, referred to as "single variants," "double variants," and "mixed variants," are described in further detail below, along with exemplary steps for their preparation and methods of use.

[0059] As used herein, the term "affinity" refers to the strength of binding between one molecule (e.g., an antibody) and another molecule (e.g., a target or antigen) at a single site, such as the monovalent binding of an antigen to an individual antigen-binding site of an antibody.

[0060] As used herein, the term "avidity" refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen-binding sites of an antibody that simultaneously interact with a target, or between an antibody and C1q. When multiple binding interactions are present, the two structures will only dissociate when all of the binding sites have dissociated; therefore, the dissociation rate will be lower than for individual binding sites, resulting in a greater effective total binding strength (avidity) compared to the strength of binding (affinity) of the individual binding sites.

[0061] As used herein, the term "oligomer" refers to a molecule consisting of a multiple, but limited, number of monomer units (e.g., an antibody), as opposed to a polymer, which, at least in principle, consists of an infinite number of monomers. Exemplary oligomers include dimers, trimers, tetramers, pentamers, and hexamers. Greek letter prefixes are often used to indicate the number of monomer units in an oligomer, e.g., a tetramer is composed of four units, and a hexamer is composed of six units.

[0062] The term "oligomerization," as used herein, refers to the process of converting monomers to a finite degree of polymerization. It has been observed herein that oligomerization of Fc domains occurs preferably, but not exclusively, on the cell surface after target binding by a polypeptide containing an Fc domain, such as an antibody. Antibody oligomerization can be assessed, for example, using a cell surface C1q binding assay (as described in Examples 4 and 9), a C1q activity assay (as described in Example 5), and complement-dependent cytotoxicity as described in Examples 6, 10, and 19.

[0063] The term "C1q binding," as used herein, is intended to refer to the binding of C1q in the context of C1q binding to an antibody bound to its antigen. Antibody bound to its antigen, in the context described herein, shall be understood to occur both in vivo and in vitro. C1q binding can be assessed, for example, by using immobilized antibodies on artificial surfaces (e.g., plastic in ELISA plates, as described in Example 3) or by binding to a predetermined antigen on the surface of cells or virions (as described in Examples 4 and 9). C1q binding to antibody oligomers, as used herein, shall be understood as a multivalent interaction that results in high avidity binding.

[0064] As used herein, the term "complement activation" refers to activation of the classical complement pathway, triggered by the binding of complement component C1q to an antibody bound to its antigen. C1q is the first protein in the early events of the classical complement cascade and participates in a series of cleavage reactions that culminate in the formation of an enzyme activity called C3 convertase, which cleaves complement component C3 into C3b and C3a. C3b covalently binds to membrane-bound C5 to form C5b, which in turn triggers the later event of complement activation, in which terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade ultimately leads to pore formation, which causes cell lysis, also known as CDC. Complement activation can be assessed by using C1q activity (as described in Example 5), CDC kinetics (as described in Examples 28, 29, and 30), CDC assays (as described in Examples 6, 10, 19, 25, 27, and 33), or by the C3b and C4b cell deposition method described in Beurskens et al., April 1, 2012, vol. 188, no. 7, 3532-3541.

[0065] The term "complement-dependent cytotoxicity" ("CDC"), as used herein, is intended to refer to the process of antibody-mediated complement activation that leads to lysis of antibodies bound to their targets on cells or virions as a result of intramembrane pores created by MAC assembly. CDC can be assessed by in vitro assays, such as CDC assays using normal human serum as a complement source, as described in Examples 6, 10, 19, 25, 27, and 33, or by C1q activity assays, as described in Example 5, in which normal human serum is C1q-limited.

[0066] The term "antibody-dependent cell-mediated cytotoxicity" ("ADCC"), as used herein, is intended to refer to the mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of the bound antibody. ADCC can be determined using methods such as, for example, the ADCC assay described in Example 12.

[0067] The term "antibody-dependent cellular phagocytosis" ("ADCP"), as used herein, is intended to refer to the mechanism of elimination of antibody-coated target cells or virions by internalization by phagocytes. The internalized antibody-coated target cells or virions are housed within vesicles called phagosomes, which subsequently fuse with one or more lysosomes to form phagolysosomes. ADCP can be assessed by using an in vitro cytotoxicity assay using macrophages as effector cells and video microscopy, as described by van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685. Alternatively, it can be assessed, for example, as described in Example 14 for the phagocytosis of Staphylococcus aureus (S. aureus) by PMNs.

[0068] The term "complement-dependent cytotoxicity" ("CDCC"), as used herein, is intended to refer to the mechanism of killing of target cells or virions by cells expressing complement receptors that recognize complement third component (C3) cleavage products covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC can be assessed in a manner similar to that described for ADCC.

[0069] The term "downmodulation," as used herein, is intended to refer to the process of reducing the number of molecules, such as antigens or receptors, on the surface of a cell, for example, by binding of an antibody to the receptor.

[0070] The term "internalization," as used herein, is intended to refer to any mechanism by which an antibody or Fc-containing polypeptide undergoes internalization from the cell surface and / or surrounding medium into a cell expressing the target, e.g., via endocytosis. Antibody internalization can be assessed using a direct assay that measures the amount of internalized antibody (e.g., the lysosomal co-localization assay described in Example 26).

[0071] The term "antibody-drug conjugate," as used herein, refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that connects the drug to, for example, the antibody. The linker may or may not be cleavable in the presence of malignant cells; the antibody-drug conjugate kills the malignant cells.

[0072] The term "antibody-drug conjugate uptake," as used herein, refers to the process by which an antibody-drug conjugate binds to a target on a cell and then undergoes uptake / engagement by the cell membrane, resulting in its internalization within the cell. The uptake of antibody-drug conjugates can be assessed as "Antibody-Mediated Internalization and Cell Killing by Anti-TF ADC in an In Vitro Killing Assay," as described in WO 2011 / 157741.

[0073] The term "apoptosis," as used herein, refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events lead to characteristic cellular changes (morphology) and cell death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Binding of antibodies to certain receptors can induce apoptosis.

[0074] Fc receptor binding can also be measured indirectly as described in Example 12.

[0075] The term "FcRn," as used herein, is intended to refer to the neonatal Fc receptor, an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from maternal milk across the intestinal epithelium of newborn rodents into the bloodstream of the newborn. Further studies revealed a similar receptor in humans. However, in humans, it has been found to help facilitate the transport of maternal IgG to the developing fetus in the placenta and has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at an acidic pH of 6.0–6.5 but not at neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal lumen (inside the intestine), which has a somewhat acidic pH, ensuring efficient unidirectional transport to the basolateral side (inside the body), which has a neutral to basic pH (pH 7.0–7.5). This receptor also plays a role in IgG recycling in adults through its presence in the endocytic pathway in endothelial cells. FcRn receptors in acidic endosomes bind IgG internalized by pinocytosis, recycle it to the cell surface, and release it at the basic pH of blood, thereby preventing it from undergoing lysosomal degradation. This mechanism may explain the longer half-life of IgG in blood compared to other isotypes. Example 13 describes an assay demonstrating IgG binding to FcRn at pH 6.0 in an ELISA.

[0076] The term "Protein A," as used herein, is intended to refer to a 56-kDa MSCRAMM surface protein first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cell osmolality, and a two-component system called ArIS-ArIR. It has found use in biochemical research due to its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain can bind proteins from many mammalian species, most notably IgG. It binds to the heavy chain Fc region of most immunoglobulins (which overlaps with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind to bacteria not only via their Fab region but also via their Fc region, thereby disrupting bacterial opsonization, complement activation, and phagocytosis.

[0077] The term "Protein G," as used herein, is intended to refer to an immunoglobulin-binding protein expressed in group C and group G streptococci that is very similar to Protein A, except for its different specificity. It is a 65 kDa (G148 Protein G) and 58 kDa (C40 Protein G) cell surface protein that has been applied to purify antibodies through its binding to the Fc region.

[0078] The term "CH2 region" or "CH2 domain," as used herein, is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system.

[0079] The term "CH3 region" or "CH3 domain," as used herein, is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341 to 447 according to the EU numbering system.

[0080] The term "allosteric mutation," as used herein, is intended to refer to modifications (e.g., insertions, substitutions, and deletions) of amino acids P247 and E430 in a polypeptide containing an Fc domain, as numbered by the EU index as set forth in Kabat.

[0081] The term "hydrophobic knob mutation," as used herein, is intended to refer to modifications (e.g., insertions, substitutions, and deletions) of amino acids 1253, S254, and Q311 in a polypeptide containing an Fc domain, as numbered according to the EU index as set forth in Kabat. Hydrophobic knobs are described by Delano WL, et al., Science 287, (2000), pages 1279-1283, e.g., page 1281.

[0082] The term "N-terminal CH3 helix mutation", as used herein, is intended to refer to modifications (e.g., insertions, substitutions, and deletions) of amino acids R355, D356, E356, E357, M358, L358, and T359, more specifically D356, E356, and T359, in a polypeptide containing an Fc domain, as numbered according to the EU index as set forth in Kabat.

[0083] The term "C-terminal CH3 β-strand mutation", as used herein, is intended to refer to modifications (e.g., insertions, substitutions, and deletions) of amino acids Y436, T437, Q438, K439, S440, and L441, more specifically Y436, K439, and S440, in a polypeptide containing an Fc domain, as numbered according to the EU index as set forth in Kabat.

[0084] Methods for affecting antibody effector functions It should be understood that all embodiments described herein with reference to a parent antibody (first parent antibody or second parent antibody) should also be interpreted as embodiments relating to a parent peptide (first parent peptide or second parent polypeptide) comprising an immunoglobulin Fc domain and a binding region.

[0085] In one aspect, the present invention relates to a method for enhancing the effector function of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, the method comprising the step of introducing a mutation into the parent polypeptide at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0086] In one embodiment, the parent polypeptide may be an antibody.

[0087] Thus, the present invention relates to a method for enhancing the effector function of a parent antibody, comprising the step of introducing a mutation into the parent antibody at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0088] Reference to "D / E356" in the present context refers to an allotypic variant in the sequence of human IgG1. In the IgG1m(za) allotype of human IgG1 amino acid, position 356 is D, whereas in the IgG1m(f) allotype of human IgG1 amino acid, position 356 is E.

[0089] The introduction of mutations into a parent antibody by the methods or uses of the invention results in a mutant or variant antibody. Thus, the methods of the invention can be performed to obtain any mutant or variant antibody as described herein.

[0090] The mutant antibodies obtained by the methods or uses of the present invention have enhanced effector function compared to the parent antibody. Typically, the effect of an antibody on effector function can be determined by the EC50 value, which is the concentration of antibody required to achieve half-maximal lysis.

[0091] Maximum lysis is the lysis obtained when a saturating amount of antibody is used, where saturation is intended to refer to an amount of antibody where all of the antigen for the antibody is bound by the antibody.

[0092] The term "enhancing effector function" or "improving effector function," in the context of the present invention, refers to a reduction in the EC50 value of the variant antibody compared to the parent antibody. The reduction in the EC50 value may be, for example, at least or about two-fold, e.g., at least or about three-fold, or at least or about five-fold, or at least or about ten-fold. Alternatively, "enhancing effector function" or "improving effector function" may also mean an increase in the maximum amount of cells lysed (when the total amount of cells is set to 100%) from, for example, 10% to 100% of the total cells, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, under conditions in which the parent antibody lyses less than 100% of the total cells.

[0093] Mutants can be tested for enhanced or improved effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the variant and testing its activity in a CDC assay, as described for Daudi (Example 6) and Wien (Example 10). When using the IgG1-7D8 HC variable domain and Daudi cells, enhancement is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-7D8 under the test conditions, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower. When using the IgG1-005 HC variable domain and Daudi cells, enhancement is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-005 under the conditions tested, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower. When using the IgG1-7D8 HC variable domain and Wien133 cells, enhancement is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-7D8 under the conditions tested, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower. When using IgG1-005 HC variable domain and Wien133 cells, enhancement is defined by an increase in maximal lysis ranging from 10% to 100% of total cells, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. Enhanced CDC activity can also be defined by an EC50 that is more than two-fold lower than the EC50 of IgG1-005 under the conditions tested, e.g., about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower (the concentration at which 50% lysis is observed under conditions where lysis of Wien133 cells is detectable).

[0094] The present inventors have surprisingly found that mutations at these specific positions have an improved effect on the effector function of mutant antibodies obtained by introducing mutations into a parent antibody according to the methods of the present invention (e.g., as shown in Example 19). Without being bound by theory, it is believed that substituting at least one amino acid from the group of positions mentioned above stimulates oligomerization. The antibodies bind with higher avidity (as exemplified by Example 2; direct labeling of IgG-7D8-E345R resulted in enhanced binding to Daudi cells compared to IgG-7D8-WT), which allows the antibodies to bind to cells for a longer period of time, thereby enabling various effector functions, such as, for example, enhanced C1q binding, C1q-activated CDC, ADCC, internalization, ADCP, and / or in vivo activity. These effects are exemplified by Example 4 (binding in cells), Example 5 (C1q activity in CDC assay), Examples 6, 7, 27, 28 and 29 (CDC assay), Example 12 (ADCC), Example 26 (internalization) and Examples 21 and 22 (in vivo activity). Therefore, mutations in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain can also be referred to as "single mutant" aspects or "effector-enhancing mutations" in the context of the present invention.

[0095] In another aspect, the present invention also provides a method for determining the effector functions of an antibody when bound to its antigen, e.g., on a cell, cell membrane, virion, or another particle, such as (i) C1q binding, (ii) complement activation, (iii) CDC, (iv) oligomer formation, (v) oligomer stability, (vi) antibody-dependent cell-mediated cytotoxicity (ADCC), (vii) FcRn binding, (viii) Fc-γ receptor binding, (ix) protein A binding, (x) protein G binding, (xi) antibody-dependent cellular phagocytosis (ADCP), (xii) complement-dependent cytotoxicity (CDCC), (xiii) complement-enhanced cytotoxicity, (xiv) antibody-mediated cytotoxicity. Also provided is the use of one or more mutations in Table 1, such as mutations in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc region of a human IgG1 heavy chain, to enhance one or more of: (xv) mediated opsonizing antibody binding to a complement receptor; (xvi) internalization; (xvi) downmodulation; (xvii) induction of apoptosis; (xviii) opsonization; and (xix) a combination of any of (i)-(xviii). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, in addition to or alternatively to at least one other effector function of the antibody, such as C1q binding, complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc-gamma receptor binding, Protein A binding, Protein G binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, antibody-mediated binding of opsonized antibodies to complement receptors, antibody-mediated phagocytosis (ADCP), internalization, apoptosis, and / or binding of opsonized antibodies to complement receptors, particularly FcRn binding, ADCC, Fc-gamma receptor binding, Protein A binding, Protein G binding, ADCP, CDCC, complement-enhanced cytotoxicity, opsonization, and any combination thereof, is enhanced.

[0096] In one embodiment, the effector function of the parent antibody is enhanced when the parent antibody binds to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion.

[0097] The inventors of the present invention have also shown that introducing mutations into a parent antibody at amino acid residues corresponding to either K439 or S440 in the Fc region of the human IgG1 heavy chain reduces the effector function of the parent antibody (Examples 5, 6, and 10).

[0098] In another aspect, the present invention relates to a method for reducing an effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the step of introducing into the parent polypeptide a mutation at one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W, for example, the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R.

[0099] In one embodiment, the parent polypeptide may be an antibody.

[0100] Accordingly, in another aspect, the present invention also relates to a method for reducing the effector function of a parent antibody, comprising the step of introducing into the parent antibody a mutation at one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; for example, the mutation at the position corresponding to K439 in the Fc region of human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of human IgG1 heavy chain is S440K / H / R.

[0101] As shown in Example 6, amino acid substitutions at positions K439E or S440K as "single mutants" reduced CDC compared to any of the first mutations according to the method of the present invention.

[0102] The mutant antibody obtained by the above-described method of reducing effector function has reduced effector function compared to the parent antibody. Typically, the effect of an antibody on effector function can be measured by the EC50 value, which is the concentration of antibody required to achieve half-maximal lysis.

[0103] Maximum lysis is the lysis obtained when a saturating amount of antibody is used, where saturation is intended to refer to an amount of antibody where all of the antigen for the antibody is bound by the antibody.

[0104] The term "reducing effector function," in the context of the present invention, refers to an increase in the EC50 value of the variant antibody compared to the parent antibody. The increase in EC50 value may be, for example, at least or about 2-fold, e.g., at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, "reducing effector function" can also mean a reduction in the maximum amount of cells lysed, e.g., from 10% to 100% of total cells, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, under conditions in which the parent antibody lyses less than 100% of total cells.

[0105] Mutants can be tested for reduced effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the mutant and testing its activity in a CDC assay, as described for Daudi (Example 6) and Wien (Example 10). When using the IgG1-7D8 HC variable domain and Daudi cells, a reduction is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-7D8 under the test conditions, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower. When using the IgG1-005 HC variable domain and Daudi cells, a reduction is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-005 under the conditions tested, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower than the EC50 of IgG1-005 under the conditions tested. When using the IgG1-7D8 HC variable domain and Wien133 cells, a reduction is defined by an EC50 value (concentration at which 50% lysis is observed) that is more than two-fold lower than the EC50 of IgG1-7D8 under the conditions tested, for example, about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower than the EC50 of IgG1-7D8 under the conditions tested. When using IgG1-005 HC variable domain and Wien133 cells, a reduction is defined by an increase in maximal lysis ranging from 10% to 100% of total cells, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. A reduction in CDC activity can also be defined by an EC50 that is more than two-fold lower than the EC50 of IgG1-005 under the conditions tested, e.g., about two-fold, about three-fold, about five-fold, about ten-fold, or more than ten-fold lower (the concentration at which 50% lysis is observed under conditions where lysis of Wien133 cells is detectable).

[0106] In one embodiment, the parent antibody has reduced effector function when it binds to its antigen on an antigen-expressing cell, on a cell membrane or on a virion.

[0107] Thus, in another aspect, the present invention provides the use of at least one further mutation in an antibody mutant comprising a mutation in one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, to restore the effector function of the antibody mutant when binding to its antigen on an antigen-expressing cell, on a cell membrane or on a virion, with the proviso that the mutation at S440 is neither S440Y nor S440W, the first mutation is at an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second mutation is at an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain; or The first mutation is at an amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain, and the second mutation is at an amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain.

[0108] In one embodiment, the parent antibody is a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0109] Where the parent antibody is a monospecific antibody comprising two CH2-CH3 regions, the mutations of the invention may in principle be present in only one of the CH2-CH3 regions, although for most practical purposes, the mutations of the invention that enhance or reduce effector function will be present in both CH2-CH3 regions.

[0110] Where the parent antibody is a bispecific antibody comprising two CH2-CH3 regions, the mutations of the invention may in principle be present in only one of the CH2-CH3 regions; i.e., in either the first or second CH2-CH3 region, although for most practical purposes, the mutations of the invention that enhance or reduce effector function will be present in both the first and second CH2-CH3 regions.

[0111] Suitable examples of monospecific, bispecific or multispecific antibodies include any of those described herein.

[0112] In one particular embodiment, the parent antibody or the first and / or second antibody may be a bispecific antibody, such as the heterodimeric protein described in WO 11 / 131746, which is incorporated herein by reference.

[0113] In one embodiment, the parent antibody is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or on different antigens.

[0114] In a further embodiment, said first CH2-CH3 region comprises an additional amino acid substitution at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of a human IgG1 heavy chain; and said second CH2-CH3 region comprises an additional amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and said additional amino acid substitution in the first CH2-CH3 region is different from said additional amino acid substitution in the second CH2-CH3 region.

[0115] In a further embodiment, the first CH2-CH3 region comprises an amino acid substitution at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain; and the second CH2-CH3 region comprises an amino acid substitution at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain.

[0116] In one embodiment, the method comprises introducing a mutation into at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain for each of the first and second CH2-CH3 regions, provided that the mutation at S440 is S440Y or S440W.

[0117] In a further embodiment, the mutation introduced into the first and second CH2-CH3 regions at at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W, may be at the same amino acid residue position or at different positions. In a further embodiment, it may be the same or different mutations at the same amino acid residue position.

[0118] In another embodiment, the method comprises introducing into the first or second CH2-CH3 region a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W.

[0119] Any of the mutations listed in Table 1 can be introduced into a bispecific antibody. Example 24 shows that introducing the E345R mutation into a bispecific CD20xEGFR antibody enhances CDC activity. Examples 23, 29 and 30 also describe some of the various bispecific antibodies that contain mutations of the invention.

[0120] In one embodiment, the method comprises introducing a mutation at one or more positions other than S440 and K447, and further introducing a mutation at: (i) at the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, respectively, provided that the mutation at S440 is neither S440Y nor S440W; (ii) amino acid residues corresponding to K447 and 448 in the Fc region of a human IgG1 heavy chain, for example, K447K / R / H and 448E / D in the Fc region of a human IgG1 heavy chain, preferably K447K and 448E in the Fc region of a human IgG1 heavy chain, or (iii) Amino acid residues corresponding to K447, 448, and 449 in the Fc region of a human IgG1 heavy chain, for example, K447D / E, 448K / R / H, and 449P in the Fc region of a human IgG1 heavy chain, preferably K447E, 448K, and 449P in the Fc region of a human IgG1 heavy chain.

[0121] In one embodiment, the method comprises introducing a mutation at one or more positions other than S440, and further introducing a mutation at each of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the further mutation at S440 is not S440Y or S440W.

[0122] The introduction of mutations into both amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain in a parent antibody, provided that the mutation at S440 is neither S440Y nor S440W, is also referred to herein as a "double mutant" aspect. The S440Y and S440W mutations were found to enhance effector function when introduced into a parent antibody, as described elsewhere.

[0123] As also described elsewhere, the inventors of the present invention found that introducing identified mutations into amino acid residues corresponding to either K439 or S440 in the Fc region of human IgG1 heavy chains resulted in reduced effector function (Examples 5, 6, and 10). However, introducing inhibitory mutations into both amino acid residues corresponding to K439 and S440 in the Fc region of human IgG1 heavy chains reverses the reduced effector function, resulting in an effector function similar to that of a parent antibody that does not have mutations at the K439 and S440 mutation sites. However, without being bound by any theory, it is believed that the presence of the K439 and S440 mutations limits the induction of effector function for oligomeric complexes composed solely of antibodies containing both the K439 and S440 mutations. Therefore, without being bound by any theory, it is believed that when the K439 and S440 mutations are included in a therapeutic antibody, when such a therapeutic antibody is administered to a patient, the induction of effector function will be limited to oligomeric antibody complexes that contain the therapeutic antibody containing the K439 / S440 mutations, but not the patient's own antibodies that do not contain the K439 and S440 mutations, thereby limiting side effects that may be caused by interactions between the therapeutic antibody and the patient's own antibodies.

[0124] Mutations at positions K439 and / or S440 combined with the first mutation result in enhanced CDC and enhance the specificity of the CDC.

[0125] Thus, in another aspect, the present invention provides a method for increasing the specificity of a combination of at least first and second parent polypeptides comprising an immunoglobulin Fc domain and a binding region, the method comprising: A) (i) introducing a mutation into a first parent polypeptide at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing a mutation into the second parent polypeptide at an amino acid residue at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W. B) (i) introducing a mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and (ii) introducing mutations into the second parent polypeptide at amino acid residues at positions corresponding to K447K / R / H and 448P in the Fc region of human IgG1 heavy chain; or C) (i) introducing a mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and (ii) introducing mutations into the second parent polypeptide at amino acid residues at positions corresponding to K447K / R / H, 448K / R / H, and 449P in the Fc region of human IgG1 heavy chain; The present invention relates to a method comprising:

[0126] In one embodiment, the parent polypeptide, the first parent polypeptide and the second parent polypeptide may each be an antibody.

[0127] Thus, in a further aspect, the present invention also provides a method for increasing the specificity of a combination of at least a first and a second parent antibody, comprising the steps of: (i) introducing a mutation into the first parent antibody at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing a mutation into the second parent antibody at an amino acid residue at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W. The present invention also relates to a method comprising:

[0128] The first and second mutant antibodies appear to have a preference for oligomerization with each other compared to any wild-type or native antibody, as shown in Example 10.

[0129] The enhancement of specificity relates to the “induction of effector function.” Thus, in one embodiment, the method is a method for increasing the specificity of the induction of effector function by the combination of at least the first and second parent antibodies.

[0130] By carrying out a method to increase the specificity of the effector function or the specificity of the induction of effector function, the combination of at least a first and a second parent antibody results in a combination of a first variant and a second mutant antibody.

[0131] By introducing a mutation into either K439 or S440 of a parent antibody, the resulting mutant antibody has reduced effector function compared to the parent antibody. However, as also described elsewhere herein, mutations at K439 and S440 can complement each other or restore the effector function of an antibody containing both mutations. This ability of mutations at K439 and S440 to complement each other can be similarly exploited in two antibodies. Thus, if a mutation at K439 is introduced into a first parent antibody and a mutation at S440 is introduced into a second parent antibody, or vice versa, the reduced effector function is no longer observed when the first and second mutant antibodies are used in combination. The terms "increasing specificity" or "improving specificity" in this context refer to the effector response induced by the combination of a first mutant antibody comprising a mutation at K439 and a second mutant antibody comprising a mutation at S440 being higher than the effector response induced by either the first mutant antibody comprising a mutation at K439 or the second mutant antibody comprising a mutation at S440.

[0132] Introduction of both amino acid substitutions at K439 and S440 increases the specificity of oligomerization.

[0133] Mutations at positions K439 and / or S440 combined with the first mutation result in enhanced CDC and enhance the specificity of the CDC.

[0134] In one embodiment, at least the first and second parent antibodies bind to the same epitope.

[0135] In one embodiment, at least the first and second parent antibodies bind to different epitopes on the same antigen.

[0136] In one embodiment, at least the first and second parent antibodies bind to different epitopes on different targets.

[0137] In one embodiment, the first and second parent antibodies have the same or different VL and VH sequences.

[0138] In one embodiment, the combination of at least a first and a second parent antibody comprises one first parent antibody and one second antibody.

[0139] In one embodiment, specificity is increased when the combination of the first and second parent antibodies binds to the antigen on an antigen-expressing cell, on a cell membrane or on a virion.

[0140] Therefore, in another aspect, the present invention also relates to the use of mutations in two or more amino acid residues of an antibody to increase the specificity of the antibody (e.g., the specificity of the effector function induced by the antibody) when binding to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion, wherein The first mutation is at the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain; The second mutation is at the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain.

[0141] In a further aspect, the present invention provides a method for enhancing the effector function of a combination of at least a first and a second parent polypeptide, wherein the at least a first and a second parent polypeptide each comprise an Fc domain and a binding region of an immunoglobulin, said method comprising: (i) introducing mutations into at least the first and / or second parent polypeptide at one or more amino acid residues selected from the group consisting of: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain The present invention relates to a method comprising:

[0142] In one embodiment, each of the first and / or second parent polypeptides may be an antibody.

[0143] Thus, in one embodiment, the present invention provides a method for enhancing the effector function of a combination of at least a first and a second parent antibody, wherein the at least first and second parent antibodies each comprise an Fc domain and an antigen-binding region of an immunoglobulin, said method comprising: (i) mutating at least the first and / or second parent antibody at one or more amino acid residues selected from the group consisting of: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain The present invention relates to a method comprising:

[0144] By practicing this method, a combination of at least a first and a second variant antibody is obtained, which, when combined, has an enhanced effector function compared to the combination of the first and second parent antibodies.

[0145] The term "enhanced effector function" is to be interpreted as described below.

[0146] The first and / or second parent antibody may be any parent antibody as described herein.

[0147] The method of enhancing the effector function of a first and second antibody combination can be carried out, inter alia, to obtain a first and / or second mutant antibody having any of the characteristics of the mutant antibody as described herein. The inventors of the present invention have found that introducing mutations into amino acid residues selected from (a), (b), (c), (d) and / or (e) results in a first and second mutant antibody combination having enhanced effector function compared to the first and second parent antibody combination.

[0148] In one embodiment, at least the first and second parent antibodies bind to the same epitope.

[0149] In one embodiment, at least the first and second parent antibodies bind to different epitopes on the same antigen.

[0150] In one embodiment, at least the first and second parent antibodies bind to different epitopes on different targets.

[0151] In one embodiment, the first and second parent antibodies have the same or different VL and VH sequences.

[0152] In one embodiment, the combination of at least a first and a second parent antibody comprises one first parent antibody and one second antibody.

[0153] In one embodiment, the combination of at least a first and second parent antibody comprises a further parent antibody, such as a third, fourth or fifth parent antibody.

[0154] In one embodiment, (a) the amino acid residue in the CH2-CH3 region that results in an allosteric mutation is an amino acid residue selected from those corresponding to P247 or E430 in the Fc region of a human IgG1 heavy chain.

[0155] In one embodiment, (b) the amino acid residues within the hydrophobic knob of the CH2-CH3 region are amino acid residues selected from those corresponding to I253, S254 and Q311 in the Fc region of a human IgG1 heavy chain.

[0156] In one embodiment, (c) the amino acid residues within the N-terminal CH3 helix are amino acid residues selected from those corresponding to D / E356 and T359 in the Fc region of a human IgG1 heavy chain.

[0157] In one embodiment, (d) the amino acid residue within the C-terminal CH3 β-strand is an amino acid residue selected from those corresponding to Y436 and S440.

[0158] The amino acid residues in (b), (c), (d) and (e) are those located at the Fc:Fc interface of two antibodies that are in close proximity to each other (and thus the Fc portion of one antibody that can interact with the Fc portion of another antibody).

[0159] Thus, in a further embodiment, the mutation in at least the first and / or second parent antibody is in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0160] In one embodiment, (i) comprises introducing mutations into both the first and second parent antibodies.

[0161] In another embodiment, the method comprises the steps of: (i) introducing a mutation into a first parent antibody at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W. (ii) providing a second parent antibody that does not contain mutations in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain;

[0162] In one embodiment, the method comprises introducing a mutation into at least one amino acid residue other than S440, wherein the method further comprises introducing a mutation into one or more positions other than S440, and wherein the method further comprises: (i) introducing a second mutation into the first parent antibody at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing into the second parent antibody a second mutation at an amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W; wherein steps (ii) and (iii) may alternatively be: (i) introducing into the first parent antibody a second mutation at an amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; and (ii) introducing a second mutation into the second parent antibody at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain.

[0163] For embodiments of the invention in which the second parent antibody does not contain mutations at amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, the term "second mutation(s)" in step (ii) may also be the first mutation(s), e.g. the second parent antibody may contain no other mutations than those introduced in step (ii). The term "second mutation(s)" in steps (i) and (ii) is also not intended to limit the number of mutations that may be introduced into the first and / or second parent antibody.

[0164] In one embodiment, the parent antibody, the first parent antibody, and the second parent antibody may each be selected from the group consisting of, but not limited to, monospecific antibodies, bispecific antibodies, and multispecific antibodies. Bispecific antibodies may, for example, be heterodimeric proteins.

[0165] In one embodiment, the first and second parent antibodies are monospecific antibodies, e.g., they may bind to the same epitope or to different epitopes, which may be on the same or different antigens if the first and second parent antibodies bind to different epitopes.

[0166] In another embodiment, the first parent antibody is a monospecific antibody and the second parent antibody is a bispecific or multispecific antibody, or vice versa.

[0167] In another embodiment, the first and second parent antibodies are bispecific or multispecific antibodies. In one embodiment, the first and second bispecific or multispecific parent antibodies are the same or different antibodies. In one embodiment, the first and second bispecific or multispecific parent antibodies bind to different epitopes on the same or different antigens. Thus, in one embodiment, the at least first and second parent antibodies are bispecific or multispecific antibodies that bind to different epitopes on the same or different antigens.

[0168] In another embodiment, the first parent antibody is a monospecific antibody and the second parent antibody is a bispecific antibody, or vice versa. The monospecific antibody may bind to the same epitope as the bispecific antibody (part of a bispecific antibody), or the monospecific antibody and the bispecific antibody may bind to different epitopes on the same or different antigens. The bispecific antibody may bind to different epitopes on the same or different antigens.

[0169] In one embodiment, each of said at least first and second parent antibodies is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or on different antigens, and said first CH2-CH3 region comprises K409, T3 in the Fc region of a human IgG1 heavy chain. the second CH2-CH3 region comprises additional amino acid substitutions at positions selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and the additional amino acid substitutions in the first CH2-CH3 region are different from the additional amino acid substitutions in the second CH2-CH3 region.

[0170] In a further embodiment, the first CH2-CH3 region comprises an amino acid substitution at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain; and the second CH2-CH3 region comprises an amino acid substitution at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain.

[0171] In one embodiment of the methods and / or uses of the present invention, the parent antibody, whether it is a parent antibody, a first parent antibody, or a second parent antibody, may contain other mutations besides the mutations of the present invention found to affect effector function. Such other mutations may be introduced simultaneously with the mutations of the present invention that affect effector function, or they may be introduced sequentially, and the methods or uses of the present invention are not limited to either simultaneous or sequential introduction of mutations. As it is foreseen that a variety of formats may be used, the bispecific antibody may be any bispecific antibody, and the methods and uses of the present invention are not limited to any particular bispecific format.

[0172] A method of combining a first antibody containing one of the above mutations capable of enhancing effector function with a second antibody that does not contain such a mutation can enhance the effector function of the combination, as shown in Example 31. Thus, without being bound by theory, it is believed that this method can be used, for example, to combine a therapeutic antibody as the second antibody that has been proven safe but not sufficiently efficient with a first antibody containing the mutation, resulting in an effective combination.

[0173] Thus, in one embodiment, a second parent antibody that does not contain mutations in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc region of a human IgG1 heavy chain is a therapeutic antibody. In a particular embodiment, it is a therapeutic antibody with a suitable safety profile. In one embodiment, it may be a therapeutic antibody with a suitable safety profile but which is not fully effective.

[0174] Examples of suitable second antibodies that do not contain mutations at amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain include, but are not limited to, any of the following; (90Y) clivatuzumab tetraxetan; (90Y) tacatuzumab tetraxetan; (99mTc) fanolesomab; (99mTc) nofetumomab merpentane; (99mTc) pintumomab; 3F8; 8H9; abagovomab; Abatacept; Abciximab; Actoxumab; Adalimumab; Adecatumumab; Afelimomab; Aflibercept; Afutuzumab; Alacizumab pegol; Albiglutide; ALD518; Alefacept; Alemtuzumab; Alirocumab; Altumomab; Altumomab pentetate; Alvircept sudotox; Amatuximab; AMG714 / HuMax-IL15; Anatumomab mafenatox; Anrukinzumab (=IMA-638); Apolizumab; Arcitumomab; Acelizumab; Atacicept; Atinumab; Atlizumab (=Tosiri) uzumab); atrolimumab; baminocept; bapineuzumab; basiliximab; bavituximab; bectumomab; belatacept; belimumab; benralizumab; bertilimumab; besilesomab; becacizumab; bezlotoxumab; biciromab; bifalcept; bivatuzumab; bivatuzumab mertansine; blinatumomab; brosozumab; brentuximab vedotin; briakinumab; briovacept; brodalumab; canakinumab; cantuzumab mertansine; cantuzumab vedotin; caplacizumab uzumab; capromab; capromab pendetide ;Carlumab;Catumaxomab;CC49;Cedelizumab;Certolizumab pegol;Cetuximab;Ch.14.18;Sitatuzumab bogatox;Cixutumumab;Clazakizumab;Clenoliximab;Crivatuzumab tetraxetan;Conatumumab;Conbercept;CR6261;Crenezumab;Dacetuzumab;Daclizumab;Dalantercept;Daratumumab;Demcizumab;Denosumab;Detumomab;Dorlimomab alitoxin;Drozitumab;Dulaglutide;Eclomeximab;Eculizumab;Edobacomab;Edrecolomab;Efalizumab; Efungumab; Elotuzumab; Elcilimomab; Enavatuzumab; Enlimomab; Enlimomab pegol; Enokizumab; Ensituximab; Epitumomab; Epitumomab cituxetan; Elapuzumab; Erlizumab; Ertumaxomab; Etanercept; Etaracizumab; Etrolizumab; Exibirumab; Fanolesomab; Farallimomab; Faretuzumab; Fasinumab; FBTA05; Felviz Mabs; Fezakinumab; Ficlatuzumab; Fizitumumab; Framvolumab; Fontolizumab; Foralumab; Foravirumab; Fresolimumab; Furlanumab; Galiximab; Ganitumab; Gantenerumab; Gavilimomab; Gemtuzumab; Gemtuzumab ozogamicin; Gevokizumab; Girentuximab; Glembatumumab; Glembatumumab vedotin; Golimumab; Gomiliximab; GS6624; Anti-CD74 antibody; WO anti-cMet antibodies such as those disclosed in WO 2011 / 110642; anti-Her2 antibodies such as those disclosed in WO 2011 / 147986 or WO 2011 / 147982; anti-IL8 antibodies such as those disclosed in WO 2004 / 058797; anti-TAC antibodies such as those disclosed in WO 2004 / 045512; Anti-tissue factor (TF) antibodies such as those disclosed in 2011 / 157741; ibalizumab; ibritumomab tiuxetan; icrucumab; igovomab; imciromab; inlacumab; indatuximab ravtansine; infliximab; inolimomab; inotuzumab ozogamicin; intetumumab; iodine (124I) girentuximab; ipilimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; remaresomab; lensercept; relde Limumab; lexatumumab; ribivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; rumiliximab; mapatumumab; maslimomab; matuzumab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minletumomab; mirococept; mitumomab; mogamulizumab; morolimumab; motavizumab; moxetumomab; pasudotox; muromonab-CD3; nacolomabutafenatox; namilumab; naptumomab estafenatox; narutomab; natalizumab;Nebacumab; Necitumumab; Nelerimomab; Nimotuzumab; Nivolumab; Nofetumomab; Merpentan; Obinituzumab; Ocaratuzumab; Ocrelizumab; Odulimomab; Ofatumumab; Olaratumab; Olokizumab; Omalizumab; Onartuzumab; Onercept; Oportuzumab monatox; Oregovomab; O Telixizumab; Oxelumab; Ozolarizumab; Pagibaximab; Palivizumab; Panitumumab; Panobacumab; Pascolizumab; Pateclizumab; Patritumab; Pegsnercept; Pemtumomab; Pertuzumab; Pexelizumab; Pintumomab; Placulumab; Ponezumab; Priliximab; Pritumumab; PRO 140; Kirizumab; Racotumomab; Ladolezumab; Rafivirumab; Ramucirumab; Ranibizumab; Raxibacumab; Regavirumab; Reslizumab; RG1507 / HuMax-IGF1R; RG1512 / HuMax-p-selectin; Rilonacept; Rilotumumab; Rituximab; Lobatumumab; Lorezumab; Romosozumab; Rontalizumab; Rovelizumab; Ruplizumab; Samalizumab ;Sarilumab;Satumomab;Satumomab pendetide;Secukinumab;Sevirumab;Sibrotuzumab;Sifalimumab;Siltuximab;Siplizumab;Sirukumab;Solanezumab;Solitomab;Sonepcizumab;Sontuzumab;Sotatercept;Stamulumab;Sulesomab;Suvisumab;Tavalumab;Tacatuzumab tetraxetan;Tadocizumab;Talizumab;Tanezumab;Tapritumomab pap Tox; tefibazumab; terimomab-alitox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; ticilimumab (=tremelimumab); tigatuzumab; TNX-650; tocilizumab (=atlizumab); toralizumab; Trapsel; tositumomab; tralokinumab; trastuzumab; trastuzumab emtansine; TRBS07; trebananib; tregalli Ibuprofen; tremelimumab; tucotuzumab-celmoleukin; tuvilumab; ublituximab; urelumab; urtoxazumab; ustekinimab; bapaliximab; batelizumab; vedolizumab; veltuzumab; bepalimomab; besencumab; visilizumab; volociximab; borsetuzumab-mafodotin; votumumab; zalutumumab; zanolimumab; diralimumab; and zolimomab-allitoxin. ;

[0175] In one embodiment of the methods and uses of the invention, the mutation in at least one amino acid residue, or in one or more amino acid residues, corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, provided that the mutation in S440 is S440Y or S440W, may be at any of the exemplary and preferred amino acid positions listed in Table 1. Thus, each of the amino acid positions listed in Table 1 is a separate and non-limiting embodiment of a mutation in at least one amino acid.

[0176] Any of the mutations described herein or combinations thereof can be introduced according to the methods of the present invention.

[0177] Mutations selected from exemplary or preferred amino acid substitutions can be tested in suitable assays, such as those described in the Examples, that allow for detection of enhanced oligomerization of antigen-bound antibodies, as well as C1q binding, complement activation, CDC, ADCC, and / or internalization. For example, C1q binding ability can be determined using cells expressing the antigen against which the antibody mutant is directed, according to an assay similar to that described in Example 4. Exemplary CDC assays are provided in Examples 5, 6, 10, 16, 19, 22, 23, 24, or 25. Exemplary ADCC assays are provided in Example 12. Exemplary internalization assays are provided in Example 26. Furthermore, to distinguish mutations in amino acid residues directly involved in C1q binding from mutations that affect oligomerization, C1q binding in an ELISA assay, for example, according to Example 3, can be compared with C1q binding in a cell-based assay, for example, according to Example 4.

[0178] In a further embodiment, the mutations are selected from those corresponding to E345, E430, S440 and Q386 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0179] In an alternative embodiment, the mutation in at least one amino acid residue, or in one or more amino acid residues, is at amino acid residues corresponding to E382 and H433 in the Fc region of a human IgG1 heavy chain.

[0180] In one particular embodiment, one mutation is at the amino acid residue corresponding to E345 in the Fc region of the human IgG1 heavy chain.

[0181] In one particular embodiment, one mutation is at the amino acid residue corresponding to E430 in the Fc region of the human IgG1 heavy chain.

[0182] In one particular embodiment, one mutation is at the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is S440Y or S440W.

[0183] In one particular embodiment, one mutation is at the amino acid residue corresponding to Q386 in the Fc region of the human IgG1 heavy chain.

[0184] In an alternative embodiment, one mutation is at an amino acid residue corresponding to E382 or H433 in the Fc region of a human IgG1 heavy chain.

[0185] In one embodiment, the mutation in at least one amino acid residue may be an amino acid substitution, an amino acid deletion or an amino acid insertion.

[0186] In one embodiment, the mutation in at least one amino acid residue is an amino acid deletion.

[0187] In one embodiment, the mutation in at least one amino acid residue is an amino acid insertion.

[0188] In one particular embodiment, the mutation in at least one amino acid residue is an amino acid substitution.

[0189] In one embodiment, the mutation at least one amino acid residue can be selected from any of the amino acid substitutions, deletions listed in Table 1. Furthermore, each preferred amino acid substitution at each specific amino acid residue listed in Table 1 is a separate, specific, non-limiting embodiment for this use. Exemplary amino acid substitutions include replacing an E residue with an R residue, and replacing an H residue with an R residue.

[0190] In a further embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345X, E430X, S440Y or W, and Q386K in the Fc region of a human IgG1 heavy chain, where X refers to any amino acid, e.g., any natural or unnatural amino acid. X can particularly refer to any of the 20 naturally occurring amino acids.

[0191] Thus, in one embodiment, the mutation is in at least one amino acid residue selected from those corresponding to E345, E430, S440 to Y or W, and Q386 in the Fc region of a human IgG1 heavy chain, preferably the mutation is at least one of the following amino acid substitutions: E345 to R, Q, N or K, E430 to T, S or G, S440 to Y or W, or Q386 to K.

[0192] Thus, in one embodiment, E345X may be E345R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V, W or Y; particularly E345A, D, G, H, K, N, Q, R, S, T, Y or W; or more particularly E345D, K, N, Q, R or W; or even more particularly E345R, Q, N, K or Y. In another further embodiment, E430X may be E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W or Y; particularly E430T, S, G, F or H. In one preferred embodiment, the amino acid substitution is selected from the group including E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y. In a further embodiment, the mutation in at least one amino acid residue is selected from E345R and E430G. In a further embodiment, the mutation in at least one amino acid residue is E345R. In a further embodiment, the mutation in at least one amino acid residue is E430G.

[0193] In an alternative embodiment, the mutation in at least one amino acid residue is selected from those corresponding to I253, H310, Q311, E382, G385, H433, N434, Y436 and Q438 in the Fc region of a human IgG1 heavy chain, such as E382 or H433. In a further alternative embodiment, the mutation in at least one amino acid residue is selected from I253E, N, Q, S or T, such as I253N or Q; H310N, Q, W or Y, such as H310Q; Q311E or R; E382D, H, K, R, N, Q, S, T, W or Y, such as E382D, Q, K or R; G385E, H, K, N, Q, R, S, T, W or Y, such as G385D, E , K or R; H433R; N434D, E, H, K, Q, R, S, T, W or Y, such as N434H, K, Q or R; Y436A, E, F, H, I, K, L, M, N, Q, R, S, T or V, such as Y436N, Q, S or T; Q438A, E, G, H, K, N, Q, R, S, T, W or Y, or for example Q438N, S or T.

[0194] Thus, in a further alternative embodiment, the mutation in at least one amino acid residue may be an amino acid substitution selected from those corresponding to P247G, I253V, S254L / V, Q311L / W, D / E356G / R, T359R, E382L / V and Y436I in the Fc region of a human IgG1 heavy chain, such as, in particular, E382L, V, D, Q, K or R or H433R. In a further alternative embodiment, the mutation in at least one amino acid residue is selected from E382R and H433R. In an alternative embodiment, the mutation is E382R. In another alternative embodiment, the mutation is H433R.

[0195] In another embodiment, the mutation is optionally not in an amino acid residue directly involved in C1q binding, as determined by comparing C1q binding in an ELISA assay according to Example 3 with C1q binding in a cell-based assay according to Example 4.

[0196] In one embodiment, the mutation is not at an amino acid residue corresponding to I253, N434 or Q311, and optionally is not at an amino acid residue corresponding to H433, or the amino acid substitution is not H433A.

[0197] In one embodiment, the at least one mutation is a single mutation, ie, no multiple mutations are introduced into the parent antibody.

[0198] In another embodiment, the method or use of the invention comprises mutating at least two, such as two, three, four, five or more of the amino acid residues in Table 1.

[0199] Any combination of the mutations described herein can be introduced according to the methods of the present invention.

[0200] In one embodiment, the method or use of the present invention comprises the step of introducing mutations into a parent antibody at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0201] In one embodiment, the method or use of the present invention comprises the step of introducing mutations into a parent antibody at least two amino acid residues selected from those corresponding to E345, E430, Q386 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is S440Y or S440W, e.g., the mutations at the at least two amino acids are selected from the following: substitution of E345 with R, Q, N or K; substitution of E430 with T, S or G; substitution of S440 with Y or W; or substitution of Q386 with K.

[0202] In an alternative embodiment, the position of the first mutation may be selected from the group consisting of positions 1253, H310, Q311, E345, E382, G385, H433, N434, Y436 and Q438.

[0203] In one embodiment, the method further comprises introducing one additional / third mutation into the antibody at amino acid residues corresponding to E345, E430, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 or K447 in both the first and / or second Fc regions.

[0204] For example, multiple mutations, such as two, three, four, or five, particularly two or three, are introduced into the parent antibody at amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc region of the human IgG1 heavy chain. For example, at least one of the amino acid residues corresponding to E345, E430, and S440 in the Fc region of the human IgG1 heavy chain, for example, two or all of E345, E430, and S440, can be mutated, optionally in combination with mutations in one or more other amino acids listed in Table 1. The at least two mutations may be any amino acid residue substitution at position E345 in combination with any amino acid residue substitution at positions E430 or S440, or any amino acid substitution at position E430 in combination with any amino acid residue at position S440.

[0205] In a further embodiment, two or three mutations are introduced into the parent antibody at amino acid residues selected from those corresponding to E345, E430, S440 and Q386 in the Fc region of a human IgG1 heavy chain.

[0206] In one embodiment, the multiple mutations may be, in particular, amino acid substitutions.

[0207] Thus, according to the present invention, the method or use comprises introducing into the antibody at least one, for example one, two, three, four, five or six amino acid substitutions selected from the group consisting of: P247G, I253V, S254L, Q311L / W, E345X, D / E356G / R, T359R, E382L / V, Q386K, E430X, Y436I and S440Y / W. In preferred embodiments thereof, the amino acid substitutions are selected from the group consisting of E345X, E430X, S440Y / W and Q386K.

[0208] In an alternative embodiment, at least two mutations, for example two, three, four or five mutations, are at amino acid residues selected from those corresponding to H310, G385, H433, N434 and Q438 in the Fc region of a human IgG1 heavy chain.

[0209] In another alternative embodiment, at least one mutation, and optionally two or three mutations, is selected from the group consisting of E345R, E382R, and H433R. In another alternative embodiment, at least one, such as both, of the amino acid residues corresponding to E382 and H433 in the Fc region of a human IgG1 heavy chain can be mutated, optionally in combination with mutations in one or more other amino acids listed in Table 1.

[0210] In some embodiments of the methods and / or uses of the present invention, mutations at amino acid residues corresponding to K439 and / or S440 are introduced into an antibody selected from the group consisting of a parent antibody, a first parent antibody, a second parent antibody, and combinations thereof. As mentioned above, the introduction of mutations at amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain has been shown to limit intermolecular interactions between antibodies containing such mutations (Examples 4, 5, 6, 10). Depending on whether K439 and S440 are introduced into the same parent antibody or into each of the first and second parent antibodies, these aspects are also referred to as "double mutant" and "mixed mutant" aspects.

[0211] In one embodiment of the invention, the mutation at the amino acid residue corresponding to K439 in the Fc region of human IgG1 heavy chain is an amino acid substitution.

[0212] In one embodiment of the invention, the mutation at the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain is an amino acid substitution.

[0213] In all embodiments of the invention, mutations at positions corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, whether in the same polypeptide or antibody or in a first and second polypeptide or antibody, may be replaced by the following mutations: (i) mutations in each of the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, such as K447K / R / H and 448E / D in the Fc region of the human IgG1 heavy chain, preferably K447K and 448E in the Fc region of the human IgG1 heavy chain; or (ii) Mutations at each of the amino acid residues corresponding to K447, 448, and 449 in the Fc region of the human IgG1 heavy chain, for example, K447D / E, 448K / R / H, and 449P in the Fc region of the human IgG1 heavy chain, preferably K447E, 448K, and 449P in the Fc region of the human IgG1 heavy chain.

[0214] Such combinations of mutations therefore include any of those set forth in Tables 2A and 2B.

[0215] In one embodiment of the invention, the mutations in the amino acid residues corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain are both amino acid substitutions.

[0216] In one embodiment, the mutation at the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain is an amino acid substitution to an amino acid selected from E and D.

[0217] In another embodiment, the mutation is K439E.

[0218] In one embodiment, the mutation at the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain is an amino acid substitution to an amino acid selected from K, R and H.

[0219] In another embodiment, the mutation is S440K.

[0220] Thus, in a further embodiment, the mutations introduced into the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain of the parent antibody are amino acid substitutions selected from K439E and D, and S440K, R and H.

[0221] Thus, in a further embodiment, the mutations introduced into the parent antibody at amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain are the amino acid substitutions K439E and S440K.

[0222] Some methods and uses of the invention include a first and a second parent antibody.

[0223] Thus, in a further embodiment, the mutation introduced into the amino acid residue corresponding to K439 in the first parent antibody is an amino acid substitution selected from K439E and D, e.g., K439E, and the mutation introduced into the amino acid residue corresponding to S440 in the second parent antibody is an amino acid substitution selected from S440K, R and H, e.g., S440K. The mutations in the first and second parent antibodies may also be introduced in the opposite manner, i.e., a mutation at the amino acid residue corresponding to S440 may be introduced into the first parent antibody, while a mutation at the amino acid residue corresponding to K439 may be introduced into the second parent antibody, where the mutations may be preferred amino acid substitutions as described above.

[0224] In one embodiment of the method or use of the invention, the effector function is enhanced when the antibody binds to its antigen.

[0225] In a further embodiment, the effector function is enhanced when the antibody binds to its antigen, where the antigen is on an antigen-expressing cell, on a cell membrane, or on a virion. In one embodiment, the Fc region of the IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO:1.

[0226] The parent antibody may be any parent antibody as described herein. A parent antibody in this context is intended to be also a first parent antibody and a second parent antibody.

[0227] In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD or IgE antibody.

[0228] In one embodiment, the parent antibody is a human full-length antibody, such as a human full-length IgG1 antibody.

[0229] In one embodiment, the parent antibody, first parent antibody and second parent antibody are human IgG1 antibodies, such as IgG1m(za) or IgG1m(f) allotypes, optionally comprising an Fc region comprising SEQ ID NO:1 or 5.

[0230] In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO:2.

[0231] In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO:3.

[0232] In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO:4.

[0233] In one embodiment, the parent antibody is a bispecific antibody.

[0234] In one embodiment, the parent antibody is any antibody as described herein, including, for example, an antibody fragment comprising at least a portion of an Fc region, a monovalent antibody (as described in WO007059782 by Genmab); a heavy chain antibody consisting of only two heavy chains and occurring naturally, for example in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); a Thiomab (Roche, WO2011069104), a chain-exchange recombinant domain (SEED or Seed-body) which is an asymmetric bispecific antibody-like molecule (Merck, WO2007110205); a Triomab (Fresenius, Lindhofer et al.(1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), dual variable domain immunoglobulins (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); dual domain diabodies (Unilever; Sanofi Aventis, WO20100226923), di-diabodies (ImClone / Eli Lilly), knob-into-hole antibody formats (Genentech, WO9850431); duobodies (Genmab, WO 2011 / 131746); electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO 2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual targeting domain antibodies (GSK / Domantis), two-in-one antibodies recognizing two targets (Genentech, NovImmune), cross-linking MAb (Karmanos Cancer Center), CovX-body (CovX / Pfizer), IgG-like bispecifics (ImClone / Eli Lilly, Shen, X, et al. al. J Immunol Methods, 2007. 318(1-2): p.65-74), as well as DIG-body and PIG-body (Pharmabcine), and dual affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), Zybody (Zyngenia), approaches using common light chains (Crucell / Merus, US7262028) or common heavy chains (κλbody by NovImmune), as well as fusion proteins such as scFv fusions, including those in which a polypeptide sequence is fused to an antibody fragment containing an Fc domain, such as BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPION by Emergent BioSolutions / Trubion, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN No. 102250246), TvAb by Roche (WO No. 2012025525, WO No. 2012025530), mAb by f-Star. 2 (WO2008 / 003116) and double scFv fusions. The term antibody should also be understood to include, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals), such as those produced by technology developed by Symphogen and Merus (Oligoclonics), as well as antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. The antibodies produced may potentially have any isotype.

[0235] Optionally, monovalent antibodies, heavy chain antibodies, strand-exchanged recombinant domains (SEED), triomabs, dual variable domain immunoglobulins (DVD-Ig), knob-into-hole antibodies, miniantibodies, dual affinity retargeting molecules (Fc-DART or Ig-DART); LUZ-Y antibodies, biclonic antibodies, dual targeting (DT)-Ig antibodies, two-in-one antibodies, cross-linked Mabs, mAbs 2 , CovX-body, IgG-like bispecific antibody, Ts2Ab, BsAb, HERCULES antibody, TvAb, ScFv / Fc fusion antibody, SCORPION, scFv fragment fused to an Fc domain, and dual scFv fragment fused to an Fc domain.

[0236] In another embodiment, the antigen is expressed on the surface of a cell.

[0237] In another embodiment, the cell is a human tumor cell.

[0238] In a further embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

[0239] In another embodiment, the antigen is cell membrane bound.

[0240] In another embodiment, the antigen is associated with the virion, and optionally, the antigen is contained within the protein coat or lipid envelope of the virion.

[0241] In another embodiment, the antibody is a human antibody, and optionally binds to at least one antigen selected from CD20 and CD38.

[0242] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, and optionally comprises the variable heavy and / or variable light chain regions of at least one of 7D8 and 005.

[0243] In any use of the disclosed invention, an antibody that does not have any of the mutations of the invention may be any parent antibody, and therefore the use herein provides for any variant of such a parent antibody.

[0244] In a further embodiment of the invention, the effector function is an Fc-mediated effector function selected from C1q binding, complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0245] In one particular embodiment, the effector function is C1q binding, complement activation (C1q activity), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, e.g., Fc-gamma receptor binding, internalization of Fc-containing polypeptides, or any combination thereof.

[0246] In one embodiment, the effector function is C1q binding.

[0247] In one embodiment, the effector function is complement activation (C1q activity).

[0248] In one embodiment, the effector function is complement dependent cytotoxicity (CDC).

[0249] In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0250] In one embodiment, the effector function is Fc receptor binding, including, for example, Fc-gamma receptor binding.

[0251] In one embodiment, the effector function is the internalization of an Fc-containing polypeptide.

[0252] In one embodiment, the effector function is a combination of complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0253] In another embodiment, the one or more mutations enhance one additional effector function selected from FcRn binding, ADCC, Fc-gamma receptor binding, protein A binding, protein G binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, antibody-mediated opsonization antibody binding to complement receptors, and any combination thereof.

[0254] In another aspect, the present invention relates to a method for enhancing the binding ability of a parent antibody preparation to C1q, comprising mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447.

[0255] As used herein, the term "C1q binding," when used in the context of a parent antibody variant or antibody, includes any mechanism of the first component to the classical pathway of complement activation mediated by binding of the variant or antibody to host tissues or factors, including various cells of the immune system (such as effector cells). C1q binding of an antibody can be assessed using an ELISA (such as the C1q binding ELISA used in Examples 3 and 4), or C1q activity can be assessed by a CDC assay (such as the CDC assay used in Example 5). In a further embodiment, the C1q binding ability of an antibody is determined according to the assay described in Example 4.

[0256] In all disclosed methods of the invention, any parent antibody may be an antibody that does not have any of the mutations of the invention, and therefore the methods herein result in any variant of such a parent antibody.

[0257] The parent antibody, first parent antibody, second parent antibody or variants thereof obtained by the methods and / or uses of the present invention may bind to any target as described herein.

[0258] Examples of antigens or targets that may be addressed by the present invention include: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; cancer-associated antigen CTAA16.88; TIFF2024116396000005.tif29154 Claudin 4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAPα; FcγRI; FCER2; FGFR3; fibrin II β chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE joining region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor β subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, lipopolysaccharide (LPS) domain; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; nectin-4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [Escherichia coli] coli)];SLAM7;SLC44A4;SOST;Staphylococcus epidermidis lipoteichoic acid;T cell receptor α_β;TF;TGFB1;TGFB2;TMEFF2;and vimentin. ;

[0259] Methods for inducing an effector response It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent peptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0260] In one further major aspect, the present invention provides a method of inducing an effector response against a cell, cell membrane, or virion expressing a target to which a parent polypeptide comprising an immunoglobulin Fc domain and a binding region binds, comprising the steps of: (i) providing a parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide mutated according to any one of claims 1 to 24; and (ii) contacting a preparation of the mutated parent polypeptide of step (i) or a combination of at least a first mutated parent polypeptide and a second mutated parent polypeptide of step (i) with a cell, cell membrane or virion expressing the antigen in the presence of human complement or effector cells. The present invention relates to a method comprising:

[0261] In one embodiment, any or all of the parent polypeptide, the first parent polypeptide and the second parent polypeptide may be an antibody.

[0262] Thus, in one embodiment, the invention relates to methods of using the antibody variants described herein to induce an effector response, such as complement activation, CDC or other effector response, against a cell, cell membrane, virion or other particle to which one or more antigens are bound. The invention also relates to a method of inducing an effector response against a cell, cell membrane or virion expressing an antigen to which a parent antibody binds, comprising: (i) providing a parent antibody or a combination of at least a first parent antibody and a second parent antibody that has been mutated according to any of the methods described herein; and (ii) contacting the preparation of the mutated parent antibody of step (i) or a combination of at least a first mutated parent antibody and a second mutated parent antibody of step (i) with a cell, cell membrane or virion expressing the antigen in the presence of human complement or effector cells. The present invention also relates to a method comprising:

[0263] The parent antibody, first parent antibody and second parent antibody can each be selected from any of the parent antibodies described herein, particularly any of those described above in relation to methods of affecting antibody effector function.

[0264] In one embodiment, the antigen is expressed on the surface of a cell.

[0265] In one embodiment, the cell is a human tumor cell.

[0266] In a further embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

[0267] In another embodiment, the antigen is cell membrane bound.

[0268] In another embodiment, the antigen is associated with the virion, and optionally, the antigen is contained within the protein coat or lipid envelope of the virion.

[0269] In another embodiment, the antibody is a human antibody, and optionally binds to at least one antigen selected from CD20 and CD38.

[0270] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, and optionally comprises the variable heavy and / or variable light chain regions of at least one of 7D8 and 005.

[0271] In a further embodiment of the invention, the effector response induced is an Fc-mediated effector response selected from complement dependent cytotoxicity (CDC), C1q binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0272] In one particular embodiment, the effector response is C1q binding, complement activation (C1q activity), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, e.g., Fc-gamma receptor binding, internalization of an Fc-containing polypeptide, or any combination thereof.

[0273] In one embodiment, the effector response is C1q binding.

[0274] In one embodiment, the effector response is complement activation (C1q activity).

[0275] In one embodiment, the effector response is complement dependent cytotoxicity (CDC).

[0276] In one embodiment, the effector response is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0277] In one embodiment, the effector response is Fc receptor binding, including, for example, Fc-gamma receptor binding.

[0278] In one embodiment, the effector response is the internalization of an Fc-containing polypeptide.

[0279] In one embodiment, the effector response is a combination of complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0280] In another embodiment, the method enhances one additional effector function selected from FcRn binding, ADCC, Fc-gamma receptor binding, protein A binding, protein G binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, antibody-mediated opsonization antibody binding to complement receptors, and any combination thereof.

[0281] In another aspect, the present invention relates to a method for enhancing the binding ability of a parent antibody preparation to C1q, comprising mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447.

[0282] Examples of antigens or targets that may be addressed by the present invention include: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; cancer-associated antigen CTAA16.88; TIFF2024116396000006.tif29150 Claudin 4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAPα; FcγRI; FCER2; FGFR3; fibrin II β chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE joining region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor β subunit; ITGA2; ITGA2BITGB3; ITGA4ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y ; lipid A, lipopolysaccharide (LPS) domain; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; nectin-4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa aeruginosa serotype IATSO11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin IIB subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T-cell receptor α_β; tissue factor (TF); TGFB1; TGFB2;and vimentin. ;

[0283] In one embodiment, the cell is a human tumor cell or a bacterial cell.

[0284] In another embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, EpCAM, and MRP3.

[0285] In a further embodiment, the antigen is CD20 or CD38.

[0286] In another embodiment, the parent IgG1 antibody is a human IgG1 antibody.

[0287] In another embodiment, the parent antibody is selected from 7D8 and 005.

[0288] In one embodiment, the cell is a human tumor cell.

[0289] In another embodiment, the first and second antigens are independently selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), EpCAM, and MRP3.

[0290] In another embodiment, the first and second parent antibodies are fully human antibodies, and optionally the first and second parent antibodies bind to antigens independently selected from CD20 and CD38.

[0291] In a further embodiment, the first and second parent antibodies are independently selected from 7D8 and 005.

[0292] In yet another embodiment, the cell is a bacterial cell.

[0293] In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.

[0294] In another embodiment, the first and / or second antigen is lipoteichoic acid (LTA), and optionally at least one of the first and second parent antibodies is pagibaximab.

[0295] In another embodiment, the antigen is expressed on the virion.

[0296] In another embodiment, the first and second antibodies bind to the same antigen.

[0297] In another embodiment, the first and second antibodies comprise the same VH, VL, or both VH and VL sequences.

[0298] For purposes of the present invention, target cells expressing or otherwise associated with antigens can be any prokaryotic or eukaryotic cell. Exemplary antigen-expressing cells include, but are not limited to, mammalian cells, particularly human cells, such as human cancer cells; and unicellular organisms, such as bacteria, protozoa, and unicellular fungi, such as yeast cells. Cell membranes containing or otherwise associated with antigens include partial and / or disrupted cell membranes from antigen-expressing cells. Antigens bound to virion particles or virus particles can be contained within or otherwise associated with the protein coat and / or lipid envelope of the virion.

[0299] The target cells may be, for example, human tumor cells. Suitable tumor antigens include any of the targets or antigens described herein, but are not limited to erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFR, L1-CAM, AXL, tissue factor (TF), EpCAM, and MRP3. Preferred antigens include CD20, CD38, HER2, EGFR, IGFR, CD25, CD74, and CD32. Exemplary antibodies include the anti-CD20 antibody 7D8 as disclosed in WO 2004 / 035607, the anti-CD38 antibody 005 as disclosed in WO 06 / 099875, the anti-CD20 antibody 11B8 as disclosed in WO 2004 / 035607, the anti-CD38 antibody 003 as disclosed in WO 06 / 099875, and the anti-EGFr antibody 2F8 as disclosed in WO 02 / 100348. Other specific antibody examples are provided herein.

[0300] Alternatively, the target cell may be a bacterial cell, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Neisseria meningitidis, and Mycobacterium tuberculosis, etc. Exemplary antigens include lipoteichoic acid (LTA), and exemplary antibodies include pagibaximab.

[0301] Alternatively, the target may be present on the surface of a virus, fungal cell, or other particle, such as West Nile virus, dengue virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papillomavirus, Epstein-Barr virus, herpesvirus, poxvirus, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus, and Histoplasma.

[0302] In one embodiment, the contacting step (ii) is carried out in vitro.

[0303] In one embodiment, the contacting step (ii) occurs in vivo.

[0304] In another embodiment, step (ii) comprises administering the variant to the subject.

[0305] In a further embodiment, the subject suffers from cancer, bacterial infection or viral infection. The contacting step (ii) of the above-mentioned embodiment can be carried out in vitro or in vivo. In the latter case, step (ii) can further comprise administering one or more preparations to the subject, optionally to a subject suffering from cancer or bacterial infection. Further details regarding therapeutic applications are provided below.

[0306] The first and second antibodies comprise antigen-binding regions that can bind to the same epitope or to different epitopes, which can be on the same target or on different targets.

[0307] In one embodiment, the first and second antibodies bind to different epitopes on different targets. These targets may be expressed on the same cell or cell type, or on different cells or cell types. In such an embodiment, the enhancement of effector function is directed only to cells or cell types that express both targets, thereby reducing the risk of collateral damage to cells or cell types that are not responsible for the disease being treated.

[0308] Without being bound by any theory, it is believed that enhanced CDC can be restricted to target cells that simultaneously express two specific targets / antigens if the first and second antibodies bind to epitopes found on the same cell, thereby exploiting combined expression of the targets to improve the selectivity of the enhanced CDC induction.

[0309] If the targets are expressed on different cells or cell types, without being bound by theory, it is believed that administration of the first and second antibodies in any order will improve CDC enhancement and possibly other effector functions by "recruitment" of a second cell or cell type expressing the second target.

[0310] In one embodiment using a combination of a first and second antibody, step (ii) may be carried out by contacting the cells with the mutated first and second parent antibodies simultaneously, separately or sequentially in the presence of human complement and / or effector cells.

[0311] In yet another aspect, the present invention relates to a method for improving the CDC-inducing ability of a parent antibody preparation, comprising mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447.

[0312] In an alternative aspect, the present invention provides a method for inducing an effector response, optionally a CDC response, against a cell, cell membrane or virion expressing an antigen bound by a parent IgG1 antibody, comprising the steps of: (i) providing an antibody containing a mutation in at least one amino acid residue selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436, and K447 in the Fc region of an IgG1 heavy chain; and (ii) contacting the antibody preparation with cells, cell membranes, or virions in the presence of human complement or effector cells. The present invention relates to a method comprising:

[0313] In another alternative aspect, the method comprises: administering a first antibody comprising a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447 in the Fc region of the first antibody; administering a second antibody comprising a second mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447 in the Fc region of the second antibody. wherein the first and second antibodies can be administered simultaneously, separately, or sequentially. The first and second antibodies can bind to the same or different epitopes on the same or different targets. The targets can be on the same cell or cell type, or on different cells or cell types.

[0314] In another alternative aspect, the present invention relates to a method for improving the CDC-inducing ability of a parent IgG1 antibody preparation, comprising mutating at least one amino acid in the Fc region of the antibody, wherein said at least one amino acid is selected from the group consisting of E345, E382 and H433.

[0315] In another alternative aspect, the present invention provides a method for inducing an effector response, optionally a CDC response, against a cell, cell membrane or virion expressing an antigen bound by a parent IgG1 antibody, comprising the steps of: (i) providing a variant of a parent antibody comprising a mutation in at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E382, and H433; and (ii) contacting the preparation of the variant with cells in the presence of human complement or effector cells. The present invention relates to a method comprising:

[0316] In another alternative aspect, the present invention provides a method for inducing an effector response, optionally a CDC response, against cells expressing an antigen to which the parent IgG1 antibody binds, comprising the steps of: (i) providing a variant of a parent antibody comprising K439E and S440K mutations in the Fc region of the antibody; and (ii) contacting the preparation of the variant with cells in the presence of human complement or effector cells. The present invention relates to a method comprising:

[0317] In another alternative aspect, the present invention provides a method for inducing a CDC response against cells, cell membranes or virions expressing a first antigen bound by a first IgG1 parent antibody and a second antigen bound by a second parent antibody, comprising the steps of: (i) providing a first variant of a first parent antibody comprising a K439E mutation and a second variant of a second parent antibody comprising a S440K mutation; and (ii) contacting the cells with the first and second variants simultaneously, separately or sequentially in the presence of human complement and / or effector cells. The present invention relates to a method comprising:

[0318] In another alternative aspect, the present invention provides a method for inducing a CDC or other effector response against a target cell, cell membrane, virion, or other particle to which an antigen bound by an IgG1 or IgG3 antibody binds, the method comprising the steps of: (i) providing an antibody variant comprising a mutation in at least one amino acid corresponding to E345, E430, or S440 in the Fc region of an IgG1 antibody; and (ii) contacting the variant preparation with cells in the presence of human complement and / or effector cells.

[0319] In a further alternative aspect, the present invention provides a method for inducing ADCC or ADCP against, or phagocytosis of, a target cell, cell membrane, virion, or other particle to which an antigen bound by an IgG1 or IgG3 antibody is bound, the method comprising the steps of: (i) providing an antibody mutant comprising a mutation in at least one amino acid corresponding to E345, E430, or S440 in the Fc region of the IgG1 antibody; and (ii) contacting the mutant preparation with cells in the presence of effector cells.

[0320] The present invention also provides a method for inducing a CDC or other effector response against a target cell, cell membrane, virion, or other particle to which an antigen bound by an IgG1 or IgG3 antibody is bound, comprising the steps of: (i) providing an antibody mutant comprising a mutation at K439 in the Fc region of the antibody, where K439 is K439E, and a mutation at S440, where S440 is S440K or S440R; and (ii) contacting the mutant preparation with cells in the presence of human complement and / or effector cells.

[0321] The present invention also provides a method for inducing a CDC or other effector response against a target cell, cell membrane, or virion expressing a first antigen bound by a first IgG1 antibody and a second antigen bound by a second antibody, the method comprising the steps of: (i) providing a first variant, the first antibody comprising a K439E mutation, and a second variant, the second antibody comprising a S440K or S440R mutation; and (ii) contacting cells simultaneously, separately, or sequentially with preparations of the first and second variants in the presence of human complement or effector cells.

[0322] In separate and specific embodiments, the first and second antibodies bind to (i) different antigens; (ii) different epitopes on the same antigen, (iii) the same epitope on an antigen, and (iv) the same epitope on an antigen and comprise the same VH and / or VL sequence.

[0323] In one embodiment, the first and second antibodies further comprise a mutation at one or more of E345, E430 and S440, e.g., E345R. In one embodiment, the first and second antibodies further comprise a mutation at one or more of E345, E382 and H433, e.g., E345R.

[0324] Other methods In another major aspect, the present invention provides a method for identifying mutations in an antibody that enhance an effector function of an antibody that binds C1q, comprising the steps of: (i) preparing at least one antibody comprising a mutation in at least one amino acid selected from the group consisting of E345, E430, S440, K439, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447; (ii) assessing the C1q activity of the antibody when bound to the surface of an antigen-expressing cell, compared to the parent antibody; and (iii) selecting any mutant mutations that have enhanced C1q binding ability; The present invention relates to a method comprising:

[0325] In one embodiment, at least one antibody comprises at least one amino acid substitution selected from the group of E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.

[0326] In yet another major aspect, the present invention provides a method for identifying mutations in a parent antibody that enhance the ability of the antibody to induce a CDC response, comprising: (i) preparing a variant of at least one parent antibody comprising a mutation in at least one amino acid selected from the group consisting of E345, E430, S440, K439, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447; (ii) assessing the CDC response induced by the variants when bound to the surface of antigen-expressing cells in the presence of effector cells or complement, compared to the parent antibody; and (iii) selecting any mutant mutations that have an enhanced CDC response; The present invention relates to a method comprising:

[0327] In one embodiment, the at least one amino acid is selected from E345, E382 and H433.

[0328] In one embodiment, at least one antibody comprises at least one amino acid substitution selected from the group of E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.

[0329] In another aspect, the present invention relates to a method for increasing the binding ability of a parent IgG1 antibody preparation to C1q, comprising mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E382 and H433.

[0330] Antibodies of the Invention Parent antibody As described herein, the present invention particularly relates to variants of parent antibodies containing one or more mutations in the CH2 and / or CH3 regions of immunoglobulins, e.g., the heavy chain of the antibody. The "parent" antibody, which may be a wild-type antibody, used as the starting material for the present invention before modification may be produced, for example, by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse splenic B cells obtained from mice immunized with the antigen of interest, e.g., in the form of cells expressing the antigen on their surface or in the form of nucleic acids encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals (e.g., rabbits, rats, dogs, primates, etc.).

[0331] The parent antibody may be, for example, a chimeric or humanized antibody. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies can be generated using transgenic or transchromosomal mice, e.g., HuMAb mice, which possess parts of the human immune system rather than the mouse system. HuMAb mice contain human immunoglobulin gene minilocuses encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). As a result, these mice exhibit reduced expression of mouse IgM or κ, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation in response to immunization, resulting in the generation of high-affinity human IgG and κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; (Reviewed in TIFF2024116396000007.tif22152). Preparation of HuMAb mice was performed using It is described in detail in TIFF2024116396000008.tif29155. Also, See also TIFF2024116396000009.tif22153. Splenocytes from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[0332] Furthermore, human antibodies of the invention, or antibodies of the invention derived from other species, can be identified by display-type technologies, including but not limited to, phage display, retroviral display, ribosome display, mammalian display, yeast display, and other techniques known in the art, and the resulting molecules can be subjected to further maturation, such as affinity maturation, as techniques well known in the art. The specific strategy described in Example 17 can be applied to any antibody to prepare and obtain variants of the invention using phage display.

[0333] The parent antibody is not limited to a naturally occurring antibody, e.g., an antibody having a human Fc domain, but may also have other mutations besides those of the present invention, such as mutations affecting glycosylation or enabling the antibody to be a bispecific antibody. The term "native antibody" refers to any antibody that does not contain any genetically introduced mutations. Antibodies containing naturally occurring modifications, such as various allotypes, should therefore be considered "native antibodies" within the meaning of the present invention and, as a result, can be considered parent antibodies. Such antibodies can serve as templates for one or more mutations of the present invention, thereby obtaining the mutant antibodies of the present invention. An example of a parent antibody containing mutations other than those of the present invention is a bispecific antibody, such as that described in WO 2011 / 131746 (Genmab), which utilizes reducing conditions to promote half-molecule exchange between two antibodies containing IgG4-like CH3 regions, thereby forming a bispecific antibody without the concomitant formation of aggregates. Other examples of parent antibodies include, but are not limited to, bispecific antibodies such as heterodimeric bispecifics: Triomab (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); knobs-into-holes (Genentech); electrostatic steering (Amgen, Chugai, Oncomed); SEEDbody (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, human antibodies, or any combination thereof.

[0334] The parent antibody may bind to any target, and examples of such targets or antigens of the present invention may be, but are not limited to, those directed against the following: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; cancer associated antigen CTAA16.88; TIFF2024116396000010.tif29154 claudin 4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAPα; FcγRI; FCER2; FGFR3; fibulin II β chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; glypican-3; GPNM B; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE joining region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7;ITGA5;ITGAL;ITGAV_ITGB3;ITGB2;KDR;L1CAM;Lewis-y;Lipid A, lipopolysaccharide (LPS) domain;LTA;MET;MMP14;MMp15;MST1R;MSTN;MUC1;MUC4;MUC16;MUC5AC;NCA-90 granulocytic cell antigen;Nectin-4;NGF;NRP;NY-ESO-1;OX40L;PLAC-1;PLGF;PDGFRA;PD1;PDL1;PSCA;phosphatidylserine;PTK-7;Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin IIB subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T-cell receptor α_β; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and vimentin.

[0335] The parent antibody may be any human antibody of any isotype, for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgD, and optionally may be a human full-length antibody, such as a human full-length IgG1 antibody. The parent antibody may comprise a sequence according to any of SEQ ID NOs: 1, 2, 3, 4, and 5.

[0336] Monoclonal antibodies for use in the present invention, e.g., parent antibodies and / or variants, may be produced by, for example, the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries, e.g., using the techniques described in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from mouse splenic B cells obtained from mice immunized with the antigen of interest, e.g., in the form of cells expressing the antigen on their surface, or in the form of nucleic acids encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals (eg, rats, dogs, primates, etc.).

[0337] In one embodiment, the antibody is a human antibody.Human monoclonal antibodies directed against any antigen can be produced using transgenic mice or transchromosomal mice that have part of the human immune system instead of the mouse system.Such transgenic mice or transchromosomal mice include those referred to herein as HuMAb® mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice."

[0338] HuMAb® mice contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). As such, these mice exhibit reduced expression of mouse IgM or κ, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation in response to immunization, resulting in the generation of high-affinity human IgG and κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; (Reviewed in TIFF2024116396000011.tif15152). Preparation of HuMAb® mice was performed using It is described in detail in TIFF2024116396000012.tif22155. Also, See also TIFF2024116396000013.tif23155.

[0339] HCo7, HCo12, HCo17, and HCo20 mice have a JKD disruption in their endogenous light chain (κ) gene (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), and a KCo5 human κ light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, Hco7 mice carry an HCo7 human heavy chain transgene (as described in U.S. Pat. No. 5,770,429), HCo12 mice carry an HCo12 human heavy chain transgene (as described in Example 2 of WO 01 / 14424), HCo17 mice carry an HCo17 human heavy chain transgene (as described in Example 2 of WO 01 / 09187), and HCo20 mice carry an HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and kappa light chain transgenes in a background with homozygous disruptions of the endogenous mouse heavy chain and kappa light chain loci.

[0340] The KM mouse strain contains a homozygous disruption of the endogenous mouse kappa light chain gene as described in Chen et al., EMBO J. 12, 811-820 (1993), and a homozygous disruption of the endogenous mouse heavy chain gene as described in Example 1 of WO 01 / 09187. This mouse strain contains a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also contains a human heavy chain transchromosome composed of chromosome 14 fragment hCF(SC20), as described in WO 02 / 43478. HCo12-Balb / C mice can be generated by crossing HCo12 with KCo5[J / K](Balb), as described in WO / 2009 / 097006. Spleen cells from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[0341] Furthermore, any antigen-binding region can be obtained from a human antibody or an antibody from another species identified by display-type technologies, including but not limited to, phage display, retroviral display, ribosome display, and other techniques, using techniques well known in the art, and the resulting molecules can be subjected to further maturation, such as affinity maturation, using techniques well known in the art (e.g., Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display); Vaughan et al., Nature Biotech 14, 309 (1996) (phage display); Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosome display); Parmley and Smith, Gene 73, 305-318 (1988) (phage display); Scott TIBS 17, 241-245 (1992); Cwirla et al., (See PNAS USA 87, 6378-6382 (1990), Russell et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and US 5,733,743.) Where display techniques are used to generate non-human antibodies, such antibodies can also be humanized.

[0342] In another aspect, the present invention relates to a parent polypeptide comprising an Fc domain and a binding region. It will be understood that in the context of the present invention, all aspects relating to a parent antibody apply equally to a "parent polypeptide".

[0343] The mutations of the present invention may be, but are not limited to, deletions, insertions, or substitutions of one or more amino acids. Such substitutions of amino acids may be with any naturally occurring or non-naturally occurring amino acids.

[0344] "Single mutant" It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent polypeptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0345] The antibody or polypeptide variants of the "single variant" aspect of the invention comprise a mutation, typically an amino acid substitution, at at least one amino acid residue as shown in Table 1, which lists each amino acid residue numbered according to the EU index in human IgG1 antibodies, along with the amino acid at the corresponding position in IgG2, IgG3 and IgG4 parent antibodies, and "exemplary" and "preferred" amino acid substitutions. The segment of IgG2 corresponding to residues P247-K447 of IgG1, the Fc-segment of IgG3 corresponding to residues P247-K447 of IgG1, and the segment of IgG4 corresponding to residues P247-K447 of IgG1 are shown in Figure 2.

[0346] Table 1. Exemplary mutation sites and amino acid substitutions for "single mutant" aspects TIFF2024116396000014.tif105160

[0347] As can be seen in Table 1, the amino acid substitutions that resulted in enhanced cytolysis of Wien133 cells in Example 19 are included as "preferred substitutions."

[0348] In one aspect, the present invention relates to variants of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, which comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0349] In one embodiment, the mutant polypeptide may be a mutant antibody.

[0350] Thus, in another aspect, the present invention relates to a variant of a parent antibody comprising an antigen-binding region and an immunoglobulin Fc domain, comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W. Alternatively, the amino acid residue is selected from those corresponding to H310, G385, H433, N434, Q438 and K439 in the Fc region of a human IgG1 heavy chain.

[0351] Each of the amino acid residues corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, as described above, may be grouped according to the following: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) Amino acid residues corresponding to E345, E382, or Q386 in the Fc region of human IgG1 heavy chain.

[0352] The mutation of the present invention may be, but is not limited to, the deletion, insertion or substitution of one or more amino acids.Such amino acid substitution may be with any natural amino acid or non-natural amino acid.Therefore, in one embodiment, the mutation in at least one amino acid residue is deletion.In another embodiment, the mutation in at least one amino acid residue is insertion.In another embodiment, the mutation in at least one amino acid residue is substitution.

[0353] In one embodiment, the mutation in at least one amino acid residue is selected from those corresponding to E345X, E430X, S440W / Y, Q386K, P247G, I253V, S254L, Q311L / W, D / E356R, E382V, Y436I and K447D / E / deletion in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid.

[0354] In one specific embodiment, the antibody mutant comprises a mutation in at least one amino acid residue selected from E345, E430, S440 and Q386 in the Fc region of a human IgG1 heavy chain.

[0355] In a further embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid.

[0356] In one preferred embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345R, Q, N, K, A, C, D, F, G, H, I, L, M, P, S, T, V, W, Y; E430T, S, G, A, C, D, F, H, I, L, K, M, N, P, Q, R, V, W, Y; S440W, Y and Q386K in the Fc region of a human IgG1 heavy chain.

[0357] In a further preferred embodiment, the mutation in the at least one amino acid residue is an amino acid substitution selected from those corresponding to E345R / Q / N / K, E430T / S / G, S440Y / W and Q386K in the Fc region of a human IgG1 heavy chain.

[0358] Alternatively, at least one amino acid residue is selected from E382 and H433. Particular alternative amino acid substitutions include E345Y, D, W; and E430F, H; or E382D, Q, K, R; and H433R.

[0359] In one specific embodiment, the amino acid substitution is E345R. In an alternative embodiment, the mutations are I253 to E, N, Q, S, or T; H310 to N, Q, W, or Y; Q311 to E or R; E382 to D, H, K, R, N, Q, S, T, W, or Y; G385 to E, H, K, N, Q, R, S, T, W, or Y; H433 to R; N434 to D, E, H , K, Q, R, S, T, W or Y; a mutation of Y436 to A, E, F, H, I, K, L, M, N, Q, R, S, T or V; a mutation of Q438 to A, E, G, H, K, N, Q, R, S, T, W or Y; a mutation of K439 to D, H, Q, R, W or Y; and a mutation of S440 to D, E, H, F, N, Q, W or Y.

[0360] In another alternative embodiment, the mutation is selected from the group consisting of: an I253 to N or Q mutation; an H310 to Q mutation; an Q311 to E or R mutation; an E382 to D, Q, K or R mutation; an G385 to D, E, K or R mutation; an H433 to R mutation; an N434 to H, K, Q or R mutation; an Y436 to N, Q, S or T mutation; an Q438 to N, S or T mutation; an K439 to Q mutation; and an S440 to D, E or Q mutation.

[0361] In another alternative embodiment, the mutation is selected from the group consisting of: E382 to D, Q, K, or R; and H433 to R.

[0362] In one embodiment, the variant comprises an E382R mutation.

[0363] In one embodiment, the variant comprises an H433R mutation.

[0364] As shown in the Examples, variants of the CD38 antibodies HuMab-005 and -003 and / or the CD20 antibodies HuMab-7D8 and -11B8 and rituximab and / or the EGFR antibody HuMab-2F8 containing one of these amino acid substitutions had higher C1q binding, complement activation and / or CDC than wild-type HuMab 005 and 7D8, respectively.

[0365] It should be understood that a variant may include only one mutation of the "exemplary substitutions" listed in Table 1. A variant may also include multiple mutations, for example, two, three, four, five, or six, of any of the mutations listed in Table 1.

[0366] One preferred embodiment of the present invention therefore provides a variant comprising one mutation in an amino acid residue selected from those listed in the above aspects. Particular amino acid mutations are: TIFF2024116396000015.tif28164, which have enhanced cell lysis (greater than 39% in Wien133 cells) as shown in Example 19, Table 17.

[0367] In an alternative embodiment, the variant comprises a mutation at one amino acid residue selected from those corresponding to E382R, H433R, H435R and H435A.

[0368] In addition to the specified mutations, the variant may have any of the characteristics described for the parent antibody. In particular, it may be a human antibody. The variant may further be of any IgG1 subtype, excluding the mutations.

[0369] When bound to its antigen on the surface of an antigen-expressing cell, on a cell membrane, on a virion, or on another particle, or when the antigen is bound to a virion (optionally contained within the protein coat or lipid envelope of the virion), such antibody variants may have at least one of the following enhancements compared to the parent antibody: (i) C1q binding, (ii) antibody-mediated complement activation, (iii) antibody-mediated CDC, (iv) oligomer formation, (v) oligomer stability, or any combination of (i)-(v). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, the variants also, or alternatively, have other effector functions that are retained or improved, such as C1q binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0370] Without being limited to any particular theory, the effect caused by substituting an amino acid at a specified position with an amino acid residue in Table 1 may, for example, cause the effect itself, directly participate in contact with the Fc domain of another molecule, or be mutated to interact with another Fc domain, directly or indirectly affecting intermolecular Fc:Fc interactions. Thus, without being bound by theory, the substitution is believed to directly or indirectly increase the binding strength between antibody molecules in oligomeric form, thereby increasing the stability of oligomeric structures such as hexameric, pentameric, tetrameric, trimeric, or dimeric structures. For example, the amino acid substitution may promote or enhance the formation of new intermolecular Fc:Fc bonds, such as, but not limited to, van der Waals interactions, hydrogen bonding, charge-charge interactions, or aromatic stacking interactions, or promote entropy enhancement through the release of water molecules during Fc:Fc interactions. With reference to Table 1, "exemplary substitutions" can be selected based on size and physicochemical properties that mediate or stimulate intermolecular Fc:Fc interactions or intramolecular interactions (allosteric mutations). "Preferred substitutions" can be selected based on optimal size and physicochemical properties that mediate or stimulate intermolecular Fc:Fc interactions or intramolecular interactions (allosteric mutations).

[0371] "Exemplary substitutions" of the amino acids listed in Table 1 include replacing an E residue with an R residue and replacing an H residue with an R residue. Each "exemplary substitution" of an amino acid at each specific amino acid residue listed in Table 1 is a separate, specific, non-limiting embodiment of the invention. Furthermore, each "preferred substitution" at each specific amino acid residue listed in Table 1 is a separate, specific, non-limiting embodiment of the invention.

[0372] In another aspect, the present invention provides a variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, comprising: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and wherein the at least two amino acid mutations are different.

[0373] In one embodiment, the parent polypeptide may be an antibody, and consequently the variant may also be an antibody.

[0374] Thus, the present invention also provides a variant of a parent antibody comprising an antigen-binding region and an Fc domain, (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and wherein said at least two amino acid mutations are different.

[0375] Thus, variants of the above embodiments may include mutations in at least two of the amino acids in Table 1, such as two, three, four, five or more amino acids.

[0376] In any embodiment in which the variant contains such mutations in at least two amino acids, the mutations may be present in each of the heavy chains of the variant, or one of the two may be present in one of the heavy chains and the other in the other heavy chain, or vice versa.

[0377] In one embodiment, the variant comprises mutations in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W. Alternatively, the variant further comprises a mutation in at least one residue selected from the group consisting of H310, G385, H433, N434, Q438 and K439.

[0378] In one embodiment, the variant comprises mutations in at least two amino acid residues selected from those corresponding to E345X, E430X, S440W / Y, Q386K, P247G, I253V, S254L, Q311L / W, D / E356R, E382V and Y436I in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid.

[0379] For example, the antibody variant may comprise a mutation in at least one of E345, E430, S440 and Q386, or at least one of E382 and H433, e.g., at two or all of E345, E430, S440 and Q386, or at two or all of E382 and H433, and optionally further comprise a mutation in one or more other amino acids listed in Table 1. Thus, in a further embodiment, the variant comprises mutations in at least two amino acid residues selected from the group corresponding to E345X, E430X, S440W / Y and Q386K in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid.

[0380] Exemplary combinations of mutations at least two amino acid residues include E345X / E430X, E345X / S440Y or W, E345X / Q386K, E430X / S440Y or W, and E430X / Q386K.

[0381] In one embodiment, the mutations in at least two amino acid residues are deletions, insertions or substitutions. Such substitutions of amino acids may be with any naturally occurring or artificial amino acid.

[0382] In one particular embodiment, the mutations in at least two amino acid residues are: TIFF2024116396000016.tif27170.

[0383] In one preferred embodiment, the variant is a variant of the Fc region of a human IgG1 heavy chain: It comprises mutations in at least two amino acid residues which are amino acid substitutions selected from those corresponding to TIFF2024116396000017.tif10152.

[0384] Or, further mutations include: It is selected from the one corresponding to TIFF2024116396000018.tif22154.

[0385] In one preferred embodiment, the mutations in at least two amino acid residues are amino acid substitutions selected from those corresponding to E345R / Q / N / K, E430T / S / G, S440Y / W and Q386K in the Fc region of a human IgG1 heavy chain. It is selected from the one corresponding to TIFF2024116396000019.tif16158.

[0386] Exemplary specific combinations of mutations in at least two amino acid residues include: There is TIFF2024116396000020.tif16154.

[0387] In one specific embodiment, the mutation is not at an amino acid residue corresponding to 1253, N434, or Q311. In a further or alternative embodiment, the mutation is not at H433 or the amino acid substitution is not H433A.

[0388] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that it does not contain a mutation corresponding to S440 in the Fc region of the human IgG1 heavy chain; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain wherein the at least three amino acid mutations are different.

[0389] In one particular embodiment, the variant comprises mutations in amino acid residues corresponding to amino acid substitutions E345R, Q396K and E430G, which may be in one or both heavy chains of the variant.

[0390] The mutations in at least three amino acid residues may be individually selected from the substitutions listed in Table 1. Non-limiting examples of mutants containing at least three mutations are: The file is TIFF2024116396000021.tif52153.

[0391] In addition to mutations at one or more amino acids in Tables 1 or 2A and B, the IgG heavy chain may include additional mutations known in the art, such as mutations that further improve effector function, including known mutations that enhance CDC, Fc-γ receptor binding or FcRn binding, and / or improve Fc-γ receptor-mediated effector function.

[0392] In one embodiment, the variants of the invention further comprise known CDC-enhancing modifications, such as, for example, exchanging segments between IgG isotypes to create chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72); one or more amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138), and / or one or more amino acid substitutions within or near the C1q binding site in the CH2 domain, centered around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99 / 51642). For example, in one embodiment, the variants of the present invention further comprise any combination of the amino acid substitutions S267E, H268F, S324T, S239D, G236A, and I332E, which result in enhanced effector function via CDC or ADCC (Moore et al., 2010 mAbs 2(2), 181-189). Other Fc mutations that affect binding to Fc receptors (as described in WO 2006 / 105062, WO 00 / 42072, U.S. Pat. No. 6,737,056, and U.S. Pat. No. 7,083,784) or physical properties of the antibody (as described in WO 2007 / 005612 A1) can also be used in the variants of the present invention.

[0393] In one embodiment, the variants of the present invention further comprise modifications that enhance Fc-γ receptor binding and / or Fc-γ receptor-mediated effector function, such as (i) reducing the amount of fucose in CH2-linked glycosylation (glycoengineering) (Umana P, et al., Nat Biotechnol 1999; 17: 176-80; Niwa R, et al., Clin Cancer Res 2004; 10: 6248-55.)), and (ii) site-directed mutagenesis of amino acids in the hinge or CH2 region of the antibody (protein engineering) (Lazar GA, et al., Proc Natl Acad Sci USA 2006; 103: 4005-10).

[0394] In one embodiment, the variants of the present invention are further engineered at the FcRn-binding site, e.g., to extend the half-life (t) of an IgG antibody. Such modifications include (i) N434A and T307A / E380A / N434A mutations (Petcova et al. Int Immunol. 2006 Dec; 18(12): 1759); (ii) substitution of one or more of Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, and Asn434 with alanine residues to improve FcRn binding (Shields RL, et al. J. Biol. Chem. 2001; 276: 6591); and (iii) alanine substitutions in IgG1 that enhance affinity for FcRn. TIFF2024116396000022.tif28158 (Dall'Acqua et al., supra).

[0395] "Double mutant" It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent polypeptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0396] As mentioned above and further explained below, the present invention also relates to a "double mutant" aspect in which two mutations, each individually reducing effector function, but together restoring effector function to the level of the parent antibody, enhance the specificity of the mutant when used together. The antibody mutant of the "double mutant" aspect contains two mutations (typically amino acid substitutions) at K439 and S440, a specific interacting pair of amino acid residues.

[0397] Thus, in one aspect, the present invention provides a variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, comprising: (i) a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is neither S440Y nor S440W, e.g., the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R; (ii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain; The present invention relates to a variant comprising:

[0398] In one embodiment, the parent polypeptide may be an antibody, and as such the variant may also be an antibody.

[0399] Thus, in one aspect, the present invention relates to variants of a parent antibody comprising an antigen-binding region and an immunoglobulin Fc domain, which comprise a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W, e.g., the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R. Tables 2A and B show "exemplary" and "preferred substitutions" for "double mutant" aspects (Table A) and "mixed mutant" aspects (Table 2B).

[0400] Table 2A: Exemplary mutation sites and amino acid substitutions for the "double mutant" aspect TIFF2024116396000023.tif17158

[0401] (Table 2B) Exemplary mutation sites and amino acid substitutions for the "mixed mutant" aspect (Ab1 + Ab2) TIFF2024116396000024.tif17158

[0402] In one embodiment of the variant, the mutation is at a position other than S440 and K447, and the variant is (i) a mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation in S440 is not S440W or S440Y; (ii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) a mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain; Further includes:

[0403] Thus, the invention provides antibody variants comprising a first mutation at a residue corresponding to K439 in the CH2 and / or CH3 region of a human IgG1 heavy chain, and a second mutation at a residue corresponding to S440 in the CH2 and / or CH3 region of a human IgG1 heavy chain.

[0404] It is contemplated by the present invention that a variant may contain only one of the amino acid residue substitutions, e.g., either K439E or S440K, e.g., a variant containing one mutation at K439 and optionally no mutation at S440.

[0405] In one embodiment, the invention relates to a variant in which the mutation at K439 is an amino acid substitution to an amino acid selected from E and D (eg, K439E).

[0406] In another embodiment, the mutant comprises a mutation at S440, optionally without a mutation at K439.

[0407] In one embodiment, the invention relates to a variant in which the mutation at S440 is an amino acid substitution to an amino acid selected from K, R and H (eg, S440K).

[0408] In one embodiment, the mutant contains mutations at both K439 and S440.

[0409] In another embodiment, the mutation at K439 is selected from K439 to D, E or R, and the mutation at S440 is selected from S440 to D, E, K, H and R.

[0410] In another embodiment, the mutation at K439 is selected from K439D and K439E, and the mutation at S440 is selected from S440K, S440R and S440H.

[0411] In another embodiment, the variant comprises a K439E and a S440K mutation.

[0412] As described in Examples 4-6, antibody variants containing only one of the K439E and S440K mutations exhibited reduced K binding to C1q. D The HuMAb 7D8 and 005 antibody variants containing both mutations had a dramatic increase in C1q binding or CDC, reflecting a reduction in complement activation and / or CDC ability. Surprisingly, antibody variants of HuMAb 7D8 or 005 containing both mutations were found to have restored or enhanced C1q binding or CDC. Without being bound to any particular theory, the underlying mechanism can likely be explained by the steric compensation of each mutation for the other, as illustrated in Figures 4 and 5.

[0413] Any "double mutant" as described herein can also be used in combination with a mutation that alone can enhance effector function. Thus, a "double mutant" aspect can be combined with a "single mutant" aspect, for example, the mutant may further comprise a mutation at any of the amino acid positions listed in Table 1 or in any other embodiment described with respect to the "single mutant" aspects above. Thus, in one embodiment, there is a mutation at a position other than S440, and the mutant further comprises a mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440W nor S440Y.

[0414] In one aspect, the present invention provides a variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, comprising: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that it does not contain a mutation corresponding to S440 in the Fc region of the human IgG1 heavy chain; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a variant comprising a mutation in at least one amino acid residue selected from the group consisting of: wherein the mutant is (i) an additional mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation in S440 is not S440W or S440Y; (ii) an additional mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) additional mutations in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain; Includes.

[0415] In one embodiment, the parent polypeptide may be an antibody, and consequently the variant may also be an antibody.

[0416] Thus, in one aspect, the present invention provides a variant of a parent antibody comprising an antigen-binding region and an Fc domain of an immunoglobulin, comprising: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that it does not contain a mutation corresponding to S440 in the Fc region of the human IgG1 heavy chain; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a variant comprising a mutation in at least one amino acid residue selected from the group consisting of: wherein the mutant is (i) an additional mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is neither S440W nor S440Y; (ii) an additional mutation in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) additional mutations in at least one amino acid residue corresponding to K447D / E or K447K / R / H and 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain; Includes:

[0417] In one embodiment, the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.

[0418] In a further embodiment, the variant comprises a mutation in at least one amino acid residue which is an amino acid substitution selected from those corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440W or S440Y.

[0419] In one embodiment, the variant comprises amino acid mutations at both positions corresponding to K439 and S440 in the Fc region of the IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W.

[0420] In a further embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R.

[0421] In a further embodiment, the first mutation is at an amino acid residue selected from those corresponding to E345, E430, Q386 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W; and the second and third mutations are amino acid substitutions at positions K439E or S440K.

[0422] In one embodiment, the first mutation is a deletion, insertion or substitution. Such a substitution may be with any naturally occurring or unnatural amino acid.

[0423] In a further embodiment, the first mutation is selected from the group of E345R,Q,N,K,A,F,G,H,I,L,M,P,S,T,V,W,Y,C,D; E430T,S,G,A,F,H,L,P,R,V,C,D,I,K,M,N,Q,W,Y; and S440W,Y,D; and the second and third mutations are amino acid substitutions at positions K439E or S440K.

[0424] In one preferred embodiment, one mutation is selected from the group of E345R,Q,N,K,Y; E430T,S,G,F,H; S440W,Y; and Q386K.

[0425] As another example, in one embodiment of the invention, the mutant comprises E345R, K439E and S440K mutations, thereby resulting in mediation of a more enhanced and more specific CDC response.

[0426] In one embodiment, the variant comprises mutations in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of the human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain, with the proviso that the mutation in S440 is neither S440W nor S440Y.

[0427] In a further embodiment, the variant comprises mutations in at least two amino acid residues which are amino acid substitutions selected from those corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, where X is any amino acid, such as a naturally occurring amino acid, and the variant comprises an additional mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440W or S440Y.

[0428] In one embodiment, the variant comprises amino acid mutations at both positions corresponding to K439 and S440 in the Fc region of the IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W.

[0429] In a further embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R.

[0430] In a further embodiment, the first and second mutations are at amino acid residues selected from those corresponding to E345, E430, Q386 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W; and the third and fourth mutations are amino acid substitutions at positions K439E or S440K.

[0431] In another embodiment, a variant comprising a mutation at both positions K439 and S440 as described herein has enhanced Fc-mediated effector function selected from C1q binding, complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof, compared to a parent antibody or an antibody variant comprising a mutation at only one of K439 and S440.

[0432] The present invention also provides for the use of the K439E and S440K mutations in an antibody to restore one or more of: (i) C1q avidity, (ii) antibody-mediated complement activation, (iii) antibody-mediated CDC, (iv) oligomer formation, (v) oligomer stability, or any combination of (i)-(v), compared to a parent antibody, which may be, for example, a wild-type antibody, or an antibody variant comprising only one of the K439E or S440K mutations. In one embodiment of (iv) or (v), the oligomer is a hexamer.

[0433] In one embodiment, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0434] Mixed mutants It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent polypeptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0435] As mentioned above, the inventors of the present invention have also discovered that there are mutations that, when used alone, reduce effector function but, when used together, restore effector function, such as mutations at positions K439 and S440 in the Fc region of the human IgG1 heavy chain. Thus, this concept can be used to ensure pairing of two different antibodies by introducing K439 into one antibody and S440 into the other. Thus, antibody variants in the "mixed variant" aspect contain a single mutation, which typically results in a reduced or significantly reduced Fc:Fc interaction between identical Fc molecules. However, the "mixed variant" antibody variants of the present invention can pair with each other; thereby, for example, restoring or even enhancing C1q binding, complement activation, CDC, oligomerization, and / or oligomer stability for a specific pair of antibody variants compared to each variant alone or a mixture of one or more parent antibodies. In one embodiment of the present invention, the oligomer is a hexamer. In one embodiment, the pair of antibody variants additionally or alternatively have other effector functions that are retained or improved, such as C1q binding, complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding including Fc-gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof. This aspect of the invention allows for numerous applications in which the strength as well as the selectivity of C1q binding, complement activation, CDC, or other effector functions can be modulated.

[0436] Exemplary mutation sites for each antibody mutant in a "mixed mutant" pair are shown in Table 2. Specifically, the present invention provides antibody mutants comprising an antigen-binding region and an immunoglobulin Fc domain, wherein the mutants comprise a mutation at a residue corresponding to one of K439 and S440 in the Fc region of a human IgG1 heavy chain. In one embodiment, the mutation is at K439, which is an amino acid substitution to an amino acid selected from E or D, e.g., K439E. In one embodiment, the mutation is at S440, which is an amino acid substitution to an amino acid selected from K, R, or H, e.g., S440K.

[0437] Thus, in one embodiment, the present invention also provides (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain A mutation in at least one amino acid residue selected from and mutations in the amino acid residue corresponding to K439 in the Fc region of human IgG1 heavy chain The present invention also relates to variants comprising:

[0438] In another aspect, the present invention also provides (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain A mutation in at least one amino acid residue selected from and mutations in the amino acid residue corresponding to S440 in the Fc region of human IgG1 heavy chain The present invention also relates to variants comprising:

[0439] In one embodiment, the above two embodiments may be combined according to the "mixed mutant" pair aspect of the present invention.

[0440] Each variant in a "mixed variant" pair may further comprise a mutation in an amino acid listed in Table 1.

[0441] In one embodiment of the invention, a "mixed variant" pair comprises a first parent antibody variant and a second parent antibody variant, wherein the first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the first variant comprises (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within its C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen-binding region of the immunoglobulin, and the second variant comprises (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within its C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; Includes:

[0442] Other exemplary "mixed mutant" pairs may further include, but are not limited to, any of the following pairs: a first mutant comprising the mutation K447E and a second mutant comprising the mutations K447 / P448; a first mutant comprising the mutation K447E and a second mutant comprising the mutations K447 / K448 / P449.

[0443] In one embodiment, the mutation is a deletion, insertion or substitution. Such substitution of amino acids may be with any naturally occurring amino acid or non-natural amino acid. In one embodiment, the mutation is a deletion. In another embodiment, the mutation is an insertion. In another embodiment, the mutation is an amino acid substitution.

[0444] In one particular embodiment, the first variant and / or the second variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0445] In one particular embodiment, the first variant and / or the second variant is TIFF2024116396000025.tif33156, wherein the first variant comprises a second mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and the second variant comprises a second mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain.

[0446] For example, in one embodiment, one variant in a "mixed variant" pair comprises E345R and K439E mutations, while the other variant comprises E345R and S440K mutations, thereby resulting in enhanced and more specific C1q avidity, complement activation, CDC, oligomerization, oligomer stability, and / or other effector-associated functions, such as FcRn binding, ADCC, Fc-gamma receptor binding, protein A binding, protein G binding, ADCP, CDCC, complement-enhanced cytotoxicity, antibody-mediated phagocytosis, internalization, apoptosis, binding of opsonizing antibodies to complement receptors, and / or combinations thereof.

[0447] The "mixed variant" aspect may also include two variants, each comprising multiple mutations listed in Table 1 in the Fc region of the human IgG1 heavy chain, e.g., a first variant comprising the mutations S440K / K447E and a second variant comprising the mutations K439E / K447 / P448; e.g., a first variant comprising the mutations K439E / K447E and a second variant comprising the mutations S440K / K447 / P448.

[0448] The multiple variants in a "mixed variant" pair as described herein may be derived from the same parent antibody or from different parent antibodies. Furthermore, the "mixed variant" aspect can also be used in bispecific or asymmetric antibodies. Furthermore, the first, second, and third antibodies may bind to different epitopes on the same or different targets.

[0449] Furthermore, the "mixed variant" aspect can provide a CDC or other effector response that is more specifically directed against tumor cells expressing two specific tumor antigens by utilizing a first antibody against a first antigen with a K439E mutation and a second antibody against a second antigen with an S440K or S440R mutation. Utilizing a "mixed variant" aspect that includes three variants, optionally a bispecific antibody, can provide a CDC or other effector response that is more specifically directed against tumor cells expressing at least two, e.g., two, three, four, five, or six, specific tumor antigens.

[0450] In one embodiment of any of the "single variant", "double variant" and "mixed variant" aspects, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0451] In any embodiment of the "mixed variant" aspect, the first, second and / or third variant may comprise the same or different mutations of any of the amino acid substitutions listed in Table 1.

[0452] multispecific antibodies It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent polypeptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0453] It should be understood that any embodiment of the "single mutant", "double mutant" and "mixed mutant" aspects described herein may be used in the multispecific antibody aspects described below.

[0454] In one major aspect, the invention relates to a variant that is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or on different antigens; The first and second CH2-CH3 regions each comprise a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0455] In one embodiment, the mutation is a deletion, insertion or substitution. Such substitution of an amino acid may be with any naturally occurring or non-naturally occurring amino acid.

[0456] The bispecific antibody of the present invention is not limited to a particular format, and it may be any of those described above and herein.

[0457] In one embodiment of the invention, the first and second polypeptides comprise one first mutation at an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

[0458] In one particular embodiment, the mutation in at least one amino acid residue is TIFF2024116396000026.tif34152.

[0459] In one particular embodiment, the bispecific antibody has the format described in WO 2011 / 131746. Thus, in one embodiment, the variant is a bispecific antibody, wherein the first polypeptide comprises further mutations at amino acid residues selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of a human IgG1 heavy chain; and The second polypeptide comprises further mutations at amino acid residues selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and wherein said further mutations in the first polypeptide are different from said further mutations in the second polypeptide.

[0460] In one specific embodiment, the bispecific antibody has a first polypeptide containing an additional mutation at an amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain, and a second polypeptide containing an additional mutation at an amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain. Such bispecific antibodies of the present invention can be produced as described in Example 22. Furthermore, the effect of the produced heterodimeric protein on CDC killing can also be tested by an assay such as that used in Example 23.

[0461] In one particular embodiment, the bispecific antibody comprises first and second polypeptides, wherein the first polypeptide comprises a mutation at an amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain; the second polypeptide comprises a mutation at an amino acid residue corresponding to F405 in the Fc region of the human IgG1 heavy chain; and the first and / or second polypeptide comprises a further mutation at an amino acid residue corresponding to the amino acid substitution E345R in the Fc region of the human IgG1 heavy chain.

[0462] In one particular embodiment, the bispecific antibody comprises first and second polypeptides, wherein the first polypeptide comprises a mutation at an amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain; the second polypeptide comprises a mutation at an amino acid residue corresponding to F405 in the Fc region of the human IgG1 heavy chain; and the first and / or second polypeptide comprise further mutations at amino acid residues corresponding to the amino acid substitutions E345R and Q386K in the Fc region of the human IgG1 heavy chain, respectively. The further mutations may be present in both the first and second polypeptides, or E345R may be present in the first polypeptide and Q386K in the second polypeptide; or vice versa.

[0463] In one particular embodiment, the bispecific antibody comprises first and second polypeptides, wherein the first polypeptide comprises a mutation at an amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain; the second polypeptide comprises a mutation at an amino acid residue corresponding to F405 in the Fc region of the human IgG1 heavy chain; and the first and / or second polypeptide each comprise further mutations at amino acid residues corresponding to the amino acid substitutions E345R, Q386K, and E430G in the Fc region of the human IgG1 heavy chain. The mutations may be present in both the first and second polypeptides, or the first polypeptide may comprise the mutations E345R and E430G and the second polypeptide may comprise the mutation Q386K; or vice versa.

[0464] Bispecific antibodies can comprise, for example, the antigen-binding region of a CD20 antibody and the antigen-binding region of a CD38 antibody, and amino acid substitutions at one or more amino acids listed in Tables 1 and / or 2. Exemplary CD20 binding regions include those of ofatumumab (2F2), 7D8, and 11B8, described in WO 2004 / 035607, which is incorporated herein by reference in its entirety, and rituximab (WO 2005 / 103081). Exemplary CD38 binding regions include those of 003 and daratumumab (005), described in WO 2006 / 099875, which is incorporated herein by reference in its entirety.

[0465] In one embodiment, the bispecific antibodies bind to different epitopes on the same or different targets.

[0466] In another embodiment, the first mutation in the first and second polypeptides can be the same or different.

[0467] In one embodiment of the "single mutant", "double mutant", "mixed mutant" and multispecific antibody aspects, the variant is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally a human full-length antibody such as a human full-length IgG1 antibody.

[0468] In any of the "single mutant", "double mutant", "mixed mutant" and multispecific antibody aspects, C1q binding of the antibody is determined according to the assay described in Example 4, CDC is determined according to the assay described in Examples 5, 6 or 10, the mutation is not in an amino acid residue directly involved in C1q binding, which is optionally determined by comparing C1q binding in an ELISA assay according to Example 3 with C1q binding in a cell-based assay according to Example 4, and ADCC is determined according to the assay described in Example 12.

[0469] Furthermore, the present invention provides preparations of variants of any of the above "single mutant," "double mutant," "mixed mutant," and multispecific antibody aspects or embodiments. The present invention also provides compositions, e.g., pharmaceutical compositions, comprising variants of any of the above "double mutant" aspects and embodiments. The present invention also provides the use of any such variants, preparations, or compositions as a medicament.

[0470] The above-described "single mutant," "double mutant," "mixed mutant," and multispecific antibody aspects of the invention are particularly applicable to human antibody molecules having an IgG1 heavy chain comprising the relevant segment P247-K447 corresponding to the underlined residues 130-330 of the human IgG1 heavy chain constant region (UniProt Accession No. P01857; SEQ ID NO:1). TIFF2024116396000027.tif39128

[0471] The present invention can also be applied to antibody molecules having a human IgG2 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 126 to 326 of the IgG2 heavy chain constant region (Accession No. P01859; SEQ ID NO:2). TIFF2024116396000028.tif35128

[0472] The present invention is also applicable to antibody molecules having a human IgG3 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to residues 177 to 377 of the IgG3 heavy chain constant region (UniProt Accession No. P01860, SEQ ID NO:3), which are underlined below. TIFF2024116396000029.tif39128

[0473] The present invention can also be applied to antibody molecules having a human IgG4 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 127 to 327 of the IgG4 heavy chain constant region (Accession No. P01859, SEQ ID NO:4). TIFF2024116396000030.tif39128

[0474] The present invention can also be applied to antibodies having a heavy chain portion of the human IgG1m(f) allotype. Amino acid sequence of the IgG1m(f) allotype (CH3 sequence underlined) - SEQ ID NO:5 TIFF2024116396000031.tif39128

[0475] An alignment of each segment of the IgG1, IgG2, IgG3, IgG4 and IgG1m(f) constant regions is shown in Figure 2. Thus, any mutation in an amino acid listed in Table 1 or Tables 2A and B can be introduced into the equivalent position in IgG2, IgG3, IgG4 and / or IgG1m(f) defined by the alignment to obtain a variant of the invention.

[0476] In one embodiment, the invention provides a variant of a full-length IgG1, IgG2, IgG3, or IgG4 antibody comprising one or more amino acid substitutions of any of the above aspects.

[0477] In any of the "single mutant," "double mutant," "mixed mutant," and multispecific antibodies, the Fc region of the IgG1 heavy chain can comprise the sequence of residues 130-330 of SEQ ID NO:1, residues 126-326 of SEQ ID NO:2, residues 177-377 of SEQ ID NO:3, or residues 127-327 of SEQ ID NO:4.

[0478] In one embodiment, the parent antibody comprises a sequence selected from SEQ ID No.:1-5, for example SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, SEQ ID No.:4 or SEQ ID No.:5.

[0479] In one embodiment, the Fc region of the IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO:1.

[0480] The parent antibody may be any parent antibody as described herein. A parent antibody in this context is intended to be also a first parent antibody and a second parent antibody.

[0481] In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD or IgE antibody.

[0482] In one embodiment, the parent antibody is a human full-length antibody, such as a human full-length IgG1 antibody.

[0483] In one embodiment, the parent antibody, first parent antibody and second parent antibody are human IgG1 antibodies, such as IgG1m(za) or IgG1m(f) allotypes, optionally comprising an Fc region comprising SEQ ID NO:1 or 5.

[0484] In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO:2.

[0485] In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO:3.

[0486] In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO:4.

[0487] In particular embodiments of any of the "single mutant", "double mutant", "mixed mutant" and multispecific antibody aspects, the variant comprises an amino acid sequence having a degree of identity of at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% to amino acids P247 to K447 of SEQ ID Nos: 1, 2, 3, 4 and 5, excluding mutations introduced in accordance with the invention.

[0488] Thus, a variant may comprise a sequence according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 or SEQ ID No:5, excluding any mutations defined herein.

[0489] Any of the above "single mutant", "double mutant", "mixed mutant" and multispecific aspects of the invention may be construed to include the following embodiments.

[0490] In one embodiment, the first and / or second parent antibody is an antibody fragment, which is optionally a monovalent antibody, a heavy chain antibody, a chain-exchanged recombinant domain (SEED), a triomab, a dual variable domain immunoglobulin (DVD-Ig), a knob-into-hole antibody, a miniantibody, a dual affinity retargeting molecule (Fc-DART or Ig-DART); a LUZ-Y antibody, a biclonic antibody, a dual targeting (DT)-Ig antibody, a two-in-one antibody, a cross-linked Mab, a mAb 2 , CovX-body, IgG-like bispecific antibody, Ts2Ab, BsAb, HERCULES antibody, TvAb, ScFv / Fc Fusion antibody, SCORPION, scFv fragment fused to an Fc domain, and dual scFv fragment fused to an Fc domain.

[0491] In a further embodiment, the first and second parent antibodies both bind to an antigen expressed on the surface of a human tumor cell.

[0492] In a further embodiment, the antigens for the first and second parent antibodies are independently selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

[0493] In a further embodiment, the first and second parent antibodies are fully human antibodies.

[0494] In a further embodiment, the antigens for the first and second parent antibodies are selected, in any order, from CD20 and CD38, and optionally the first and second parent antibodies are selected, in any order, from 7D8 and 005.

[0495] In a further embodiment, the first antibody and the second antibody both bind to an antigen expressed on the surface of a bacterial cell or virion.

[0496] In another embodiment, the bacterial cell is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Neisseria meningitidis, and Mycobacterium tuberculosis.

[0497] In a further embodiment, the first and second parent antibodies bind to the same antigen.

[0498] In another embodiment, the first and second parent antibodies are the same antibody.

[0499] In another embodiment, the parent antibody is selected from 7D8 and 005.

[0500] composition It should be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody should also be interpreted as embodiments relating to a parent polypeptide, first parent polypeptide or second parent polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0501] The present invention also relates to compositions comprising the variants, wherein the parent antibody may be any of the variants and parent antibodies as described herein. Specific aspects and embodiments are described below. Moreover, such variants may be obtained by any of the methods described herein.

[0502] In one aspect, the invention provides a composition comprising a first variant of a parent polypeptide and a second variant of the parent polypeptide, wherein the first variant comprises a first Fc domain and binding region of an immunoglobulin, and the second variant comprises a second Fc domain and binding region of an immunoglobulin, and (i) the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W; (ii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation at a position corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises mutations at positions corresponding to K447K / R / H, 448K / R / H, and 449P in the Fc region of a human IgG1 heavy chain. It relates to a composition.

[0503] In one embodiment, the first or both of the variant of the parent polypeptide and the second variant of the parent polypeptide may be an antibody.

[0504] Thus, in one aspect, the present invention provides a composition comprising a first variant of a parent antibody and a second variant of the parent antibody, wherein the first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin; (i) the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is neither S440Y nor S440W; (ii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation at a position corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises mutations at positions corresponding to K447K / R / H, 448K / R / H, and 449P in the Fc region of a human IgG1 heavy chain. It relates to a composition.

[0505] In one embodiment, the composition comprises a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, and the second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, and wherein said first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and wherein the second variant comprises a mutation at a position corresponding to S440, with the proviso that the mutation at S440 is neither S440Y nor S440W.

[0506] In one embodiment, the composition comprises a first variant of a parent antibody and a second variant of the parent antibody, wherein the first variant comprises a first Fc domain and an antigen binding region of an immunoglobulin, and said first variant comprises: (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen-binding region of the immunoglobulin, and the second variant comprises (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; Includes:

[0507] In another embodiment, the composition comprises a first variant of an antibody and a second variant of a parent antibody, wherein the first variant is (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and The second mutant contains a mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain, which is neither S440Y nor S440W.

[0508] In another embodiment, the composition comprises a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen binding region of the immunoglobulin, said second variant comprising: (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; Includes.

[0509] In one embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E, and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / H / R.

[0510] In another aspect, the present invention provides a composition comprising a first variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, and a second variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, The first variant comprises a first Fc domain and a first antigen-binding region of an immunoglobulin, and the first variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a first mutation in at least one amino acid residue selected from the group The second mutant is (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain The present invention relates to a composition that does not contain a mutation in an amino acid residue selected from the group consisting of:

[0511] In one embodiment, the first and / or second parent polypeptide may be an antibody.

[0512] The present invention also provides an embodiment of a composition, comprising: a first mutant (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and It also relates to an embodiment in which the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, which is neither S440Y nor S440W.

[0513] The present invention also provides, in one embodiment, a composition, wherein the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen binding region of an immunoglobulin, and the second variant comprises: (i) (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is neither S440Y nor S440W; The present invention also relates to an embodiment comprising:

[0514] In another aspect, the present invention provides a composition comprising a first variant of an antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and a first antigen-binding region of an immunoglobulin, and wherein the first variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain and a first mutation in at least one amino acid residue selected from the group The second variant comprises a second Fc domain and a second antigen-binding region of the immunoglobulin, and the second variant comprises: (a) Amino acid residues within the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) an amino acid residue within the C-terminal CH3 β-strand, provided that in the case of a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation is S440Y or S440W; and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain does not contain a mutation in an amino acid residue selected from the group It relates to a composition.

[0515] In embodiments in which the second variant does not comprise any of the recited mutations described herein, such second variant may comprise any of the examples of suitable second antibodies listed above in relation to the effector function methods.

[0516] In one embodiment, the first and second variants comprise a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

[0517] In one embodiment, the first variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0518] In one particular embodiment, the first variant comprises mutations at amino acid residues corresponding to E345R and Q386K in the Fc region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.

[0519] In one particular embodiment, the first variant comprises mutations at amino acid residues corresponding to E345R, Q386K and E430G in the Fc region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.

[0520] In one embodiment, at least one first mutation in the first and second variants is different.

[0521] In one embodiment, the first variant and the second variant are each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally each a human full length IgG1 antibody, for example each a human full length antibody.

[0522] In one embodiment, the first variant and the second variant are selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0523] In a further embodiment, the first and second variants bind to different epitopes on the same antigen or different antigens.Therefore, in an embodiment where the first and second antibodies are bispecific, each antibody can bind to two different epitopes.At least two bispecific antibodies can be the same or different.Therefore, when the bispecific antibodies are different, the composition can include targeting up to four different epitopes on the same or different targets.

[0524] In a further embodiment, one or both of the first and second mutants are conjugated to a drug, toxin or radiolabel, e.g., one or both of the first and second mutants are conjugated to a toxin via a linker.

[0525] In a further embodiment, one or both of the first variant and the second variant are part of a fusion protein.

[0526] In another aspect, the invention relates to a composition comprising any variant, any bispecific antibody, or any composition described herein and a pharmaceutically acceptable carrier.

[0527] It is also contemplated that any of the embodiments of the "mixed variant" aspect may be included within any of the composition embodiments.

[0528] In one embodiment, the first and second parent antibody variants bind to antigens expressed on the same cell.

[0529] In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 with an amino acid selected from E and D.

[0530] In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.

[0531] In another embodiment, the variant of the second parent antibody comprises an amino acid substitution at S440 with an amino acid selected from K, R and H.

[0532] In another embodiment, the amino acid substitution in the variant of the second parent antibody is S440K.

[0533] In an alternative embodiment, the variant of the first and / or second antibody further comprises a mutation at a residue selected from the group consisting of H310, G385, H433, N434 and Q438.

[0534] In a further alternative embodiment, the variant of the first and / or second parent antibody further comprises a mutation selected from: E345 to D, K, N, Q, R or W; E382 to D, Q, K or R; and H433 to R.

[0535] In a further embodiment, the variants of the first and second parent antibodies further comprise a mutation selected from E345R, E382R and H433R, eg E345R.

[0536] In another aspect, the invention relates to a pharmaceutical composition comprising a first parent antibody variant and a second parent antibody varian...

Claims

1. A method for reducing an effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the step of introducing a mutation into the parent polypeptide at one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, wherein the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / R, and further wherein the effector function is C1q binding or complement-dependent cytotoxicity (CDC).

2. The method of claim 1 , wherein the parent polypeptide is a parent antibody.

3. The method of claim 2, wherein the parent antibody is a monospecific, bispecific or multispecific antibody.

4. The method of claim 3, wherein the parent polypeptide or antibody is bound to an antigen of the parent polypeptide or antibody on the surface of an antigen-expressing cell, cell membrane or virion.

5. A method for increasing the specificity of a combination in oligomerization of at least a first and a second parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the steps of: A） (i) introducing a K439D / E mutation into the first parent polypeptide at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing into said second parent polypeptide an S440K / R mutation at an amino acid residue at a position corresponding to S440 in the Fc region of human IgG1 heavy chain.

6. 6. The method of claim 5, wherein said first and second parent polypeptides are first and second parent antibodies, comprising the steps of: (i) introducing a K439D / E mutation into the first parent antibody at an amino acid residue at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and (ii) introducing into said second parent antibody an S440K / R mutation at an amino acid residue at a position corresponding to S440 in the Fc region of human IgG1 heavy chain.

7. The method according to claim 5 or 6, comprising introducing a mutation into the parent antibody at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of human IgG1 heavy chain.

8. The method of claim 7, comprising introducing a mutation into the parent antibody at least one amino acid residue selected from those corresponding to E345, E430, and Q386 in the Fc region of a human IgG1 heavy chain, wherein the mutation in the at least one amino acid is selected from mutation of E345 to R, Q, N or K, mutation of E430 to T, S or G, or mutation of Q386 to K.

9. 9. The method of any one of claims 1 to 8, wherein said parent antibody or said combination of a first parent antibody and a second parent antibody is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE, optionally a human full length antibody, a human full length IgG1 antibody.

10. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and binding region, (i) a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R; Including, variants.

11. The variant of claim 10, wherein the parent polypeptide is a parent antibody comprising an antigen-binding region and an immunoglobulin Fc domain, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, wherein the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R.

12. 12. The variant of claim 11, wherein the parent antibody comprises an antigen-binding region and an Fc domain, and wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

13. The mutant of claim 12, wherein the mutation in the at least one amino acid residue is selected from those corresponding to E345R / Q / N / K, E430T / S / G, and Q386K in the Fc region of a human IgG1 heavy chain.

14. 14. The variant of any one of claims 10 to 13, which is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

15. The variant of any one of claims 10 to 14, which is conjugated to a drug, toxin or radiolabel, and which is conjugated to a toxin via a linker.

16. The variant of any one of claims 10 to 14, which is part of a fusion protein.

17. 17. The variant of any one of claims 10 to 16, which is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally a human full length antibody, a human full length IgG1 antibody.

18. A composition comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide, the first variant comprising a first Fc domain and binding region of an immunoglobulin, the second variant comprising a second Fc domain and binding region of an immunoglobulin, and (i) the first mutant comprises a K439D / E mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second mutant comprises a S440K / R mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain.

19. The composition of claim 18, wherein said first variant of a parent polypeptide is a first variant of a parent antibody and said second variant of a parent polypeptide is a second variant of a parent antibody, wherein said first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin and said second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin.

20. 20. The composition of claim 19, comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc domain and antigen-binding region of an immunoglobulin, and the second variant comprises a second Fc domain and antigen-binding region of an immunoglobulin, the first variant comprising a K439D / E mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second variant comprising a S440K / R mutation at a position corresponding to S440.

21. The first variant comprises a first Fc domain and an antigen-binding region of an immunoglobulin, （i） (a) Amino acid residues in the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix, and (d) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; and a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, （i） (a) Amino acid residues in the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; and a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W.

21. The composition of claim 20, comprising:

22. The first mutant （i） (a) Amino acid residues in the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix, and (d) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; and a first mutation, other than the mutation at K439, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and the second mutant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, the mutation being neither S440Y nor S440W; 21. The composition of claim 20.

23. the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and The second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, （i） (a) Amino acid residues in the CH2-CH3 region that result in allosteric mutations; (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region; (c) amino acid residues within the N-terminal CH3 helix; (d) amino acid residues within the C-terminal CH3 β-strand, and (e) an amino acid residue corresponding to E345, E382, or Q386 in the Fc region of a human IgG1 heavy chain; and a first mutation, other than the mutation at S440, in at least one amino acid residue selected from the group consisting of: (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W.

21. The composition of claim 20, comprising:

24. The composition of any one of claims 18 to 23, wherein the first and second mutants comprise a mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

25. 25. The composition of claim 24, wherein the first and second mutants comprise a mutation in at least one amino acid residue selected from those corresponding to E345R / Q / N / K, E430T / S / G, and Q386K in the Fc region of a human IgG1 heavy chain.

26. 25. The composition of claim 24, wherein the mutations in the first and second mutants are different.

27. 27. The composition of any one of claims 18 to 26, wherein said first variant and said second variant are human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibodies, respectively, optionally each being a human full length antibody, each being a human full length IgG1 antibody.

28. 28. The composition of any one of claims 18 to 27, wherein the first variant and the second variant are selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

29. The composition of any one of claims 18 to 28, wherein the first and second variants bind to different epitopes on the same antigen or on different antigens.

30. 30. The composition of any one of claims 18 to 29, wherein one or both of the first and second mutants are conjugated to a drug, a toxin or a radiolabel.

31. 30. The composition of any one of claims 18 to 29, wherein one or both of the first variant and the second variant are part of a fusion protein.

32. A composition comprising a variant according to any one of claims 10 to 17 or a composition according to any one of claims 18 to 31 and a pharma- ceutically acceptable carrier.

33. A kit-of-parts comprising a first variant and a second variant as defined in any one of claims 10 to 17 for simultaneous, separate or sequential use in therapy.

34. The variant, composition or kit-of-parts according to any one of claims 10 to 33 for the treatment of a disease.

35. The variant, composition or kit-of-parts according to any one of claims 10 to 33 for the treatment of cancer.

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