Antibody variants and uses thereof

Abstract

Described herein are polypeptides and related antibodies comprising a variant Fc domain. The variant Fc domain provides stabilized Fc: Fc interactions when the polypeptide, antibody or antibodies are attached to its target, antigen or antigens on the surface of a cell, thereby providing improved effector effects, such as a CDC response.

Description

[0001] The present invention is a divisional application based on the Chinese invention patent application with application date of July 6, 2012, application number “202010835726.2”, and invention name “Antibody variants and their uses”. Field of the Invention

[0002] The present invention relates to polypeptides comprising variant Fc domains and related antibodies. More specifically, the present invention relates to antibodies or polypeptides comprising Fc domains having modified effector functions brought about by one or more amino acid modifications in the Fc domain. Background of the Invention

[0004] Effector functions mediated by the antibody Fc domain allow for the destruction of foreign entities, such as killing pathogens and clearance and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by binding of the Fc region to cells with Fc receptors (FcRs), while complement-dependent cytotoxicity (CDC) is initiated by binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

[0005] Each IgG antibody contains two C1q binding sites, one in each heavy chain constant (Fc) region. However, because monomeric IgG has a very weak affinity for C1q (K d ~10 -4 M), a single IgG molecule in solution does not activate complement (Sledge et al., 1973 J. Biol. Chem. 248, 2818-13; Hughes-Jones et al., 1979 Mol. Immunol. 16, 697-701). Antigen-driven IgG association can lead to tighter binding of multivalent C1q molecules (K d ~10 -8 M) and complement activation (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM exists naturally as covalently linked pentamers or hexamers, and after binding to cell-expressed or immobilized antigens, IgM pentamers and hexamers can effectively cause CDC. Antigen binding is a necessary condition for inducing conformational changes in IgM to expose the C1q binding site (Feinstein et al., 1986, Immunology Today, 169-174).

[0006] It has been proposed that IgG can also complete complement activation by forming a hexameric ring structure through the interaction of the CH2 / CH3 domains of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69). Evidence supporting the existence of this hexameric IgG structure has been found in two-dimensional (Reidler et al., 1986 I Handbook of Experimental Immunology 4th Edition. (Weir, DM ed.), pp17.1-17.5. Blackwell, Edinburgh; Pinteric et al., 1971 Immunochem. 8, 1041-5) and three-dimensional crystals, as well as for IgG1, IgG2a and IgG4 in solution and human Fc (Kuznetsov et al., 2000 J Structure. Biol. 131, 108-115). The formation of the hexameric ring was also observed in the crystal structure of the b12 human IgG1κ antibody against HIV-1 gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug 10; 293(5532), 1155-9). In the b12 hexameric ring, six accessible C1q binding sites are located on the surface of the hexamer, one from each of the six antibodies, while the other six binding sites face downward.

[0007] C1q resembles a tulip bundle with six bulbs, containing antibody binding regions attached to six collagen rods [Perkins et al., 1985 Biochem J. 228, 13-26; Poon et al., 1983 J Mol Biol. 168, 563-77; Reid et al., 1983 Biochem Soc Trans 11, 1-12; Weiss et al., 1986 J. Mol. Biol. 189, 573-81]. C1q was found to fit into the b12 hexamer assembly of the 1HZH crystal structure so that each of the six globular heads contacts one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., 5-10 July 2010; "Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector Function", Erica Ollmann Saphire, the Scripps Research Institute, La Jolla, California. November 2000). Mutations of selected amino acids in the Fc interface observed between symmetry-related b12 antibodies in the crystal structure were found to reduce C1q binding affinity, indicating the contribution of these amino acids to intermolecular Fc:Fc interactions.

[0008] US2011 / 0123440 describes an altered antibody Fc region and its use. The altered Fc region has one or more amino acid substitutions.

[0009] US2008 / 0089892 describes polypeptide Fc region variants and compositions comprising these Fc region variants.

[0010] US2010 / 0184959 describes methods for providing Fc polypeptide variants with altered Fc ligand recognition and / or effector functions.

[0011] US 2010 / 015133 describes methods for producing polypeptides by modulating polypeptide association.

[0012] US2010 / 105873 describes a comprehensive approach to generating multi-domain protein therapeutics.

[0013] US 6,737,056 describes polypeptide variants with altered effector function.

[0014] Efforts have been made to identify antibody Fc variants with enhanced effector functions or other modified properties. Such studies have focused on, for example, exchanging fragments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72), or amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) or at or near the C1q binding site of the CH2 domain, 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, Moore et al. (2010 mAbs 2 (2), 181-189) described the enhanced effector effects of detecting various combinations of S267E, H268F, S324T, S239D, I332E, G236A and I332E by CDC or ADCC. Other Fc mutations that affect binding to Fc receptors (WO 2006 / 105062, WO00 / 42072, U.S. Pat. No. 6,737,056 and U.S. Pat. No. 7,083,784) or antibody physical properties (WO 2007 / 005612A1) have also been proposed.

[0015] Despite these and other advances in the art, however, there remains a need for new and improved antibody-based therapeutics. SUMMARY OF THE INVENTION

[0017] The invention provides polypeptides and antibody variants having enhanced effector effects compared to its parent polypeptide / antibody. Without being limited to theory, it is believed that variants can have more stable connection interactions between two polypeptide / antibody molecule Fc regions, thus providing a more affine surface, which can cause enhanced effector effects, such as improved or more specific CDC responses. The feature of specific variants is also that improved ADCC responses, ADCP responses and / or other enhanced effector effects. As described herein, this subtle mechanism of polypeptide / antibody engineering can be applied to, for example, improving effectiveness or specificity based on antibody therapy.

[0018] Therefore, one aspect of the invention relates to variants of a parent polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, 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.

[0019] The invention also provides the use of at least one such mutation to enhance the effector function mediated by the polypeptide or antibody when the polypeptide or antibody binds to its antigen, for example on the surface of an antigen-expressing cell, a cell membrane or a viral particle.

[0020] In one aspect of the invention, referred to as a "single mutant", the variant has an improved effector function compared to the parent polypeptide or antibody.

[0021] In one aspect of the invention referred to as a "double mutant", the variant comprises at least two mutations in said fragment and has improved effector function compared to a variant comprising only one of the two mutations, the parent polypeptide or antibody, or both.

[0022] In one aspect of the invention referred to as a "mixed mutant", the variant, when used in combination with a second variant of the same or different polypeptide or antibody comprising a mutation at a different amino acid residue in the segment, provides improved effector function compared to one or more of the variant, the second variant, and the parent polypeptide or antibody alone.

[0023] Typically, the mutation is an amino acid substitution, such as a mutation that exchanges a parent amino acid residue for an amino acid with a different size and / or physicochemical properties that promotes the formation of a new intermolecular Fc:Fc bond or increases the strength of an existing pair of interactions. Exemplary amino acid residues for mutations according to the invention are shown in Tables 1 and 2A and B, along with exemplary amino acid substitutions. Non-limiting examples of various aspects of the invention are shown in Figure 1 Available in.

[0024] These and other aspects of the invention, particularly the various uses and therapeutic applications of the antibody variants, are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1:(A) Schematic representation of hexameric IgG molecules. The dotted circle shows two adjacent Fc:Fc interaction pairs of two adjacent IgG molecules. The arrows in the box show the direction of viewing the diagrams in B, C and D: two adjacent Fc molecules are rotated 90° (in the plane of the figure) and viewed from the Fab arm in the direction of the CH3 domain. (B) The effect of the observed oligomerization enhancing mutations on CDC. The schematic representation shows the Fc:Fc interaction pairs with enhanced efficacy according to the single mutant and double mutant aspects of the present invention. (C) The effect of the observed oligomerization inhibiting mutations on CDC. The schematic representation shows how at least two oligomerization inhibiting mutations that compensate for each other are combined into one molecule (double mutant aspect) or separated on two molecules (mixed mutant aspect) according to the double mutant and mixed mutant aspects of the present invention to restore or improve Fc:Fc interactions. The mixed mutant achieves a specific effector effect that depends on the combination of two antibodies, which can recognize different targets. (D) The theoretical effect of the C1q binding inhibiting mutation on CDC. Schematic representation of the Fc:Clq interaction shows that if mutations inhibit Clq binding, they cannot be combined or mixed to restore CDC activity due to the inability of Clq to compensate for the defect introduced into the antibody.

[0027] Figure 2 : Alignment of human IgG1, IgG1f, IgG2, IgG3 and IgG4 Fc fragments corresponding to residues P247 to K447 in the IgG1 heavy chain, using Clustel 2.1 software, numbered according to the EU index as listed in Kabat. The sequences shown represent residues 130 to 330 of the human IgG1 heavy chain constant region (SEQ ID NO: 1; UniProt accession number P01857) and allotypic variants of IgG1m(f) (SEQ ID NO: 5); residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID NO: 2; UniProt accession number P01859); residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID NO: 3; UniProt accession number P01860); and residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO: 4; UniProt accession number P01861).

[0028] Figure 3Aand 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) frameworks. The amino acid numbering according to Kabat and according to the EU index is described (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0029] Figure 4 : Detailed view of the K439 / S440 interaction between Fcs of neighboring molecules (Fc and Fc', respectively) in a multimeric (eg, hexameric) arrangement, showing the interaction between wild-type, unmodified Fc and Fc' molecules.

[0030] Figure 5 : Detailed view of K439 / S440 interactions between adjacent Fc molecules (Fc and Fc', respectively) in a multimeric (eg, hexameric) arrangement, showing the interaction between variant Fc and Fc' molecules comprising K439E and S440K mutations.

[0031] Figure 6 : C1q binding ELISA with 7D8 Fc:Fc mutants. Serial concentrations of the indicated antibodies were coated onto microtiter plate wells and incubated with a fixed concentration of C1q. With the exception of I253D, all coated variants bound C1q with comparable efficiency to wild-type 7D8. Schematic of at least 3 experiments is shown.

[0032] Figure 7 : CDC of CD20 positive Raji cells mediated by 7D8 variants. Raji cells were incubated with 7D8 variants (K439E, S440K, K439E / S440K double mutant, K439E+S440K mixed) and a series of concentrations of C1q to test CDC efficacy by measuring cell lysis. A schematic diagram of repeated experiments is shown.

[0033] Figure 8 : CDC mediated by 7D8 variants (7D8-WT, K439E, S440K, K439E / S440K double mutant, K439E+S440K mixed) on CD20-positive Daudi cells. The efficacy of CDC induction of a series of concentrations of 7D8 variants was tested.

[0034] Figure 9: CD38 antibody HuMAb005 mutant-mediated CDC on CD38-positive cells. (A) CDC efficacy of 005 mutant series concentrations on Daudi cells. (B) CDC efficacy of HuMAb 005 mutant series concentrations on Raji cells. (C) CDC efficacy of E345R mutant of HuMAb 005 with 20% or 50% NHS on Wien133 cells. (D) CDC efficacy of E345R mutant of HuMAb 005 and 7D8 with 20% or 50% NHS on Raji cells.

[0035] Test unpurified antibody samples isolated from transient transfections. Use supernatant from mock-transfected cells as a negative control.

[0036] Figure 10: CDC of wild-type and E345R mutants of CD38 antibody HuMAb 005 (A) and CD20 antibody HuMAb 7D8 (B) in competition experiments with Fc-binding peptides. Cell lysis was measured after CDC on antibody-opsonized Daudi cells incubated with a concentration series of Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO: 7). Unpurified antibody samples isolated from transient transfections were used. Supernatants of mock-transfected cells were used as negative controls.

[0037] Fig.11 : ADCC of CD38 expressing Daudi cells by wild-type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R. Expressed as % lysis, ADCC of one donor PBMC is shown.

[0038] Fig. 12A -C: Binding of wild-type IgG1-7D8 and variant IgG1-7D8-E345R to human, monkey and mouse FcRN at pH 6 as determined by ELISA.

[0039] Fig.13 : Plasma concentrations of wild-type IgG1-7D8 and -E354R, -S440K and K322A variants after intravenous injection in SCID mice.

[0040] Fig.14A , B, C and D: CDC on CD20 and CD38 positive Wien133 cells.

[0041] Fig.15A and B: Evaluation of the in vivo efficacy of IgG1-7D8-E345R in a subcutaneous transplant tumor model using Raji-luc#2D1 cells.

[0042] Fig.16Aand B: Evaluation of the in vivo efficacy of IgG1-005-E345R in a subcutaneous transplant tumor model using Raji-luc#2D1 cells.

[0043] Fig.17 : CDC of CD38-positive, EGFR-negative Wien133 cells by a CD38 / EGFR bispecific antibody with E345R mutation.

[0044] Fig.18A and B: CDC of CD20 / CD38 bispecific antibodies with or without E345R mutation on CD20-positive, CD38-negative Wien133 cells or Raji cells.

[0045] Fig.19 : CDC of EGFR-positive A431 cells by EGFR antibody 2F8 with E345R mutation.

[0046] Fig. 20 : E345R mutant antibody-mediated CDC.

[0047] Fig.21 : Colocalization analysis of TF antibody (FITC) with the lysosomal marker LAMP1 (APC).

[0048] Fig.22A -D: Introduction of the E345R mutation resulted in enhanced CDC-mediated killing compared to wild-type rituximab tested in different B cell lines.

[0049] Fig.22E : Introduction of E345R resulted in an increase in maximal CDC-mediated killing compared to wild-type rituximab, which was independent of the expression levels of the complement regulatory proteins CD46 (A), CD55 (B), or CD59 (C) in different B cell lines with comparable CD20 expression levels.

[0050] Fig.23 : CDC kinetics. Compared with the wild-type antibody, the E345R antibody resulted in more rapid and greater target cell lysis by CDC.

[0051] Fig.24 : CDC kinetics. Introduction of the E345R mutation in the bispecific CD38xCD20 antibody resulted in more rapid and more extensive CDC-mediated lysis of target cells.

[0052] Fig.25 : CDC kinetics. Introduction of the E345R mutation into the bispecific antibodies CD38xEGFR (A) and CD20xEGFR (B) that monovalently bind to EGFR-negative Raji cells results in more rapid and more extensive CDC-mediated lysis of target cells.

[0053] Fig.26 : CDC of the combination of wild-type antibody and mutant antibodies containing (AC) E345R and Q386K or (DF) E345R, E430G and Q386K on Wien 133 cells. The IgG1-b12 mutant did not bind to Wien 133 cells and was used as a negative control antibody.

[0054] Fig. 27 : CDC efficacy of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing the E345R mutation.

[0055] Fig.28 : Introduction of the Fc-Fc stabilizing E345R mutation into the wild-type CD38 antibody 005 resulted in enhanced killing of primary CLL cells in an ex vivo CDC assay (mean ± standard deviation of the mean). DETAILED DESCRIPTION OF THE INVENTION

[0057] As described in the present invention, surprisingly, mutations in amino acids that are not directly involved in Fc: C1q binding can still improve the CDC of the antibody and can also 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 are subsequently linked by C1q. In addition, although certain mutations were found to reduce CDC induction, certain combinations of such mutations in the same or different antibody molecules led to the restoration of CDC induction and showed more specificity for antibody oligomerization, thereby promoting more specific CDC induction. As shown in the examples, specific mutations that improve CDC response are also characterized by improved ADCC response, improved affinity, improved internalization and in vivo efficacy in a mouse tumor model system. These findings allow for new antibody-based therapies with enhanced CDC induction ability, more selective CDC induction, and / or other improved effector functions.

[0058] The antibody variants of the present invention all comprise an antigen binding region and a full or partial Fc region, which comprises at least one mutation in the segment corresponding to amino acid residues P247 to K447 in IgG1. Without being limited by theory, based on Figure 1 Based on three different principles, schematically shown in FIG. 1 and referred to in the present invention as “single mutants”, “double mutants” and “mixed mutants”, it is believed that the identified mutations lead to more efficient and / or more specific CDC induction.

[0059] The improved C1q and / or CDC effects resulting from the variants of the invention are primarily detectable only in assays that allow the formation of antibody oligomers, such as cell-based assays where the antigen is not immobilized but located on a fluid membrane. Furthermore, these effects resulting from more stable antibody oligomers rather than from modifications of the direct binding site of C1q can be determined based on Figure 1 C shows the proof of principle.

[0060] definition

[0061] The term "single mutant" is to be understood as a variant of the invention having an improved effector function compared to the parent polypeptide or antibody.

[0062] The term "double mutant" is to be understood as a variant comprising at least two mutations in said fragment and having an improved effector function compared to a variant comprising only one of the two mutations, the parent polypeptide or antibody, or both.

[0063] The term "mixed mutant" is to be understood as a variant that, when used in combination with a second mutant of the same or different polypeptide or antibody comprising a mutation at a different amino acid residue of the segment, provides an improved effector function compared to one or more of the variants, the second variant, and the parent polypeptide or antibody alone.

[0064] 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, which can bind to any molecule, such as a polypeptide, located on a cell, a bacterium, or a virus particle, for example. The Fc domain of an immunoglobulin is defined as an antibody fragment usually generated after digestion of an antibody with papain (which is known to those skilled in the art), which includes two CH2-CH3 regions of an immunoglobulin and a connecting region, such as a hinge region. The constant region of the antibody heavy chain defines the antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE. The Fc domain mediates the effector action of antibodies with cell surface receptors and complement system proteins known as Fc receptors. The binding region can be a polypeptide sequence that can be attached to a cell, a bacterium, a virus particle, such as a protein, a protein ligand, a receptor, an antigen binding region or a ligand binding region. If 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 an immunoglobulin Fc domain and the binding region. If the binding region is an antigen binding region, the "polypeptide comprising an immunoglobulin Fc domain and a binding region" may be an antibody, like a human antibody or a heavy chain-only antibody or a ScFv-Fc-fusion. A polypeptide comprising an immunoglobulin Fc domain and a binding region generally includes a connecting region, such as a hinge region, and two CH2-CH3 regions of an immunoglobulin heavy chain, and thus a "polypeptide comprising an immunoglobulin Fc domain and a binding region" may be a "polypeptide comprising at least an immunoglobulin Fc domain and a binding region". The term "immunoglobulin Fc domain" in the context of the present invention means the presence of a connecting region, such as a hinge, and an immunoglobulin CH2 and CH3 region depending on the antibody subtype, such as human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2 or IgE.

[0065] The term "CH2 region" or "CH2 domain" used in the present invention refers 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 also be any other subtype described herein.

[0066] The term "CH3 region" or "CH3 domain" used in the present invention means 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 CH3 region may also be any other subtype described herein.

[0067] The term "immunoglobulin" refers to a class of structurally related glycoproteins, which are composed of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which are internally connected by disulfide bonds. The structure of immunoglobulins has been fully characterized. See, for example, Fundamental Immunology Ch.7 (Paul, W., ed. 2nd ed. Raven Press, NY (1989)). In short, each heavy chain is usually composed of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region. The heavy chain constant region is usually composed of three domains, CH1, CH2, and CH3. The heavy chains are connected by disulfide bonds in the so-called "hinge region". Each light chain is usually composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region. The light chain constant region is usually composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions in the form of sequence and / or structurally defined loops), also known as complementarity determining regions (CDRs), which are interspersed with more conserved regions, known as framework regions (FRs). Each VH and VL is usually composed of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise indicated or contradictory to the context, the amino acids of the constant region sequences in the present invention are numbered according to the EU-index (described in Kabat, EA et al., Sequences of proteins of immunological interest. Fifth Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)).

[0068] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, an immunoglobulin molecule fragment, or a derivative of the two, which has the ability to specifically bind to an antigen under typical physiological conditions, and has a half-life of a significant period of time, such as 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, about 48 hours, about 3, 4, 5, 6, 7 or more days, etc., followed by any other relevant function-defined period of time (such as a time sufficient to induce, promote, enhance and / or regulate the physiological response associated with antibody binding to antigen, and / or a time sufficient to allow the antibody to recruit effector activity). The antibody of the present invention comprises an immunoglobulin Fc domain and a binding region. Antibodies generally contain two CH2-CH3 regions and a connecting region, such as a hinge region, such as at least an Fc domain. Therefore, the antibody of the present invention may comprise an Fc region and an antigen binding region. The heavy chain and light chain variable regions of the immunoglobulin molecule comprise a binding domain that interacts with an antigen. The constant or "Fc" domain of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component of the classical pathway of complement activation. Antibodies can also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term "bispecific antibody" refers to an antibody having specificity for at least two different, usually non-overlapping epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different target sites, such targets can be on the same cell or on different cells or cell types. As described above, unless otherwise indicated or clearly contradictory to the context, in the present invention, the term antibody includes antibody fragments that include at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be provided by any known technology, such as enzymatic cleavage, peptide synthesis, and recombinant expression technology. It has been shown that the antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "Ab" or "antibody" include, without limitation, monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, which consist of only two heavy chains and occur naturally in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), chain exchange engineered domains (SEEDs or Seed bodies), which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomabs (Fresenius, Lindhofer et al. (1995 J Immunol 155:219));FcΔAdp (Regeneron, WO2010151792), AzymetricScaffold (Zymeworks / Merck, WO2012 / 058768), mAb-FV (Xencor, WO2011 / 028952), dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent 7,612,181); dual domain biface antibody (Unilever; SanofiAventis, WO20100226923), diface antibody (ImClone / Eli Lilly), knob-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO2011 / 131746); electrostatically controlled antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2);Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), Dual-targeting domain antibodies (GSK / Domantis), Two-in-one antibodies recognizing two targets (Genentech, Novlmmune), Cross-linking MAbs (Karmanos Cancer Center), CovX bodies (CovX / Pfizer), IgG-like bispecifics (ImClone / EliLilly, Shen, J., et al. J Immunol Methods, 2007.318(1-2):p.65-74), and DIG bodies and PIG bodies (Pharmabcine), as well as dual affinity retargeting molecules (Fc-DART or Ig-DART of Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), Zybodies (Zyngenia), common light chains (Crucell / Merus, US7262028) or common heavy chains (κλ bodies of Novlmmune), and fusion proteins comprising polypeptide sequences fused to antibody fragments containing an Fc domain, such as scFv fusions, such as BsAb of ZymoGenetics / BMS, HERCULES of Biogen Idec (US007951918), SCORPIONS of Emergent BioSolutions / Trubion, Ts2Ab (Medlmmune / AZ Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion of Novartis, scFv fusion of Changzhou Adam Biotech Inc (CN 102250246), TvAb of Roche (WO2012025525, WO2012025530), mAb of f-Star; 2 (WO2008 / 003116) and double scFv-fusions. It should also be understood that the term antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal) such as those produced by the technology developed by Symphogen and Merus (Oligoclonics), and antibody-like polypeptides such as chimeric antibodies and humanized antibodies. The antibodies produced can potentially have any isotype.

[0069] The term "full length antibody", when used in the present invention refers to an antibody (eg, a parent or variant antibody) that comprises all heavy and light chain constant and variable domains corresponding to those domains normally found in a wild-type antibody of that isotype.

[0070] The term "human antibody" used in the present invention includes antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or point-specific mutations in vitro or by somatic mutations in vivo), however, the term "human antibody" used in the present invention is not intended to include antibodies wherein derived from another mammalian species germline, such as antibodies in which the CDR sequences of mice have been transplanted to human framework sequences.

[0071] As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb" or the like refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be generated by hybridoma cells comprising B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, the B cells comprising a human heavy chain transgenic portion and a light chain transgenic portion that are rearranged to produce a functional human antibody and fused to an immortal cell.

[0072] As used herein, "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE or IgM or any allotype thereof such as IgG1m(za) and IgG1m(f)). In addition, each heavy chain allotype can be combined with a kappa or lambda light chain.

[0073] The term "monovalent antibody" in the context of the present invention means an antibody molecule which is only capable of binding to an antigen with only one antibody binding domain, e.g. has a single antigen-antibody interaction, and is therefore not capable of antigen cross-linking.

[0074] The term "target" as used in the present invention is to be understood in the context of the present invention as a molecule to which the binding region of a polypeptide comprising an Fc domain and a binding region binds, and when used in the context of antibody binding includes any antigen to which the proposed antibody is directed. The terms "antigen" and "target" can be used interchangeably with respect to antibodies and constitute the same meaning or purpose for any aspect or embodiment of the present invention.

[0075] The term "binding" as used in the present invention in the context of the binding of an antibody to a predetermined antigen is generally binding, when using the antigen as a ligand and the antibody as an analyte, the binding is measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument. -6 M or smaller, e.g. 10 -7 M or smaller, such as 10 -8 M or smaller, such as 10 -9 M or smaller, about 10 -10 M or smaller, or about 10 -11 M or even smaller K D The affinity of the binding agent is at least ten-fold lower than the affinity of the binding agent when binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen, such as at most 1 / 100, such as at most 1 / 1000, such as at most 1 / 10000, such as at most 1 / 100000. D The amount of affinity is determined by the antibody's K D , so that when the antibody K D When the affinity for the antigen is very low (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is lower than the affinity for the nonspecific antigen may be at least 10,000 times. D ” (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0076] A "variant" or "antibody variant" or "parent antibody variant" of the present invention is an antibody molecule comprising one or more mutations compared to a "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" comprising one or more mutations compared to a "parent polypeptide comprising an immunoglobulin Fc domain and a binding region". Different terms can be used interchangeably and constitute the same meaning or purpose for any aspect or embodiment of the present invention. Exemplary parent antibody forms include, but are not limited to, wild-type antibodies, full-length antibodies or antibody fragments containing Fc, bispecific antibodies, human antibodies, or any combination thereof. Exemplary mutations include amino acid deletions, insertions or amino acid substitutions in the parent amino acid sequence. Amino acid substitutions can replace a natural amino acid with another naturally occurring amino acid, or a non-naturally occurring amino acid derivative. Amino acid substitutions can be conservative or non-conservative. In the context of the present invention, conservative substitutions are defined by substitutions between amino acid types reflected in one or more of the following three tables:

[0077] Types of conservatively substituted amino acid residues

[0078] Acidic residue Asp(D) and Glu(E) Basic residues Lys(K), Arg(R), and His(H) Hydrophilic uncharged residues Ser(S), Thr(T), Asn(N) and Gln(Q) Aliphatic uncharged residue Gly(G), Ala(A), Val(V), Leu(L) and Ile(I) Nonpolar uncharged residues Cys(C), Met(M), and Pro(P) Aromatic residues Phe(F), Tyr(Y), and Trp(W)

[0079] Optional conservative amino acid residue substitution types

[0080] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0081] Optional physical and functional classification of amino acid residues

[0082]

[0083]

[0084] In the context of the present invention, substitutions in variants are indicated as:

[0085] Original amino acid-position-substituted amino acid

[0086] Amino acid residues are represented using three letter codes or single letter codes, including the codes Xaa and X. Thus, the notation "E345R" or "Glu345Arg" means that the variant comprises a substitution of arginine for glutamic acid at the amino acid position corresponding to the amino acid at position 345 of the parent antibody. When the two are aligned as shown below,

[0087] When the position itself does not exist in the antibody, but the variant contains an amino acid insertion, e.g.

[0088] Position - substituted amino acid; use a label, e.g. "448E".

[0089] Such labels are particularly relevant to modifications within a homologous polypeptide or antibody series.

[0090] Similarly, when the identity of the substituted amino acid residue is not important:

[0091] Original amino acid-position; or "E345".

[0092] The original amino acid and / or the substituted amino acid may contain more than one, but not all, amino acid modifications, glutamic acid at position 345 is substituted with arginine, lysine or tryptophan:

[0093] "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.

[0094] In addition, the term "substitution" includes substitution with any of the other 19 natural amino acids, or substitution with other amino acids, such as non-natural amino acids. For example, the substitution of amino acid E at position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T, 345V, 345W and 345Y. Incidentally, this is equivalent to the name 345X, where X refers to any amino acid. These substitutions may also be referred to as E345A, E345C, etc., or E345A, C, etc. or E345A / C / , etc. The same applies to each similar situation and each position involved in the present invention, and any of such substitutions is specifically included in the present invention.

[0095] An amino acid or fragment in one sequence that "corresponds to" an amino acid or fragment in another sequence is an amino acid or fragment that (i) aligns with the other amino acid or fragment 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 amino acid or fragment can be aligned using Figure 2 The sequences shown in and 3 were aligned to identify any amino acid in the IgG2, IgG3, or IgG4 Fc sequence that corresponds to a specific amino acid in the IgG1 Fc sequence.

[0096] The present invention relates to variants, i.e., parent antibodies, and / or variant antibodies, which have a certain degree of identity with amino acids P247 to K447 of SEQ ID Nos: 1, 2, 3, 4 and 5, such parent and / or variant antibodies are hereinafter referred to as "homologous antibodies".

[0097] For the purposes of the present invention, the degree of identity between two amino acid sequences and the degree of identity between two nucleotide sequences are determined by the program "Alignment" of the Needleman-Wunsch alignment (i.e., global alignment). This program is used for the alignment of polypeptides and nucleotide sequences. The default score matrix BLOSUM50 is used for polypeptide alignment, while the default identity matrix is ​​used for nucleic acid sequence alignment, and the first residue penalty of the gap is polypeptide-12 and nucleotide-16, and the penalty of the other residues of the gap is polypeptide-2 and nucleotide-4.

[0098] "Alignment" is part of the FASTA package version v20u6 (see WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and WR Pearson (1990) "Rapid and Sensitive Sequence Comparaison with FASTP and FASTA", Methods in Enzymology 183:63-98). FASTA protein alignment uses the Smith-Waterman algorithm with no restrictions on gap size (see "Smith-Waterman algorithm", TF Smith and MS Waterman (1981) J. Mol. Biolo. 147:195-197).

[0099] The term "effector cell" used in the present invention refers to an immune cell that participates in the effector phase of an immune response relative to the recognition and activation phase of an immune response. Exemplary immune cells include cells of bone marrow 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. Some effector cells express Fc receptors (FcRs) or complement receptors and perform specific immune functions. In some embodiments, effector cells such as, for example, natural killer cells can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells expressing FcRs participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens. In some embodiments, antibodies that can obtain accumulation of target cell activation C3 fragments drive classical complement activation can further enhance ADCC. C3 cleavage products are complement receptors (CRs) expressed on myeloid cells, such as ligands of CR3. Complement fragment recognition of CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells can engulf target antigens, target particles or target cells. The expression of specific FcR or complement receptors on effector cells may be regulated by humoral factors such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFNγ) or G-CSF. Such enhanced expression increases the cytotoxicity of cells carrying FcγRI to targets. Effector cells can engulf target antigens or engulf or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct engulfment by effector cells or indirectly engulf by enhancing antibody-mediated cell engulfment.

[0100] The term "vector" used in the present invention means a nucleic acid molecule that can induce transcription of a nucleic acid fragment connected to the vector. One type of vector is a "plasmid", which is a circular double-stranded DNA ring form. Another type of vector is a viral vector, in which a nucleic acid fragment can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (such as non-episomal mammalian vectors) can be integrated into the host genome when introduced into the host cell, and thus replicate with the host genome. In addition, certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to as "recombinant expression vectors" (or "expression vectors" for short) in the present invention. In general, expression vectors used in recombinant DNA technology are usually in the form of plasmids. In this specification, since plasmids are the most commonly used vector forms, "plasmids" and "vectors" can be used interchangeably. However, the present invention is intended to include such other forms of expression vectors that play equivalent functions, such as viral vectors (such as replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0101] The term "recombinant host cell" (or simply "host cell") as used in the present invention means 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 specific subject cell, but also to the progeny of such cells. Due to mutations or environmental influences, certain modifications may occur in the offspring, so such progeny may not actually be the same as the parent cell, but are still included in the scope of the term "host cell" as used in the present invention. Recombinant host cells include, for example, transfected cells such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli, and other eukaryotic hosts such as plant cells and fungi.

[0102] The term "transfected cells" used in the present invention includes recombinant host cells expressing antibodies or target antigens, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells or fungi, including yeast cells.

[0103] The term "preparation" refers to a preparation of antibody variants and mixtures of different antibody variants that may have an increased ability to form oligomers when interacting with an antigen in contact with an associated cell (e.g., an antigen expressed on a cell surface), a cell membrane, a viral particle, or other structure, thereby resulting in increased C1q binding, complement activation, CDC, ADCC, ADCP, other Fc-mediated effector functions, internalization, downregulation, apoptosis, antibody-drug conjugate (ADC) uptake, affinity, or any combination thereof. Exemplary assays are provided in the Examples, e.g., C1q binding affinity (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25); ADCC (Example 12) and in vivo efficacy (Examples 20, 21). Mutants referred to as "single mutants", "double mutants", and "mixed mutants" according to the present invention, as well as exemplary preparation processes and methods of use thereof are described in more detail below.

[0104] The term "affinity" as used herein refers to the strength of binding of one molecule, such as an antibody, to another, such as a target or antigen, at a single site, such as the monovalent binding of an antibody to an individual antigen binding site of an antigen.

[0105] The term "avidity" as used in the present invention is the strength of binding between two structures, such as multiple antigen binding sites of an antibody that interact with a target simultaneously, or multiple binding sites such as an antibody and C1q. When there is more than one binding interaction, the two structures will dissociate only when all binding sites dissociate, and therefore, the dissociation rate will be lower than that of a single binding site, thereby providing a more effective overall binding strength (avidity) compared to the strength of binding (affinity) of a single binding site.

[0106] The term "oligomer" as used in the present invention refers to a molecule consisting of more than one but limited number of monomeric units (such as antibodies), compared to a polymer which, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Greek prefixes are often used to indicate the number of monomeric units in an oligomer, such as a tetramer consisting of four units and a hexamer consisting of six units.

[0107] The term "oligomerization" as used in the present invention means the process of converting monomers into polymers of limited degree. In the present invention, it is observed that oligomerization of Fc domain occurs after the binding of the polypeptide containing Fc domain, such as antibody, preferably but not limited to on the cell surface, to the target. Oligomerization of antibodies can be evaluated, for example, using cell surface C1q binding assay (as described in Examples 4 and 9), C1q efficacy assay (as described in Example 5), and complement dependent cytotoxicity as described in Examples 6, 10 and 19.

[0108] The term "Clq binding" as used herein means that in the context of Clq binding, Clq binds to an antibody bound to its antigen. Antibody binding to its antigen is understood to occur in vivo and in vitro in the context described herein. Clq binding can be assessed, for example, by using antibodies immobilized on an artificial surface (e.g., plastic plates for ELISA as described in Example 3), by using binding to a predetermined antigen on a cell or virus surface (as described in Examples 4 and 9). Binding of Clq to an antibody oligomer is understood to be a multivalent interaction resulting in high affinity binding.

[0109] The term "complement activation" as used in the present invention refers to the activation of the classical complement pathway, which is activated by the binding of complement component C1q to an antibody to which its antigen is attached. C1q is the first protein in the early events of the classical complement cascade, which includes a series of cleavage reactions that ultimately lead to the formation of an enzymatic activity called C3 convertase, which cleaves complement component C3 into C3b and C3a. C3b covalently binds to C5 on the membrane to form C5b, which in turn activates the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8 and C9 assemble into a membrane attack complex (MAC). The complement cascade leads to the formation of pores, which cause cell lysis, also known as CDC. Complement activation can be assessed using C1q potency (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 cellular deposition methods described in Beurskens et al., April 1, 2012, vol. 188 no. 7 3532-3541.

[0110] As used herein, the term "complement dependent cytotoxicity" ("CDC") refers to the process of antibody-mediated complement activation that results in lysis of cells or virus particles following attachment of antibodies to their antigens on cells or virus particles due to pores in the membrane created by MAC assembly. CDC can be evaluated by in vitro assays such as the CDC assays described in Examples 6, 10, 19, 25, 27 and 33, in which normal human serum is used as a complement source, or the C1q potency assay described in Example 5, in which normal human serum has been trapped in C1q.

[0111] The term "antibody-dependent cell-mediated cytotoxicity" ("ADCC") as used herein refers to a mechanism of killing of antibody-coated target cells or viral particles by cells expressing Fc receptors that recognize the constant region of the attached antibody. ADCC can be determined using methods such as the ADCC assay described in Example 12.

[0112] The term "antibody-dependent cellular phagocytosis" ("ADCP") used in the present invention refers to a mechanism of elimination of antibody-coated target cells or viral particles by internalization by phagocytic cells. The internalized antibody-coated target cells or viral particles are contained in a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP can be evaluated by using an in vitro cytotoxicity assay using macrophages as effector cells and visual microscopy as described in van Bij et al. Journal of Hepatology Volume 53, Issue 4, October 2010, pp. 677-685, or, for example, PMN phagocytosis of Staphylococcus aureus as described in Example 14.

[0113] As used herein, the term "complement dependent cellular cytotoxicity" ("CDCC") refers to a mechanism of target cell or viral particle killing by cells expressing complement receptors that recognize complement 3 (C3) cleavage products that are covalently attached to the target cell or viral particle as a result of antibody-mediated complement activation. CDCC can be evaluated in a manner similar to that described for ADCC.

[0114] The term "downregulation" as used in the present invention means a process of reducing the number of molecules such as antigens or receptors on the surface of cells, for example, by binding of antibodies to receptors.

[0115] The term "internalization" as used in the present invention means that an antibody or Fc-containing polypeptide is internalized from the cell surface and / or from the surrounding medium, such as by endocytosis, into any mechanism of the target expressing cell. The internalization of the antibody can be evaluated using a direct detection method (such as, for example, the lysosomal co-localization assay described in Example 26) that measures the amount of internalized antibody.

[0116] The term "antibody-drug conjugate" used in the present invention refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cells, a drug, and a linker that connects the drug to, for example, the antibody. The linker is cleavable or non-cleavable in the presence of malignant cells; wherein the antibody-drug conjugate kills the malignant cells.

[0117] The term "antibody-drug conjugate uptake" as used in the present invention refers to the process by which the antibody-drug conjugate is attached to a target of a cell and then taken up / engulfed by the cell membrane and thereby absorbed into the cell. As described in WO 2011 / 157741, antibody-drug conjugate uptake can be evaluated as "antibody-mediated internalization and cell killing of anti-TF ADC in an in vitro killing assay".

[0118] The term "apoptosis" used in the present invention refers to a programmed cell death (PCD) process that can occur in cells. Biochemical events lead to characteristic cell changes (morphology) and death, which include vacuolization, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosome DNA fragmentation. Antibodies can induce apoptosis by binding to certain receptors.

[0119] Fc receptor binding can be measured indirectly as described in Example 12.

[0120] The term "FcRn" used in the present invention means a neonatal Fc receptor that is an Fc receptor. It was first discovered in rodents as a unique receptor that can transport IgG from breast milk to the bloodstream of newborn animals through the intestinal epithelial cells of newborn rodents. Further studies have revealed similar receptors in humans. However, in humans, it was found in the placenta to help promote the delivery of maternal IgG to the growing fetus, and it has also been shown to play a role in detecting IgG turnover. FcRn binds IgG at a pH of 6.0-6.5, but not at a neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal cavity (inside of the intestine) at a slightly acidic pH and ensure effective unidirectional transport to the basal side of a pH neutral to alkaline (pH7.0-7.5). This receptor also plays a role in IgG adult rescue through its occurrence in the endothelial cell endocytosis pathway. The FcRn receptor in the acidic endosome binds IgG internalized by pinocytosis, recirculates it to the cell surface, and releases it into the blood at an alkaline pH, thereby preventing it from undergoing lysosomal degradation. This mechanism also provides an explanation for the higher half-life of IgG in blood compared to other isotypes.Example 13 describes an assay showing IgG binding to FcRn at pH 6.0 in an ELISA.

[0121] The term "protein A" used in the present invention means a 56kDa MSCRAMM surface protein originally found on the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cell osmotic pressure, and a two-component system called ArlS-ArlR. Due to its ability to bind immunoglobulins, its use in biochemical research has also been found. It consists of 5 homologous Ig binding domains folded into a three-helix bundle. Each domain can bind to proteins from many mammalian species, most notably IgGs. It binds to the heavy chain Fc region of most immunoglobulins (overlapping 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 bacteria through their Fc regions rather than simply through their Fab regions, thereby bacterial destruction conditioning, complement activation and phagocytosis.

[0122] The term "Protein G" as used in the present invention means an immunoglobulin binding protein expressed in group C and G Streptococcus bacteria, which is very similar to Protein A but has a different specificity. It is a 65 kDa (G148 Protein G) and 58 kDa (C40 Protein G) cell surface protein and has found its use in purifying antibodies through its binding to the Fc region.

[0123] The term "CH2 region" or "CH2 domain" used in the present invention means 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.

[0124] The term "CH3 region" or "CH3 domain" used in the present invention means the CH3 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system.

[0125] The term "allosteric mutation" used in the present invention means modification, such as insertion, substitution and deletion, of amino acids P247 and E430 in a polypeptide containing an Fc domain according to the EU index numbering as set forth in Kabat.

[0126] The term "hydrophobic knob mutation" used in the present invention means modification, such as insertion, substitution and deletion, of amino acids I253, S254, and Q311 in a polypeptide containing an Fc domain according to the EU index numbering listed in Kabat. The hydrophobic knob is described by Delano WL, et al., Science 287, (2000), pp. 1279-1283, such as p. 1281.

[0127] The term "N-terminal CH3 helix mutation" as used in the present invention means modifications, such as insertions, substitutions and deletions, of amino acids R355, and D356, and E356, and E357, and M358, and L358, and T359, more specifically D356, and E356 and T359 in an Fc domain-containing polypeptide according to the EU index numbering as listed in Kabat.

[0128] The term "C-terminal CH3 β chain mutation" used in the present invention means modifications, such as insertions, substitutions and deletions, of amino acids Y436, and T437, and Q438, and K439, and S440 and L441, more specifically Y436 and K439, and S440 in a polypeptide containing an Fc domain according to the EU index numbering as listed in Kabat.

[0129] Methods of influencing antibody effector functions

[0130] It is to be understood that all embodiments described in the present invention concerning a parent antibody, a first parent antibody or a second parent antibody are also to be understood as embodiments concerning a parent, a first parent or a second polypeptide comprising an immunoglobulin Fc domain and a binding region.

[0131] In one aspect, the present invention relates to a method for improving the effector function of a parent polypeptide, said parent polypeptide comprising an immunoglobulin Fc domain and a binding domain, the method comprising introducing into the parent polypeptide a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, 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.

[0132] In one embodiment the parent polypeptide may be an antibody.

[0133] Therefore, the present invention relates to a method for improving the effector function of a parent antibody, comprising introducing a mutation into at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain in the parent antibody, with the proviso that the mutation in S440 is S440Y or S440W.

[0134] Reference to "D / E356" in the context of the present invention refers to an allotypic variant in the human IgG1 sequence. In the IgG1m(za) allotype of human IgG1, the amino acid at position 356 is D, while in the IgG1m(f) allotype of human IgG1, the amino acid at position 356 is E.

[0135] According to the method or use of the present invention, introducing mutations into a parent antibody results in a variant or variant antibody.Thus, the method of the present invention can be performed to obtain any variant or variant antibody described in the present invention.

[0136] The variant antibodies obtained from the method or use of the invention have an improved effector function compared to the parent antibody. Generally, the effect of an antibody on the effector function can be determined by the EC50 value, which is the antibody concentration required to obtain the maximum lysis half value.

[0137] Maximum lysis is the lysis obtained when a saturating amount of antibody is used, wherein saturation means the amount of antibody at which all antigens of the antibody are bound by the antibody.

[0138] The term "increasing effector function" or "improving effector function" in the context of the present invention refers to a decrease in the EC50 value of a variant antibody compared to a parent antibody. The reduction in EC50 value can be, for example, at most or about 1 / 2, such as at most or about 1 / 3, or at most or about 1 / 5, or at most or about 1 / 10. Optionally, "increasing effector function" or "improving effector function" means that under conditions where the parent antibody lysis is less than 100% of all cells, the maximum amount of cells lysed increases (wherein the total amount of cells is set to 100%), for example from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%.

[0139] Variants can be tested for improved or enhanced effector function by cloning IgG1-005 or IgG1-7D8 heavy chain variable domains into the variants and testing their potency in a CDC assay, as described for Daudi (Example 6) and Wien (Example 10). Using IgG1-7D8 HC variable domains and Daudi cells, enhancement is defined as less than 1 / 2 EC50, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 EC50 values ​​compared to the EC50 of IgG1-7D8 under the conditions studied, EC50 being the concentration at which half of the maximum lysis is observed. Using IgG1-005 HC variable domains and Daudi cells, enhancement is defined as less than 1 / 2 EC50, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 EC50 values ​​compared to the EC50 of IgG-005 under the conditions studied, EC50 being the concentration at which half of the maximum lysis is observed. Using IgG1-7D8 HC variable domains and Wien133 cells, an increase is defined as an EC50 of less than 1 / 2 compared to the EC50 of IgG1-7D8 under the conditions of the study, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 of the EC50 value, where EC50 is the concentration at which half of the maximum lysis is observed. Using IgG1-005 HC variable domains and Wien133 cells, an increase is defined as an increase in maximum lysis from 10% to 100% of all cells, such as an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. An increase in CDC potency can also be defined as an EC50 of less than 1 / 2, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 compared to the EC50 of IgG-005 under the conditions studied, where the EC50 is the concentration at which half-maximal lysis is observed under conditions where lysis of Wien 133 cells can be detected.

[0140] The inventors of the present invention surprisingly found that mutations at these specific positions have an improved effect on the effector function of variant antibodies obtained by introducing mutations into the parent antibody according to the method of the present invention (e.g., as shown in Example 19). Without being limited to theory, it is believed that substitution of at least one amino acid from the above position groups stimulates oligomerization. The antibody binds with higher affinity (exemplified in Example 2; IgG-7D8-E345R direct labeling resulted in improved binding to Daudi cells compared to IgG-7D8-WT), which allows the antibody to bind to cells for a longer time and thereby enable different effector functions, such as improved C1q binding, C1q efficacy CDC, ADCC, internalization, ADCP, and / or in vivo efficacy. These effects have been exemplified by Example 4 (C1q binding on cells), Example 5 (C1q efficacy 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 efficacy).

[0141] 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 may also be referred to as "single mutations" or "effect enhancing mutations" in the context of the present invention.

[0142] In another aspect, the present invention also provides 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, which can enhance effector functions, such as (i) C1q binding, (ii) complement activation, (iii) CDC, (iv) oligomer formation, (v) oligomer stability, (v) oligomer kinase activity, (vi) oligomer kinase activity, (vii) oligomer kinase activity, (viii) oligomer kinase activity, (viv ... vi) antibody-dependent cell-mediated cytotoxicity (ADCC), (vii) FcRn binding, (viii) Fc-gamma receptor binding, (ix) protein A binding, (x) protein G binding, (xi) antibody-dependent cellular phagocytosis (ADCP), (xii) complement-dependent cellular cytotoxicity (CDCC), (xiii) complement-enhanced cytotoxicity, (xiv) antibody-mediated complement receptor binding with opsonized antibodies, (xv) internalization, (xvi) downregulation, (xvii) induction of apoptosis, (xviii) opsonization, and one or more of any combination of (xix) (i) to (xviii). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, 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-γ receptor binding, protein A binding, protein G binding, ADCP, complement dependent cellular cytotoxicity (CDCC), complement enhanced cytotoxicity, antibody-mediated complement receptor binding to the opsonized antibody, antibody-mediated cellular phagocytosis (ADCP), internalization, apoptosis, and / or complement receptor binding to the opsonized antibody is also or optionally increased, such as specifically FcRn binding, ADCC, Fc-γ receptor binding, protein A binding, protein G binding, ADCP, CDCC, complement enhanced cytotoxicity, opsonization and any combination thereof.

[0143] 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, cell membrane or viral particle.

[0144] The inventors of the present invention have also shown that introducing a mutation into the amino acid residue corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain of the parent antibody reduces the effector function of the parent antibody (Examples 5, 6 and 10).

[0145] Another aspect of the invention relates to a method for reducing the effector function of a parent polypeptide, said parent polypeptide comprising an immunoglobulin Fc domain and a binding domain, the method comprising introducing a mutation into at least one amino acid residue in the parent polypeptide 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 not S440Y or S440W, such as, wherein the mutation in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E, and / or the mutation in the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R.

[0146] In one embodiment the parent polypeptide may be an antibody.

[0147] Therefore, in another aspect, the present invention relates to a method for reducing an effector function of a parent antibody, comprising introducing into the parent antibody 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 not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain is K439D / E, and / or the mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain is S440K / H / R.

[0148] 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 invention.

[0149] The variant antibody obtained from the method of reducing effector function has reduced effector function compared to the parent antibody.Usually, the effect of an antibody on effector function can be measured by the EC50 value, which is the antibody concentration required to obtain the maximum lysis half value.

[0150] Maximum lysis is the lysis obtained when a saturating amount of antibody is used, wherein saturation means the amount of antibody at which all antigens of the antibody are bound by the antibody.

[0151] The term "reduced effector function" in the context of the present invention refers to an increase in the EC50 value of a variant antibody compared to a parent antibody. The increase in the EC50 value can be, for example, at least or about 2 times, such as at least or about 3 times, at least or about 5 times, at least or about 10 times. Optionally, "reduced effector function" means that under conditions where the parent antibody lysis is less than 100% of all cells, the maximum amount of cells lysed is reduced, such as from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%.

[0152] Variants can be tested for reduced effector effects by cloning IgG1-005 or IgG1-7D8 heavy chain variable domains into the variants and testing their potency in CDC assays, as described for Daudi (Example 6) and Wien (Example 10). Using IgG1-7D8 HC variable domains and Daudi cells, reduction is defined as less than 1 / 2 EC50, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 EC50 values ​​compared to the EC50 of IgG1-7D8 under the conditions studied, EC50 being the concentration at which half of the maximum lysis is observed. Using IgG1-005 HC variable domains and Daudi cells, reduction is defined as less than 1 / 2 EC50, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 EC50 values ​​compared to the EC50 of IgG-005 under the conditions studied, EC50 being the concentration at which half of the maximum lysis is observed. Using IgG1-7D8 HC variable domains and Wien133 cells, a reduction is defined as an EC50 of less than 1 / 2 compared to the EC50 of IgG1-7D8 under the conditions of the study, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 of the EC50 value, where the EC50 is the concentration at which half of the maximum lysis is observed. Using IgG1-005 HC variable domains and Wien133 cells, a reduction is defined as a reduction in maximum lysis from 10% to 100% of all cells, such as a reduction of 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 potency can also be defined as an EC50 of less than 1 / 2, such as about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10 or less than 1 / 10 of the EC50 of IgG-005 under the conditions studied, where the EC50 is the concentration at which half of the maximum lysis is observed under conditions where lysis of Wien 133 cells can be detected.

[0153] In one embodiment, the effector function is reduced when the parent antibody binds to its antigen on an antigen expressing cell, cell membrane or viral particle.

[0154] Thus, in another aspect, the present invention relates to the use of at least more mutations in an antibody variant comprising a mutation in one of the amino acid residues selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W, which restores the effector function of the antibody variant when bound to its antigen on an antigen-expressing cell, cell membrane or virus particle.

[0155] 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

[0156] The first mutation is at an amino acid residue corresponding to S440 in the Fc region of a human IgGl heavy chain, and the second mutation is at an amino acid residue corresponding to K439 in the Fc region of a human IgGl heavy chain.

[0157] In one embodiment, the parent antibody is a monospecific, bispecific, or multispecific antibody.

[0158] If the parent antibody is a monospecific antibody comprising a CH2-CH3 region, the mutation according to the invention may in principle be present in only one of the CH2-CH3 regions, although for most practical purposes the mutations according to the invention that increase or decrease the effector function are present in both CH2-CH3 regions.

[0159] If the parent antibody is a bispecific antibody comprising a CH2-CH3 region, the mutations according to the invention may in principle be present in only one of the CH2-CH3 regions; i.e., in the first or the second CH2-CH3 region, although for most practical purposes the mutations according to the invention that increase or decrease the effector function are present in both the first and the second CH2-CH3 region of the bispecific antibody.

[0160] Suitable examples of monospecific, bispecific, or multispecific antibodies include any of those described herein.

[0161] In a specific embodiment, the parent or first and / or second antibody may be a bispecific antibody, such as a heterodimeric protein as described in WO 11 / 1311746, which is hereby incorporated by reference into the present invention.

[0162] In one embodiment, the parent antibody is a bispecific antibody, which includes 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.

[0163] In a further embodiment said first CH2-CH3 region comprises more amino acid substitutions at a position selected from those positions corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; and wherein said first CH2-CH3 region comprises more amino acid substitutions at a position selected from those positions corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and wherein said more amino acid substitutions in the first CH2-CH3 region are different from said more amino acid substitutions in the second CH2-CH3 region.

[0164] 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.

[0165] In one embodiment, the method comprises introducing a mutation at at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, into each of the first and second CH2-CH3 regions, with the proviso that the mutation at S440 is S440Y or S440W.

[0166] In a further embodiment, a mutation is 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, which may be at the same amino acid residue position or at a different position. In a further embodiment, it may be the same or a different mutation at the same amino acid residue position.

[0167] In another embodiment, the method comprises introducing a mutation at at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain into the first or second CH2-CH3 region, with the proviso that the mutation at S440 is S440Y or S440W.

[0168] Any mutation listed in Table 1 can be introduced into the bispecific antibody. Example 24 shows that the introduction of the E345R mutation into the bispecific CD20xEGFR antibody enhances CDC efficacy. Examples 23, 29 and 30 also describe some different bispecific antibodies comprising mutations according to the invention.

[0169] 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 the following positions

[0170] (i) 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 in S440 is not S440Y or S440W,

[0171] (ii) at each of the amino acid residues corresponding to K447 and 448 in the Fc region of a human IgG1 heavy chain, such as K447K / R / H and 448E / D on the Fc region of a human IgG1 heavy chain, preferably K447K and 448E on the Fc region of a human IgG1 heavy chain, or,

[0172] (iii) at each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of a human IgG1 heavy chain, such as K447D / E, 448K / R / H and 449P on the Fc region of a human IgG1 heavy chain, preferably K447E, 448K and 449P on the Fc region of a human IgG1 heavy chain.

[0173] 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 mutation at S440 is not S440Y or S440W.

[0174] Mutations are introduced into the two amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain of the parent antibody, provided that the mutation on S440 is not S440Y or S440W, also referred to as "double mutant" in the present invention. As described elsewhere, S440Y or S440W mutations have been found to improve effector function when introduced into the parent antibody.

[0175] It has also been described elsewhere that the inventors have found that introducing the identified mutations at the amino acid residues corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain results in a reduction in effector function (Examples 5, 6, 10). However, when inhibitory mutations are introduced at two amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, the reduction in effector function is restored, thereby making it similar to the effector function of the parent antibody without mutations at the K439 and S440 mutations. However, without being limited to any theory, it is believed that the presence of the K439 and S440 mutations limits the induction of effector functions to oligomeric complexes consisting only of antibodies containing the two mutations of K439 and S440 alone. Therefore, without being limited to any theory, if K439 and S440 mutations are included in a therapeutic antibody, it is believed that when such a therapeutic antibody is administered to a patient, the induction of effector function is limited to an oligomeric antibody complex comprising the therapeutic antibody containing the K439 / S440 mutations but not the patient's own antibodies, which do not contain the K439 and S440 mutations, thereby limiting any potential side effects caused by the interaction of the therapeutic antibody with the patient's own antibodies.

[0176] When mutations at positions K439 and / or S440 are combined with the first mutation, enhancement of CDC is obtained and the specificity of CDC is improved.

[0177] Therefore in another aspect the invention relates to a method for improving the specificity of a combination of at least a first and a second parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising:

[0178] A)

[0179] (i) introducing a mutation into the first parent polypeptide at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain; and

[0180] (ii) introducing a mutation into the second parent polypeptide at the amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W,

[0181] B)

[0182] (i) introducing a mutation into the first parent polypeptide at the amino acid residue corresponding to the position K447D / E in the Fc region of a human IgG1 heavy chain; and

[0183] (ii) introducing mutations into the second parent polypeptide at the amino acid residues corresponding to positions K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or

[0184] C)

[0185] (i) introducing a mutation into the first parent polypeptide at the amino acid residue corresponding to the position K447D / E in the Fc region of a human IgG1 heavy chain; and

[0186] (ii) introducing mutations into the second parent polypeptide at amino acid residues corresponding to positions K447K / R / H, 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

[0187] In one embodiment, the parent polypeptide, the first parent polypeptide and the second parent polypeptide may each be an antibody.

[0188] Therefore, in a further aspect, the present invention also relates to a method for improving the specificity of a combination of at least a first and a second parent antibody, comprising

[0189] (i) introducing a mutation into the amino acid residue at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain of the first parent antibody; and

[0190] (ii) introducing a mutation into the second parent antibody at an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W.

[0191] As shown in Example 10, the first and second variant antibodies have a preference to oligomerize with each other compared to any wild-type or naturally occurring antibody.

[0192] The increase in specificity is with respect to "induction of effector functions". Thus in one embodiment the method is a method for increasing the specificity of induction of effector functions by the combination of at least a first and a second parent antibody.

[0193] A method for improving specificity, or inducing specificity of effector function, by combining at least a first and a second parent antibody is performed, obtaining a combination of at least a first mutant and a second mutant antibody.

[0194] By introducing mutations at K439 or S440 of the parent antibody, the variant antibodies thus obtained have reduced effector effects compared to the parent antibody. However, the present invention also describes elsewhere that mutations at K439 and S440 can compensate for each other to restore the effector effects of antibodies comprising two mutations. The ability of mutations at K439 and S440 to compensate for each other can be used similarly in two antibodies. Therefore, when the mutation at K439 is introduced into the first parent antibody and the mutation at S440 is introduced into the second parent antibody, or vice versa, the reduction in effector effects will no longer be seen when the first and second mutant antibodies are used in combination. The term "increasing specificity" or "improving specificity" in this context refers to the effector response induced by the combination of a first variant antibody comprising a mutation at K439 and a second variant antibody comprising a mutation at S440, which is higher than the effector response induced by the first variant antibody comprising a mutation at K439 or the second variant antibody comprising a mutation at S440.

[0195] The specificity of the oligomer was improved by introducing two amino acid substitutions at K439 and S440.

[0196] When mutations at positions K439 and / or S440 are combined with the first mutation, enhancement of CDC is obtained and the specificity of CDC is improved.

[0197] In one embodiment at least the first and the second parent antibody bind to the same epitope.

[0198] In one embodiment at least the first and second parent antibodies bind to different epitopes on the same antigen.

[0199] In one embodiment at least the first and second parent antibodies bind to different epitopes on different targets.

[0200] In one embodiment the first and second parent antibodies have the same or different VL and VH sequences.

[0201] In one embodiment the combination of at least a first and a second parent antibody comprises one first parent antibody and one second parent antibody.

[0202] In one embodiment, the specificity is improved when the combination of the first and second parent antibodies is attached to its antigen on an antigen expressing cell, cell membrane or viral particle.

[0203] Therefore, in another aspect, the present invention also relates to the use of mutations at two or more amino acid residues of an antibody, which mutations increase the specificity of effector functions induced by the antibody, for example, when the antibody is bound to its antigen on an antigen-expressing cell, cell membrane or virus particle, wherein

[0204] The first mutation is at an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain;

[0205] The second mutation is at the amino acid residue corresponding to S440 in the Fc region of a human IgGl heavy chain.

[0206] In a further aspect, the present invention relates to a method for increasing the effector function of a combination of at least a first and a second parent polypeptide, wherein at least the first and the second parent polypeptide each comprises an immunoglobulin Fc domain and a binding region, wherein the method comprises

[0207] (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:

[0208] (a) providing allosteric mutations to amino acid residues within the CH2-CH3 region,

[0209] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0210] (c) amino acid residues within the N-terminal CH3 helix,

[0211] (d) an amino acid residue within the C-terminal CH3 beta 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

[0212] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0213] In one embodiment, the first and / or second parent polypeptide may each be an antibody.

[0214] Thus in one embodiment the invention relates to a method for increasing the effector function of a combination of at least a first and a second parent antibody, wherein at least the first and the second parent antibody each comprises an immunoglobulin Fc domain and an antigen binding region, wherein the method comprises

[0215] (i) introducing mutations into at least the first and / or second parent antibody at one or more amino acid residues selected from the group consisting of:

[0216] (a) providing allosteric mutations to amino acid residues within the CH2-CH3 region,

[0217] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0218] (c) amino acid residues within the N-terminal CH3 helix,

[0219] (d) an amino acid residue within the C-terminal CH3 beta 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

[0220] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0221] By performing this method, a combination of at least a first and a second variant antibody is obtained. The at least a first and a second variant antibody obtained by this method has an improved effector function when combined compared to the combination of the first and the second parent antibody.

[0222] The term "enhanced effector function" is to be understood as described in the present invention.

[0223] The first and / or second parent antibody may be any parent antibody described herein.

[0224] In particular, a method for improving the effector function of a combination of a first and a second antibody may be performed to obtain a first and a second antibody having any of the characteristics of a variant antibody described herein. The inventors have found that a combination of a first and a second variant antibody resulting from introduction of mutations into an amino acid residue selected from (a), (b), (c), (d) and / or (e) has an improved effector function compared to a combination of the first and second parent antibodies.

[0225] In one embodiment at least the first and the second parent antibody bind to the same epitope.

[0226] In one embodiment at least the first and second parent antibodies bind to different epitopes on the same antigen.

[0227] In one embodiment at least the first and second parent antibodies bind to different epitopes on different targets.

[0228] In one embodiment at least the first and the second parent antibodies have identical or different VL and VH sequences.

[0229] In one embodiment the combination of at least a first and a second parent antibody comprises one first parent antibody and one second parent antibody.

[0230] In one embodiment the combination of at least a first and a second parent antibody comprises further parent antibodies, such as a third, fourth or fifth parent antibody.

[0231] In one embodiment, (a) the amino acid residue within the CH2-CH3 region providing the 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.

[0232] In one embodiment, the amino acid residues within the hydrophobic knobs of the (b) CH2-CH3 regions are amino acid residues selected from those corresponding to I253, S254 and Q311 in the Fc region of a human IgG1 heavy chain.

[0233] In one embodiment, the (c) amino acid residue within the N-terminal CH3 helix is ​​an amino acid residue selected from those corresponding to D / E356 and T359 in the Fc region of a human IgG1 heavy chain.

[0234] In one embodiment, the (d) amino acid residue within the C-terminal CH3 beta strand is an amino acid residue selected from those corresponding to Y436 and S440.

[0235] The amino acid residues in (b), (c), (d) and (e) are located at the Fc:Fc interface of two antibodies, so that the Fc portion of an antibody can interact with the Fc portion of another antibody and the two antibodies are close to each other.

[0236] 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, 1253, 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.

[0237] In one embodiment (i) comprises introducing mutations in the first and second parent antibodies.

[0238] In another embodiment the method comprises:

[0239] (i) introducing into the first parent antibody a mutation at at least one of those amino acid residues corresponding to E345, E430, S440, Q386, P247, 1253, 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,

[0240] (ii) providing a second parent antibody which does not comprise a mutation at an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc region of a human IgG1 heavy chain.

[0241] In one embodiment, the method comprises introducing a mutation at at least one amino acid residue other than S440, wherein the method further comprises the step of introducing a mutation at one or more positions other than S440, and wherein the method further comprises the step of

[0242] (i) introducing a second mutation into the first parent antibody at an amino acid residue corresponding to the position K439 in the Fc region of a human IgG1 heavy chain; and

[0243] (ii) introducing a second mutation into the second parent antibody at an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W; wherein steps (ii) and (iii) may alternatively be

[0244] (i) introducing a second mutation into the first parent antibody at an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W; and

[0245] (ii) introducing a second mutation into the second parent antibody at an amino acid residue corresponding to the position K439 in the Fc region of the human IgG1 heavy chain.

[0246] For those embodiments of the invention, wherein the second parent does not comprise a mutation at an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain, the term "second mutation" in step (ii) may be the first mutation, e.g., the second parent antibody comprises only the mutation introduced in step (ii). The term "second mutation" in steps (i) and (ii) is also not intended to limit the number of mutations introduced into the first and / or second parent antibody.

[0247] In one embodiment, the parent antibody, the first parent antibody and the second parent antibody may each be selected from the group including, but not limited to, monospecific, bispecific and multispecific antibodies. The bispecific may be, for example, a heterodimeric protein.

[0248] In one embodiment, the first and second parent antibodies are monospecific antibodies, which may, for example, bind to the same or different epitopes. If the first and second parent antibodies bind to different epitopes, they may be on the same or different antigens.

[0249] 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.

[0250] In another embodiment, the first and second parent antibodies are bispecific or multispecific antibodies. In one embodiment, the first and second bispecific or multispecific antibodies are the same or different antibodies. In one embodiment, the first and second bispecific or multispecific antibodies bind to different epitopes on the same or different antigens. Thus, in one embodiment, at least the first and second parent antibodies are bispecific or multispecific antibodies that bind to different epitopes on the same or different antigens.

[0251] In another embodiment, the first parent antibody is a monospecific antibody and the second parent antibody is a bispecific antibody, or vice versa. A monospecific can bind to the same epitope, while a bispecific (or partially bispecific) or a monospecific and bispecific antibody can bind to different epitopes on the same or different antigens. A bispecific antibody can bind to different epitopes on the same or different antigens.

[0252] In one embodiment, the at least first and second parent antibodies are each a bispecific antibody, the 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 or different antigens, and wherein the first CH2-CH3 region comprises a further 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 wherein the second 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 the further amino acid substitution in the first CH2-CH3 region is different from the further amino acid substitution in the second CH2-CH3 region.

[0253] 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.

[0254] In one embodiment of the methods and / or uses of the invention, the parent antibody, whether it is a parent antibody, a first parent antibody or a second parent antibody, comprises other mutations in addition to those mutations of the invention that have been found to affect effector function. Such mutations can be introduced simultaneously with the mutations of the invention that affect effector function, or they can be introduced sequentially. The methods or uses of the invention are not limited to simultaneous or sequential introduction of mutations. The bispecific antibody can be any bispecific antibody, and since it is foreseen that different formats can be used, the methods or uses of the invention are not limited to any particular bispecific antibody format.

[0255] The method of combining a first antibody comprising one of the mutations that can improve the effector function with a second antibody that does not comprise such a mutation can increase the effector function of the combination as shown in Example 31. Therefore, without being limited to any theory, it is believed that, for example, this method can be used to combine a therapeutic antibody that has been proven to be safe but not sufficiently effective as a second antibody with a first antibody comprising a mutation, and thereby obtain an effective combination.

[0256] Thus in one embodiment, the second parent antibody that does not comprise a mutation at an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, 1253, 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 specific embodiment it is a therapeutic antibody with a suitable safety profile. In one embodiment it is a therapeutic antibody with a suitable safety profile but not sufficiently effective.

[0257] Examples of suitable second antibodies that do not comprise a mutation at a selected amino acid residue among those corresponding to E345, E430, S440, Q386, P247, 1253, 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 Merpentan; (99mTc) pintumomab; 3F8; 8H9; abagovomab; abatacept; abciximab; Actoxumab; adalimumab; adecatumumab; afelimomab; aflibercept; Afutuzumab; alacizumab pegol;albiglutide;ALD518;alfacept;alemtuzumab;alirocumab;altumomab;Altumomab pentetate;alvirceptsudotox;amatuximab;AMG714 / HuMax-IL15;anatumomab mafenatox;Anrukinzumab(=IMA-638);apolizumab;arcitumomab;aselizumab;atacicept;atinumab;Atlizumab(=tocilizumab);atorolimumab;baminercept;Bapineuzumab;basiliximab;bavituximab;bectumomab;belatacept;belimumab;benralizumab;bertilimumab;besilesomab;bevacizumab;Bezlotoxumab;biciromab;bifarcept;bivatuzumab;Bivatuzumab mertansine; blinatumomab; blosozumab; brentuximab vedotin; briakinumab; briobacept; brodalumab; canakinumab; cantuzumab mertansine; cantuzumab ravtansine; caplacizumab; capromab; Capromab pendetide; carlumab; catumaxomab; CC49; cedelizumab;certolizumab pegol; cetuximab; Ch.14.18; citatuzumab bogatox; cixutumumab; Clazakizumab; clenoliximab; Clivatuzumab tetraxetan; conatumumab; conbercept; CR6261; crenezumab; dacetuzumab; dalantercept; dalotuzumab; daratumumab; Demcizumab; Detumomab; Dorlimomab aritox; drozitumab; dulaglutide; ecoromeximab; eculizumab; edobacomab; edrecolomab; efungumab; elotuzumab; elsilimomab; enavatuzumab; enlimomab; enlimomab pegol; enokizumab; ensituximab; epitumomab; epitumomab cituxetan; epratuzumab; erlizumab; ertumaxomab; etanercept; etaracizumab; etrolizumab; exbivirumab; Fanolesomab; faralimomab; farletuzumab; Fasinumab; FBT A05; felvizumab; fezakinumab; ficlatuzumab; figitumumab; flanvolumab; fontolizumab; foralumab; foravirumab; fresolimumab; fulranumab; galixim ab; ganitumab; gantenerumab; gavilimomab; gemtuzumab; gemtuzumab ozogamib; gevokizumab; girentuximab; glembatumumab; Glembatumumabvedotin; golimumab; Gomiliximab; GS6624; anti-CD74 antibody; anti-cMet antibody as disclosed in WO2011 / 110642; anti-Her2 antibody as disclosed in WO2011 / 147986 or WO2011 / 147982; anti-IL-8 antibody as disclosed in WO2004 / 058797; anti-TAC antibody as disclosed in WO2004 / 045512;Anti-tissue factor (TF) antibodies as disclosed in WO 2010 / 066803 or WO 2011 / 157741; ibalizumab; ibritumomab tiuxetan; icrucumab; igovomab; Imciromab; inclacumab; indatuximab ravtansine; infliximab; inolimomab; inotuzumabozogamcin; intetumumab; iodine (I241) girentuximab; pirimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; lemalesomab; lenercept; lerdelimumab; lexatumumab; libivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; lumiliximab; mapatumumab; maslimoma; maslimomab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minretumomab; mirococept; mitolimumab; mogamulizumab; morolimumab; motavizumab; moxetumomab; pasudotox; morolimumab-CD3; nacolomab tafenatox; namilumab; naptumomab estafenatox; narnatumab; natalizumab; nebacumab; necitumumab; nerelimomab; nimotuzumab; nivolumab; nofetumomab; merpentan; obinutuzumab; ocaratumab; ocrelizumab; odulimomab; ofatumumab; olaratumab; olokizumab; omalizumab; onartuzumab; onascept; oportuzumab monatox; oregovomab; otelixizumab; oxelumab; ozoralizumab; pagibaximab; palivizumab; panitumumab; panobacumab; pascolizumab; pateclizumab; patritumab; pegsunercept; pemtumomab; pertuzumab; pexelizumab; pintumomab; placulumab;ponezumab; priliximab; pritumumab; PRO140; quilizumab; racotumomab; radretumab; rafivirumab; ramucirumab; ranibizumab; raxibacumab; regavirumab; reslizumab; RG1507 / HuMax-IGFlR; RG1512 / HuMax-pSelectin; rilonacept; rilotumumab; rituximab; robatumumab; roledumab; romosozumab; rontalizumab; rovelizumab; ruplizumab; samalizumab; sarilumab; satumomab; Satumomab pendetide; secukinumab; sevirumab; sibrotuzumab; sifalimumab; siltuximab; siplizumab; sirukumab; solanezumab; solitomab; Sonepcizumab; sontuzumab; sotatercept; stamulumab; sulesomab; suvizumab; tabalumab; Tacatuzumab tetraxetan; tadocizumab; talizumab; tanezumab; taplitumomab paptox; tefibazumab; telimomabaritox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; Ticilimumab(=tremelimumab); tigatuzumab; TNX-650; tocilizumab(=atlizumab); toralizumab; torapsel; tositumomab; tralokinumab; trastuzumab; trastuzumab emtansine; TRBS07; trebananib; tregalizumab; tremelimumab; tucotuzumab celmoleukin; tuvirumab; ublituximab; urelumab; urtoxazumab; ustekinumab; vapaliximab; vatelizumab; vedolizumab; veltuzumab; vepalimomab; vesencumab; visilizumab;volociximab; vorsetuzumabmafodotin; votumumab; zanolimumab; ziralimumab; and zolimomab aritox.

[0258] In one embodiment of the methods and uses of the present invention, mutations at at least one amino acid residue 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, or at one or more amino acid residues, with the proviso that the mutation at S440 is S440Y or S440W, may be at any of the exemplary and preferred amino acid positions listed in Table 1. Each amino acid position listed in Table 1 is therefore a separate, non-limiting embodiment of a mutation at at least one amino acid.

[0259] Any mutation described herein or a combination thereof may be introduced according to the methods of the present invention.

[0260] Mutations selected from exemplary or preferred amino acid substitutions can be tested in appropriate assays that allow oligomer formation of antigen-bound antibodies and detect enhanced C1q binding, complement activation, CDC, ADCC and / or internalization, such as those described in the Examples. For example, C1q binding affinity can be determined according to an assay similar to the assay described in Example 4 using cells expressing the antigen of the antibody variant. Exemplary exemplary CDC assays are provided in Examples 5, 6, 10, 16, 19, 22, 23, 24 or 25. Exemplary exemplary ADCC assays are provided in Example 12. Exemplary exemplary internalization assays are provided in Example 26. Finally, to distinguish mutations in amino acid residues directly involved in C1q binding from mutations that affect oligomer formation, C1q binding in an ELISA assay according to, for example, Example 3 can be compared with C1q binding in a cell-based assay according to, for example, Example 4.

[0261] In a further embodiment, the mutation is selected from those corresponding to E345, E430, S440 and Q386 in the Fc region of a human IgGl heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.

[0262] In alternative embodiments, the mutation at the at least one amino acid residue, or one or more amino acid residues, is at amino acid residues corresponding to E382 and H433 in the Fc region of a human IgGl heavy chain.

[0263] In a specific embodiment, one mutation is at the amino acid residue corresponding to E345 in the Fc region of a human IgGl heavy chain.

[0264] In a specific embodiment, one mutation is at the amino acid residue corresponding to E430 in the Fc region of a human IgG1 heavy chain.

[0265] In a specific embodiment, one mutation is at the amino acid residue corresponding to S440 in the Fc region of a human IgGl heavy chain, with the proviso that the mutation at S440 is S440Y or S440W.

[0266] In a specific embodiment, one mutation is at the amino acid residue corresponding to Q386 in the Fc region of a human IgG1 heavy chain.

[0267] In alternative embodiments, one mutation is at an amino acid residue corresponding to E382 or H433 in the Fc region of a human IgG1 heavy chain.

[0268] In one embodiment, the mutation at at least one amino acid residue may be an amino acid substitution, an amino acid deletion or an amino acid insertion.

[0269] In one embodiment, the mutation at at least one amino acid residue may be an amino acid deletion.

[0270] In one embodiment, the mutation at at least one amino acid residue may be an amino acid insertion.

[0271] In one embodiment, the mutation at at least one amino acid residue may be an amino acid substitution.

[0272] In one embodiment, the mutation at at least one amino acid residue can be selected from any amino acid substitution, amino acid deletion listed in Table 1. In addition, each preferred amino acid substitution at each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment of this use. Exemplary exemplary amino acid substitutions include exchanging an E residue for an R residue, and exchanging an H residue for an R residue.

[0273] In a further embodiment, the mutation at 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, wherein X refers to any amino acid, such as a natural amino acid or a non-naturally occurring amino acid. X can specifically refer to any of the 20 naturally occurring amino acids.

[0274] 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 wherein 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.

[0275] Thus, in one embodiment, E345X can be E345R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V, W or Y; specifically E345A, D, G, H, K, N, Q, R, S, T, Y or W, or more specifically E345D, K, N, Q, R or W; or even more specifically E345R, Q, N, K or Y. In another further embodiment, E430X can be E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W or Y; specifically E430T, S, G, F or H. In a preferred embodiment, the amino acid substitution is selected from the group comprising E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y. In a further embodiment, the mutation at at least one amino acid residue is selected from E345R and E430G. In a further embodiment, the mutation at at least one amino acid residue is E345R. In a further embodiment, the mutation at at least one amino acid residue is E430G.

[0276] In an alternative embodiment, the mutation at at least one amino acid residue is selected from those corresponding to I253, H310, Q311, E382, G385, H433, N434, Y436, Q438 in the Fc region of a human IgG1 heavy chain, such as E382 and H433. In a further alternative embodiment, the mutation at at least one amino acid residue may be 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 those amino acid substitutions such as Q438 N, S or T.

[0277] Thus, in a further alternative embodiment, the mutation at 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, for example specifically E382L, V, D, Q, K or R or H433R. In a further alternative embodiment, the mutation at 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.

[0278] In another embodiment, optionally, 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, the mutation is not in an amino acid residue directly involved in C1q binding.

[0279] In one embodiment, the mutation is not at the amino acid residue corresponding to 1253, N434 or Q311, optionally not at the amino acid residue corresponding to H433, or the amino acid substitution is not H433A.

[0280] In one embodiment, the at least one mutation is one mutation, ie no more than one mutation is introduced into the parent antibody.

[0281] In another embodiment, the method or use according to the invention comprises introducing mutations at at least two, such as two, three, four, five or more amino acid residues of Table 1.

[0282] Any combination of mutations described in the present invention may be introduced according to the methods of the present invention.

[0283] In one embodiment, the method or use according to the invention comprises introducing mutations into the parent antibody at at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, 1253, 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.

[0284] In one embodiment, the method or use according to the invention comprises introducing mutations into the parent antibody at 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, with the proviso that the mutation at S440 is S440Y or S440W, such as wherein the mutations at at least two amino acids are selected from the following: E345 to R, Q, N or K, E430 to T, S or G, S440 to Y or W, or Q386 to K.

[0285] In alternative embodiments, 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.

[0286] In one embodiment, the method further comprises introducing an additional or third mutation into the antibody at an amino acid residue corresponding to E345, E430, P247, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436 or K447 in the first and / or second Fc region.

[0287] For example, more than one, such as two, three, four, or five, specifically two or three mutations are introduced into the parent antibody 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. For example, at least one amino acid residue corresponding to E345, E430 and S440 in the Fc region of a human IgG1 heavy chain may be mutated, such as two or all of E345, E430 and S440, optionally in combination with one or more mutations at other amino acids listed in Table 1. At least two mutations may be any amino acid substitution at position E345 in combination with any amino acid substitution at position E430 or S440, or may be any amino acid substitution at position S440 in combination with any amino acid substitution at position E430.

[0288] In a further embodiment, two or three mutations are introduced into the parent antibody at an amino acid residue selected from those corresponding to E345, E430, S440 and Q386 in the Fc region of a human IgGl heavy chain.

[0289] In one embodiment, more than one mutation may specifically be an amino acid substitution.

[0290] Therefore, according to the present invention, the method or use comprises introducing into the antibody at least one, such as 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 a preferred embodiment, the amino acid substitution is selected from the group consisting of E345X, E430X, S440Y / W and Q386K.

[0291] In alternative embodiments, at least two, such as 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 IgGl heavy chain.

[0292] In another alternative embodiment, at least one mutation, optionally two or three mutations, are selected from the group consisting of E345R, E382R and H433R. In another alternative embodiment, at least one amino acid residue, such as two, corresponding to E382 and H433 in the Fc region of a human IgG1 heavy chain may be mutated, optionally in combination with mutations at one or more other amino acids listed in Table 1.

[0293] In certain embodiments of the methods and / or uses of the invention, mutations in 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 a combination thereof. As described above, it has been shown that the introduction of mutations at amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain limits the intermolecular interactions between antibodies to those containing the mutations (Examples 4, 5, 6, 10). Depending on whether K439 and S440 are introduced into the same parent antibody or into the first second parent antibody, these aspects are also referred to as "double mutants" and "mixed mutants" aspects.

[0294] In one embodiment of the invention, the mutation at the amino acid residue corresponding to K439 in the Fc region of a human IgGl heavy chain is an amino acid substitution.

[0295] In one embodiment of the invention, the mutation at the amino acid residue corresponding to S440 in the Fc region of a human IgGl heavy chain is an amino acid substitution.

[0296] In all embodiments of the invention, the 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 a second polypeptide or antibody, may be replaced by mutations in:

[0297] (i) at each of the amino acid residues corresponding to K447 and 448 in the Fc region of a human IgG1 heavy chain, such as 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

[0298] (ii) at amino acid residues each corresponding to K447, 448 and 449 in the Fc region of a human IgG1 heavy chain, such as K447D / E, K448K / 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.

[0299] Combinations of such mutations therefore include any of those described in Tables 2A and 2B.

[0300] In one embodiment of the invention, the mutations at the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain are both amino acid substitutions.

[0301] In one embodiment, the mutation at the amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain is an amino acid substitution to an amino acid selected from E and D.

[0302] In another embodiment, the mutation is K439E.

[0303] In one embodiment, the mutation at the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain is an amino acid substitution to an amino acid selected from the group consisting of K, R and H.

[0304] In another embodiment, the mutation is S440K.

[0305] Thus in a further embodiment, the mutations introduced into the parent antibody at the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain are amino acid substitutions selected from K439E and D, and S440K, R and H.

[0306] Thus in a further embodiment, the mutations introduced into the parent antibody at the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgGl heavy chain are the amino acid substitutions K439E and S440K.

[0307] Certain methods and uses of the invention include first and second parent antibodies.

[0308] Therefore, in a further embodiment, the mutation introduced into the first parent antibody corresponding to the amino acid residue K439 is an amino acid substitution selected from K439E and D, such as K439E, and the mutation introduced into the second parent antibody corresponding to the amino acid residue S440 is an amino acid substitution selected from S440K, R and H, such as S440K. The mutations in the first and second parent antibodies can be introduced in the same reverse manner, that is, it can also be that the mutation corresponding to the amino acid residue S440 is introduced into the first parent polypeptide, and the mutation corresponding to the amino acid residue K439 is introduced into the second parent polypeptide, wherein the mutations can be the preferred amino acid substitutions described above.

[0309] In one embodiment of the method or use according to the invention the effector function is enhanced when the antibody is linked to its antigen.

[0310] In a further embodiment, the effector function is enhanced when the antibody is linked to its antigen, wherein the antigen is on an antigen expressing cell, cell membrane or viral particle. In one embodiment, the IgG1 heavy chain Fc region comprises the sequence of residues 130-330 of SEQ ID NO:1.

[0311] The parent antibody may be any parent antibody described herein. Parent antibodies in this context are also first parent and second parent antibodies.

[0312] In one embodiment the parent antibody is a human IgGl, IgG2, IgG3 or IgG4, IgAl, IgA2, IgD or IgE antibody.

[0313] In one embodiment, the parent antibody is a full length human antibody, such as a full length human IgG1 antibody.

[0314] In one embodiment, the parent antibodies, the first parent antibody and the 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.

[0315] In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO:2.

[0316] In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO:3.

[0317] In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO:4.

[0318] In one embodiment the parent antibody is a bispecific antibody.

[0319] In one embodiment, the parent antibody is any antibody described herein, e.g., an antibody fragment comprising at least part of the Fc region, a monovalent antibody (described by Genmab in WO2007059782); a heavy chain antibody, which consists of only two heavy chains and occurs naturally in, e.g., camels (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), a chain exchange engineering domain (SEED or seed body), which is an asymmetric and bispecific antibody-like molecule (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. Patent 7,612,181); dual domain biface antibody (Unilever; Sanofi Aventis, WO20100226923), diface antibody (ImClone / Eli Lilly), knob-in-hole antibody format (Genentech, WO9850431); DuoBody (Genmab, WO2011 / 131746); electrostatically controlled antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual-targeting domain antibodies (GSK / Domantis), two-in-one antibodies recognizing two targets (Genentech, Novlmmune), cross-linked MAbs (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), and DIG bodies and PIG bodies (Pharmabcine), as well as dual affinity retargeting molecules (Fc-DART or Ig-DART of Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), Zybodies (Zyngenia), methods with common light chains (Crucell / Merus, US7262028) or common heavy chains (κλ bodies of Novlmmune), and fusion proteins comprising polypeptide sequences fused to antibody fragments containing an Fc domain, such as scFv fusions, such as BsAb of ZymoGenetics / BMS, HERCULES of Biogen Idec (US007951918), SCORPIONS of Emergent BioSolutions / Trubion, Ts2Ab (Medlmmune / AZ Dimasi, N., et al. J Mol Biol, 2009.393(3):p.672-92), Novartis' scFv fusion, Changzhou Adam Biotech Inc's scFv fusion (CN 102250246), Roche's TvAb (WO 2012025525, WO2012025530), f-Star's mAb. 2 (WO2008 / 003116) and double scFv-fusions. It should also be understood that the term antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal) such as those generated by the technology developed by Symphogen and Merus (Oligoclonics), antibody-like polypeptides such as chimeric antibodies and humanized antibodies. The antibodies generated can potentially have any isotype.

[0320] Optionally selected from monovalent antibodies, heavy chain antibodies, chain exchange engineering domains (SEED), triomab, dual variable domain immunoglobulin (DVD-Ig), knob-into-hole antibodies, mini antibodies, dual affinity retargeting molecules Fc-DART or Ig-DART); LUZ-Y antibodies, biclonal antibodies, dual targeting (DT)-Ig antibodies, two-in-one antibodies, cross-linked Mab, mAb 2 , CovX body, IgG-like bispecific antibody, Ts2Ab, BsAb, HERCULES antibody, TvAb, ScFv / Fc fusion antibody, SCOPRION, scFv fragment fused to Fc domain and double scFv fragment fused to Fc domain.

[0321] In another embodiment, the antigen is expressed on the surface of a cell.

[0322] In another embodiment, the cell is a human tumor cell.

[0323] In a further embodiment, the antigen is selected from the group consisting of ErbBl (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.

[0324] In another embodiment, the antigen is associated with a cell membrane.

[0325] In another embodiment, the antigen is associated with a virion, optionally wherein the antigen is contained within the protein capsid or lipid envelope of the virion.

[0326] In another embodiment, the antibody is a human antibody, optionally binding at least one antigen selected from CD20 and CD38.

[0327] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally comprising the variable heavy and / or variable light chain regions of at least one of 7D8 and 005.

[0328] In any use according to the disclosed invention, the antibody without any mutation of the invention may be any parent antibody. Thus, the use of the invention provides any variant of such a parent antibody.

[0329] In a further embodiment of the invention, the effector function is an Fc-mediated effector function selected from the group consisting of 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 cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-regulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0330] In specific embodiments, the effector function is C1q binding, complement activation (C1q efficacy), complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, such as Fc-gamma receptor binding, internalization of an Fc-containing polypeptide, or any combination thereof.

[0331] In one embodiment, the effector function is CIq binding.

[0332] In one embodiment, the effector function is complement activation (CIq efficacy).

[0333] In one embodiment, the effector function is complement dependent cytotoxicity (CDC).

[0334] In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0335] In one embodiment, the effector function is Fc receptor binding, including, for example, Fc-gamma receptor binding. In one embodiment, the effector function is internalization of an Fc-containing polypeptide.

[0336] In one embodiment, the effector function is a combination of complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC).

[0337] In another embodiment, the one or more mutations increase more effector functions selected from FcRn binding, ADCC, Fc-gamma receptor binding, protein A binding, protein G binding, ADCP, complement dependent cellular cytotoxicity (CDCC), complement enhanced cytotoxicity, antibody mediated binding of opsonized antibodies to complement receptors, and any combination thereof.

[0338] In another aspect, the present invention relates to a method for increasing the affinity of a parent antibody preparation to C1q, comprising the step of 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.

[0339] As used herein, the term "Clq binding", when used in the context of a variant of a parent antibody or an antibody, includes any mechanism of the first component of 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 (e.g., effector cells). The Clq binding of an antibody can be evaluated by ELISA (e.g., the Clq binding ELISA used in Examples 3 and 4), or the Clq potency can be evaluated by CDC assay (e.g., the CDC assay used in Example 5). In a further embodiment, the Clq binding affinity of an antibody is determined according to the assay described in Example 4.

[0340] In all methods according to the disclosed invention, the antibody without any mutation of the invention can be any parent antibody. Therefore, the method of the invention provides any variant of such a parent antibody.

[0341] The parent antibody, the first parent antibody, the second parent antibody, or a variant thereof obtained by the method and / or use of the present invention can be linked to any target described in the present invention.

[0342] Examples of antigens or targets to which the present invention is directed are: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA72-4; cancer associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; C D72;CD74;CD79a;CD79b;CD80;CD86;CD98;CD137;CD147;CD138;CD168;CD200;CD248;CD254;CD257;CDH3;CEA;CEACAM5;CEACAM6;CEACAM8;Claudin4;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;FOLH 1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; gD glycoprotein of herpes simplex virus; HGF; HIV-1; HIV-1IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus; glycoprotein F; ICAM-1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE linker region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3;ITGA4ITGB7;ITGA5;ITGAL;ITGAV_ITGB3;ITGB2;KDR;L1CAM;Lewis-y;lipid A;lipopolysaccharide LPS domain;LTA;MET;MMP14;MMpl5;MST1R;MSTN;MUC1;MUC4;MUC16;MUC5AC;NCA-90 granulocyte 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]; SLAM7; SLC44A4; SOST; lipoteichoic acid from Staphylococcus epidermidis; 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. ;

[0343] Methods for inducing effector responses

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

[0345] In a further main aspect, the present invention relates to a method of inducing an effector response to a cell, cell membrane or virus particle expressing a target to which a parent polypeptide comprising an immunoglobulin Fc domain and a binding region binds, the method comprising

[0346] (i) providing a parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide which has been mutated according to any one of claims 1 to 24; and

[0347] (ii) contacting the mutant parent polypeptide of step (i) or a preparation of a mutant combination of at least the first parent polypeptide and the second parent polypeptide of step (i) with cells, cell membranes or virus particles expressing the antigen in the presence of human complement or effector cells.

[0348] In one embodiment, any or all of the parent polypeptides, the first parent polypeptide and the second parent polypeptide may be antibodies.

[0349] Thus, in one embodiment, the invention relates to a method of inducing an effector response, such as complement activation, CDC or other effector response, to cells, cell membranes, viral particles or other particles associated with an antigen or antigens using the antibody variants described herein. The invention also relates to a method of inducing an effector response to cells, cell membranes or viral particles expressing an antigen bound by a parent antibody, comprising

[0350] (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

[0351] (ii) contacting the mutated parent antibody of step (i) or a preparation of at least the first parent antibody of step (i) combined with mutations of the second parent antibody with cells, cell membranes or virus particles expressing the antigen in the presence of human complement or effector cells.

[0352] The parent antibody, the first parent antibody and the second parent antibody may each be selected from any parent antibody described herein, in particular any of those antibodies described above with respect to methods of influencing antibody effector function.

[0353] In one embodiment, the antigen is expressed on the surface of a cell.

[0354] In one embodiment, the cell is a human tumor cell.

[0355] In a further embodiment, the antigen is selected from the group consisting of ErbBl (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.

[0356] In another embodiment, the antigen is associated with a cell membrane.

[0357] In another embodiment, the antigen is associated with a virion, optionally wherein the antigen is contained within the protein capsid or lipid envelope of the virion.

[0358] In another embodiment, the antibody is a human antibody, optionally binding at least one antigen selected from CD20 and CD38.

[0359] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally comprising the variable heavy and / or variable light chain regions of at least one of 7D8 and 005.

[0360] In a further embodiment of the invention, the induced effector response is complement dependent cytotoxicity (CDC), an Fc-mediated effector response selected from the group consisting of: 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 cellular cytotoxicity (CDCC), complement enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-regulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0361] In specific embodiments, the effector response is C1q binding, complement activation (C1q efficacy), complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, such as Fc-gamma receptor binding, internalization of an Fc-containing polypeptide, or any combination thereof.

[0362] In one embodiment, the effector response is CIq binding.

[0363] In one embodiment, the effector response is complement activation (CIq efficacy).

[0364] In one embodiment, the effector response is complement dependent cytotoxicity (CDC).

[0365] In one embodiment, the effector response is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0366] In one embodiment, the effector response is Fc receptor binding, eg, comprising Fc-gamma receptor binding.

[0367] In one embodiment, the effector response is internalization of the Fc-containing polypeptide.

[0368] In one embodiment, the effector response is a combination of complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC).

[0369] In another embodiment, the method increases a further effector response selected from the group consisting of: FcRn binding, ADCC, Fc-gamma receptor binding, protein A binding, protein G binding, ADCP, complement dependent cytotoxicity (CDCC), complement enhanced cytotoxicity, antibody mediated binding to complement receptors opsonized antibodies, and any combination thereof.

[0370] In another aspect, the present invention relates to a method for increasing the affinity of a parent antibody preparation for C1q, comprising the step of mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447.

[0371] Examples of antigens or targets against which the present invention is directed are: 5T4; ADAM-10; ADAM-12; ADAM 17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; cancer associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137 ;CD147;CD138;CD168;CD200;CD248;CD254;CD257;CDH3;CEA;CEACAM5;CEACAM6;CEACAM8;Claudin4;CS-1;CSF2RA;CSPG-4;CTLA4;Cripto;DLL4;ED-B;EFNA2;EGFR;endothelin B receptor;ENPP3;EPCAM;ERBB2;ERBB3;FAPα;FcγRI;FCER2;FGFR3;fibrin IIβ chain;FLT1;FOLH 1; 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 gpl20 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgEFc; IGF1R; IGHE linker 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, domain of lipopolysaccharide LPS;LTA;MET;MMP14;MMpl5;MST1R;MSTN;MUC1;MUC4;MUC16;MUC5AC;NCA-90 granulocyte antigen;connexin 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]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipomyramidal acid; T cell receptor α_β; tissue factor (TF); TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and vimentin. ;

[0372] In one embodiment, the cell is a tumor cell or a bacterial cell.

[0373] In another embodiment, the antigen is selected from erbBl (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, Ll-CAM, EpCAM and MRP3.

[0374] In a further embodiment, the antigen is CD20 or CD38.

[0375] In another embodiment, the IgG1 parent antibody is a human IgG1 antibody.

[0376] In another embodiment, the parent antibody is selected from 7D8 and 005.

[0377] In one embodiment, the cell is a human tumor cell.

[0378] In another embodiment, the first and second antigens are respectively selected from erbBl (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.

[0379] In another embodiment, the first and second parent antibodies are fully human, optionally wherein said first and second parent antibodies bind to an antigen selected from CD20 and CD38, respectively.

[0380] In a further embodiment, the first and second parent antibodies are selected from 7D8 and 005, respectively.

[0381] In an even further embodiment, the cell is a bacterial cell.

[0382] In another embodiment, the bacterial cell is selected from the group consisting of Staphylococcus aureus, S. Epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.

[0383] In another embodiment, the first and / or second antigen is lipoteichoic acid (LTA), optionally wherein at least one of said first and second parent antibodies is Paxiumab.

[0384] In another embodiment, the antigen is expressed on a viral particle.

[0385] In another embodiment, the first and second antibodies bind to the same antigen.

[0386] In another embodiment, the first and second antibodies comprise the same VH sequence, VL sequence, or both VH and VL sequences.

[0387] For the purposes of the present invention, the target cell expressing the antigen or otherwise binding to the antigen can be any prokaryotic or eukaryotic cell. Exemplary antigen-expressing cells include, but are not limited to, mammalian cells, especially human cells, such as human cancer cells; and unicellular organisms such as bacteria, protozoa; and unicellular fungi such as yeast cells. Cell membranes that include or otherwise bind to the antigen include partial and / or disrupted cell membranes derived from antigen-expressing cells. Antigens associated with virions or viral particles may be included in the protein coat and / or lipid envelope of the virion, or may be bound to the protein coat and / or lipid envelope of the virion.

[0388] The target cell can be, for example, a human tumor cell. Suitable tumor antigens include any target or antigen 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 anti-CD20 antibody 7D8 disclosed in WO 2004 / 035607, anti-CD38 antibody 005 disclosed in WO 06 / 099875, anti-CD20 antibody 11B8 disclosed in WO 2004 / 035607, anti-CD38 antibody 003 disclosed in WO06 / 099875, and anti-EGFr antibody 2F8 disclosed in WO 02 / 100348. The present invention provides other examples of specific antibodies.

[0389] Alternatively, the target cell can be a bacterial cell, such as, for example, Staphylococcus aureus (S.epidermidis), Staphylococcus epidermidis (S.Epidermidis), Staphylococcus pneumoniae (S.pneumonia), Bacillusanthracis (Bacillusanthracis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Chlamydia (Chlamydia), Escherichia coli (E.coli), Salmonella (Salmonella), Shigella (Shigella), Yersinia (Yersinia), Salmonella typhimurium (S.typhimurium), Neisseria meningitides (Neisseria meningitides) and Mycobacterium tuberculosis (Mycobacterium tuberculosis). Exemplary antigens include lipoteichoic acid (LTA), and exemplary antibodies include Paximab.

[0390] Alternatively, the target can be present on the surface of a virus, fungal cell or other particle, such as, for example, West Nile virus, dengue virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papillomavirus, Epstein-Barr virus, herpes virus, poxvirus, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus, and Histoplasma.

[0391] In one embodiment, the contacting step (ii) occurs in vitro.

[0392] In one embodiment, the contacting step (ii) occurs in vivo.

[0393] In another embodiment, step (ii) comprises administering the variant to the subject.

[0394] In further embodiments, the subject suffers from cancer, bacterial infection, or viral infection. The contacting step (ii) of the above embodiment can occur in vitro or in vivo. In the latter case, step (ii) can further include administering one or more preparations to the subject (optionally a subject suffering from cancer or bacterial infection). More details about therapeutic applications will be provided below.

[0395] The first and second antibodies include antigen binding regions that can bind to the same or different epitopes. Such epitopes can be on the same or different targets.

[0396] In one embodiment, the first and second antibodies bind to different epitopes on different targets. Such targets may be expressed on the same cell or cell type, or may be expressed on different cells or cell types. In such embodiments, the enhancement of effector function is only directed to cells or cell types expressing both targets, thereby reducing the risk of any collateral damage to cells or cell types that are not the cause of the disease to be treated.

[0397] Without being bound by any theory, it is generally believed that enhancement of CDC can be restricted to target cells that simultaneously express two specific targets / antigens, provided that the first and second antibodies bind epitopes found on the same cells, thereby utilizing the combined expression of targets to increase the selectivity of enhanced CDC induction.

[0398] Where the targets are expressed on different cells or cell types, it is believed, without being bound by theory, that administration of the first and second antibodies in either order will promote enhanced CDC and may also enhance other effector functions by "recruiting" a second cell or cell type expressing the second target.

[0399] In embodiments where a combination of first and second antibodies is used, step (ii) may be performed 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.

[0400] In another aspect, the present invention relates to a method for improving the CDC inducing ability of a parent antibody preparation, comprising the step of mutating at least one amino acid in the Fc region of the antibody, wherein the at least one amino acid is selected from E345, E430, S440, P247, I253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447.

[0401] In an alternative aspect, the invention relates to a method of inducing an effector response (optionally a CDC response) against a cell, cell membrane or virus particle expressing an antigen to which an IgG1 parent antibody binds, comprising

[0402] (i) providing an antibody comprising a mutation in at least one amino acid residue selected from the group consisting of E345, E430, S440, P247, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447 in the Fc region of an IgG1 heavy chain; and

[0403] (ii) contacting the antibody preparation with cells, cell membranes or viral particles in the presence of human complement or effector cells.

[0404] In another alternative embodiment, the method further comprises administering a first antibody comprising a first mutation in at least one amino acid residue selected from the group consisting of amino acid residues corresponding to E345, E430, S440, P247, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436, and K447 in the Fc region of the first antibody;

[0405] administering a second antibody comprising a second mutation in at least one amino acid residue selected from the group consisting of amino acid residues corresponding to E345, E430, S440, P247, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436, and K447 in the Fc region of the second antibody;

[0406] 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 found in the same or different cells or cell types.

[0407] In another optional aspect, the present invention relates to a method for improving the CDC inducing ability of an IgG1 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 E345, E382 and H433.

[0408] In another optional aspect, the present invention relates to a method for inducing an effector response (optionally a CDC response) against cells, cell membranes or viral particles expressing an antigen to which an IgG1 parent antibody binds, comprising the following steps:

[0409] (i) providing a variant of a parent antibody, comprising a mutation in at least one amino acid in the Fc region of the antibody, the at least one amino acid being selected from the group consisting of E345, E382 and H433; and

[0410] (ii) contacting the variant preparation with cells in the presence of human complement or effector cells.

[0411] In another optional aspect, the present invention relates to a method of inducing an effector response (optionally a CDC response) against cells expressing an antigen to which an IgG1 parent antibody binds, comprising the following steps:

[0412] (i) providing a variant of a parent antibody comprising K439E and S440K mutations in the Fc region of the antibody; and

[0413] (ii) contacting the variant preparation with cells in the presence of human complement or effector cells.

[0414] In another optional aspect, the present invention relates to a method for inducing a CDC response against a cell, cell membrane or virus particle expressing a first antigen bound by a first IgG1 parent antibody and a second antigen bound by a second parent antibody, comprising the following steps:

[0415] (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

[0416] (ii) contacting the cell simultaneously, separately or sequentially with the first and second variants in the presence of human complement and / or effector cells.

[0417] In another optional aspect, the present invention provides a method for inducing CDC or other effector responses against target cells, cell membranes, viral particles or other particles, wherein the target cells, cell membranes, viral particles or other particles are associated with an antigen bound by an IgG1 or IgG3 antibody, 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.

[0418] In another optional aspect, the present invention provides a method for inducing ADCC or ADCP against a target cell, cell membrane, virus particle or other particle, or phagocytosis of a target cell, cell membrane, virus particle or other particle, wherein the target cell, cell membrane, virus particle or other particle is associated with an antigen bound by an IgG1 or IgG3 antibody, comprising the steps of: (i) providing an antibody variant comprising a mutation in at least one amino acid corresponding to E345, E430 or S440 of the Fc region of an IgG1 antibody; and (ii) contacting the variant preparation with the cell in the presence of effector cells.

[0419] The present invention also provides a method for inducing CDC or other effector responses against target cells, cell membranes, viral particles or other particles associated with an antigen bound to an IgG1 or IgG3 antibody, comprising the steps of: (i) providing an antibody variant comprising a mutation in K439 (K439E) and a mutation in S440 (S440K or S440R) in the Fc region of the antibody; and (ii) contacting the variant preparation with cells in the presence of human complement and / or effector cells.

[0420] The present invention also provides a method for inducing CDC or other effector responses against target cells, cell membranes or virus particles, wherein the target cells, cell membranes or virus particles express a first antigen bound by a first IgG1 antibody and a second antigen bound by a second antibody, comprising the steps of: (i) providing a first variant (which is a first antibody comprising a K439E mutation) and a second variant (which is a second antibody comprising a S440K or S440R mutation); and (ii) contacting the cells with the first and second variant preparations simultaneously, separately or sequentially in the presence of human complement or effector cells.

[0421] In separate and specific embodiments, the first and second antibodies bind (i) different antigens, (ii) different epitopes on the same antigen, (iii) the same epitope on the antigen, and (iv) the same epitope on the antigen and comprise the same VH and / or VL sequence.

[0422] In one embodiment, the first and second antibodies further comprise a mutation in one or more of E345, E430 and S440, such as E345R. In one embodiment, the first and second antibodies further comprise a mutation in one or more of E345, E382 and H433, such as E345R.

[0423] Other methods

[0424] In another main aspect, the invention relates to a method for identifying antibody mutations that enhance the effector function of the antibody binding to C1q, comprising the steps of:

[0425] (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, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447;

[0426] (ii) evaluating the CIq activity of the antibody when bound to the surface of an antigen-expressing cell, compared to the parent antibody; and

[0427] (iii) selecting mutations in any variant that have increased affinity for C1q.

[0428] In one embodiment, at least one antibody comprises at least one amino acid substitution selected from E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.

[0429] In still another main aspect, the present invention relates to a method for identifying a parent antibody mutation that increases the ability of the antibody to induce a CDC response, comprising the steps of:

[0430] (i) preparing at least one parent antibody variant comprising a mutation in at least one amino acid selected from the group consisting of E345, E430, S440, K439, P247, 1253, S254, Q311, D / E356, T359, E382, Q386, Y436 and K447;

[0431] (ii) evaluating the CDC response induced by the variant when bound to the surface of an antigen-expressing cell in the presence of effector cells or complement, as compared to the parent antibody; and

[0432] (iii) Selecting mutations in any variant that increase the C1q response.

[0433] In one embodiment, at least one amino acid is selected from E345, E382 and H433.

[0434] In one embodiment, at least one antibody comprises at least one amino acid substitution selected from E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.

[0435] In another aspect, the present invention relates to a method for increasing the affinity of an IgG1 parent antibody preparation for 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 E345, E382 and H433.

[0436] Antibodies of the present invention

[0437] Parental antibody

[0438] As described herein, the present invention particularly relates to parent antibody variants comprising one or more mutations in the CH2 and / or CH3 regions of immunoglobulins (e.g., in antibody heavy chains). The "parent" antibody used as the starting material of the present invention before modification may be a wild-type antibody, which may be produced, for example, by the hybridoma method first described by Kohler et al., Nature 256,495 (1975), or by a recombinant DNA method. Using the technology described in, for example, Clackson et al., Nature 352,624 628 (1991) and Marks et al., J.Mol.Biol.222,581 597 (1991), monoclonal antibodies may also be isolated from phage antibody libraries. Monoclonal antibodies may be obtained from any suitable source. Therefore, for example, monoclonal antibodies may be obtained from hybridomas prepared from mouse spleen B cells, which are obtained from mice immunized with an antigen of interest, wherein the antigen of interest is, for example, in the following form: cells expressing antigens on the surface, or nucleic acids encoding antigens of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals (eg, rabbits, rats, dogs, primates, etc.).

[0439] The parent antibody can be, for example, a chimeric or humanized antibody. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies can be produced using transgenic or transchromosomal mice (e.g., HuMAb mice, carrying part of the human immune system rather than the mouse system). HuMAb mice contain human immunoglobulin gene minilocus encoding unrearranged human heavy chains (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations (Lonberg, N. et al., Nature 368, 856 859 (1994)) that inactivate endogenous μ and κ chain loci. Thus, the mice display reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536 546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187. According to known techniques, spleen cells from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies.

[0440] In addition, the human antibodies of the present invention or antibodies of the present invention from other species can be identified by display technology, including, but not limited to, phage display, retroviral display, ribosome display, mammalian display, yeast display and other technologies known in the art, and the obtained molecules can be subjected to additional maturation, such as affinity maturation, because such technologies are well known in the art. The specific strategy described in Example 17 is applicable to any antibody to use phage display to prepare and obtain variants of the present invention.

[0441] The parent antibody is not limited to antibodies with natural, for example, human Fc domains, but it can also be an antibody with other mutations other than those of the present invention, such as, for example, mutations that affect glycosylation or make the antibody a bispecific antibody. The term "natural antibody" means any antibody that does not include any genetically introduced mutations. Antibodies comprising naturally occurring modifications (e.g., different allotypes) are therefore understood as "natural antibodies" for the present invention, and can thus be understood as parent antibodies. Such antibodies can be used as templates for one or more mutations according to the present invention, thereby providing variant antibodies of the present invention. An example of a parent antibody comprising other mutations other than those of the present invention is a bispecific antibody described in WO2011 / 131746 (Genmab), utilizing reducing conditions to promote half-molecule replacement of two antibodies comprising an IgG4-like CH3 region, 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: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); knob-into-hole (Genentech); electrostatic steering (Amgen, Chugai, Oncomed); SEED body (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentch). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or antibody fragments containing Fc, human antibodies, or any combination thereof.

[0442] The parent antibody may bind to any target. Examples of such targets or antigens of the present invention may be, but are not limited to, those directed against: 5T4; ADAM-10; ADAM-12; ADAM 17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA72-4; cancer associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248 ;CD254;CD257;CDH3;CEA;CEACAM5;CEACAM6;CEACAM8;Claudin4;CS-1;CSF2RA;CSPG-4;CTLA4;Cripto;DLL4;ED-B;EFNA2;EGFR;endothelin B receptor;ENPP3;EPCAM;ERBB2;ERBB3;FAPα;FcγRI;FCER2;FGFR3;fibrin IIβ 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 (Candida albicans albicans)]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gpl20 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE linker; IL12B; IL13; IL15; IL17A; ILIA; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3;ITGA4ITGB7;ITGA5;ITGAL;ITGAV_ITGB3;ITGB2;KDR;L1CAM;Lewis-y;lipid A, domain of lipopolysaccharide LPS;LTA;MET;MMP14;MMpl5;MST1R;MSTN;MUC1;MUC4;MUC16;MUC5AC;NCA-90 granulocyte 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]; 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.

[0443] The parent antibody can be any human antibody of any isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE and IgD, optionally a full-length human antibody, such as a full-length human IgG1 antibody. The parent antibody can include a sequence according to any one of SEQ ID NOs: 1, 2, 3, 4 and 5.

[0444] Monoclonal antibodies used in the present invention, such as parents and / or variants, can be produced by the hybridoma method first described by Kohler et al., Nature 256,495 (1975), or by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol.Biol.222:581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Therefore, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells, which are obtained from mice immunized with an antigen of interest (e.g., in the form of cells expressing antigens on the surface, or nucleic acids encoding antigens of interest). Monoclonal antibodies can also be obtained from hybridomas of antibody-expressing cells derived from immunized humans or non-human mammals (e.g., rats, dogs, primates, etc.).

[0445] In one embodiment, the antibody is a human antibody. Transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system can be used to generate human monoclonal antibodies against any antigen. Such transgenic and transchromosomal mice are respectively included in the present invention as Mice of the KM mice and KM mice are collectively referred to as "transgenic mice" in the present invention.

[0446] The mouse contains a human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ immunoglobulin light chain sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368 , 856-859 (1994)). As a result, the mice show a reduction in mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536-546 (1995)). The preparation of mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187.

[0447] HCo7, Hco12, Hco17 and HCo20 mice have a JKD disruption in their endogenous light chain (κ) gene (described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (described in Example 1 of WO 01 / 14424) and a KCo5 human κ light chain transgene (described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). In addition, Hco7 mice have the HCo7 human heavy chain transgene (described in US 5,770,429), HCo12 mice have the HCo12 human heavy chain transgene (described in Example 2 of WO 01 / 14424), HCo17 mice have the HCo17 human heavy chain transgene (described in Example 2 of WO 01 / 09187), and HCo20 mice have the HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.

[0448] In the KM mouse strain, the endogenous mouse κ light chain gene has been homozygously disrupted as described by Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01 / 09187. This mouse strain carries the human κ light chain transgene KCo5 as described by Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome consisting of a chromosome 14 fragment hCF (SC20) as described by WO 02 / 4347814. HCo12-Balb / C mice were generated by hybridizing HCo12 with KCo5 [J / K] (Balb) as described in WO / 2009 / 097006. According to known techniques, splenocytes from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies.

[0449] In addition, any antigen binding region can be obtained from human antibodies or antibodies of other species by display technology (including but not limited to phage display, retroviral display, ribosome display and other technologies) using techniques well known in the art, and the obtained molecules can undergo additional maturation, such as affinity maturation. Such technologies are well known in the art (see, for example, 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., PNAS USA 87, 6378-6382 (1990), Russel 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). If display technology is used to generate non-human antibodies, such antibodies can be humanized.

[0450] In another aspect, the invention relates to a parent polypeptide comprising an Fc domain and a binding region.In the context of the present invention it is to be understood that all embodiments referring to a parent antibody apply analogously to a "parent polypeptide".

[0451] The mutation according to the present invention may be, but is not limited to, the deletion, insertion or substitution of one or more amino acids. Such substitution of amino acids may be substitution with any naturally occurring or non-natural amino acid.

[0452] “ Single mutant

[0453] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0454] The antibody or polypeptide variant according to the "single mutant" aspect of the invention comprises a mutation, typically an amino acid substitution, in at least one amino acid residue as shown in Table 1, which lists each amino acid residue numbered according to the EU index of a human IgG1 antibody, together with the amino acids at the corresponding positions of the IgG2, IgG3 and IgG4 parent antibodies, and "exemplary" and "preferred" amino acid substitutions. Figure 2 Shown are the IgG2 segment corresponding to residues P247 to K447, the IgG3 Fc segment corresponding to residues P247 to K447, and the IgG4 segment corresponding to residues P247 to K447 in IgG1.

[0455] Table 1 - Example mutation sites and amino acid substitutions in the "single mutant" aspect

[0456]

[0457] As can be seen in Table 1, amino acid substitutions that result in increased cell lysis of Wien 133 cells of Example 19 are included as "preferred substitutions".

[0458] In one aspect, the present invention relates to a variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from the group consisting of residues corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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.

[0459] In one embodiment, the variant polypeptide may be a variant antibody.

[0460] Therefore, in another aspect, the present invention relates to a variant of an antibody comprising an antigen binding region and an Fc domain of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from the group corresponding to the following amino acid residues: 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 of S440 is S440Y or S440W. Optionally, the amino acid residue is selected from the group corresponding to the following amino acid residues: H310, G385, H433, N434, Q438 and K439 in the Fc region of a human IgG1 heavy chain.

[0461] Each of the amino acid residues corresponding to E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain can be grouped according to the following as described above:

[0462] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0463] (b) amino acid residues within the hydrophobic knobs of the CH2-CH3 region,

[0464] (c) amino acid residues within the N-terminal CH3 helix,

[0465] (d) an amino acid residue within the C-terminal CH3 beta 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

[0466] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0467] Mutations according to 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 substitutions using any naturally occurring or non-natural amino acids. Thus, in one embodiment, the mutation of at least one amino acid residue is a deletion. In another embodiment, the mutation of at least one amino acid residue is an insertion. In another embodiment, the mutation of at least one amino acid residue is a substitution.

[0468] In one embodiment, the mutation of at least one amino acid residue is selected from the group consisting of amino acid residues 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, wherein X is any amino acid, e.g., a naturally occurring amino acid.

[0469] In a specific embodiment, the antibody variant comprises a mutation in at least one amino acid residue selected from the group consisting of E345, E430, S440 and Q386 in the Fc region of a human IgG1 heavy chain.

[0470] In a further embodiment, the mutation of at least one amino acid residue is selected from the amino acid substitutions corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid.

[0471] In a preferred embodiment, the mutation of at least one amino acid residue is selected from the group consisting of amino acid substitutions 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 IgGl heavy chain.

[0472] In a further preferred embodiment, the mutation of at least one amino acid residue is selected from the group consisting of amino acid substitutions corresponding to E345R / Q / N / K, E430T / S / G, S440Y / W and Q386K in the Fc region of a human IgG1 heavy chain.

[0473] Optionally, at least one amino acid residue is selected from E382 and H433. Specific optional amino acid substitutions include E345Y, D, W; and E430F, H. Optionally, E382D, Q, K, R; and H433R.

[0474] In a specific embodiment, the amino acid substitution is E345R. In alternative embodiments, the mutation is selected from 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; Y436 to A, E, F, H, I, K, L, M, N, Q, R, S, T or V; Q438 to A, E, G, H, K, N, Q, R, S, T, W or Y; K439 to D, H, Q, R, W or Y; and S440 to D, E, H, F, N, Q, W or Y.

[0475] In another alternative embodiment, the mutation is selected from I253 to N or Q; H310 to Q; Q311 to E or R; E382 to D, Q, K, or R; G385 to D, E, K or R; H433 to R; N434 to H, K, Q or R; Y436 to N, Q, S or T; Q438 to N, S or T; K439 to Q; and S440 to D, E or Q.

[0476] In another alternative embodiment, the mutation is selected from E382 to D, Q, K or R; and H433 to R.

[0477] In one embodiment, the variant comprises an E382R mutation.

[0478] In one embodiment, the variant comprises an H433R mutation.

[0479] As shown in the examples, variants of CD38 antibodies HuMab-005 and -003 and / or CD20 antibodies HuMab-7D8 and -11B8 and rituximab and / or EGFR antibody HuMab-2F8 comprising one of the above amino acid substitutions have higher C1q binding, complement activation and / or CDC than wild-type HuMab 005 and 7D8, respectively. It should be understood that the variant may also include only one mutation in the "exemplary substitutions" listed in Table 1. The variant may include more than one mutation, such as two, three, four, five or six of any mutation listed in Table 1.

[0480] Therefore, a preferred embodiment of the present invention provides a variant comprising a mutation in an amino acid residue selected from the group consisting of those listed in the above aspects. The specific amino acid mutation may be an amino acid substitution corresponding to any of the following amino acid substitutions: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345 Y, D / E356G, D / E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W. As shown in Example 19, Table 17, these had increased cell lysis (>39% for Wien 133 cells).

[0481] In alternative embodiments, the variant comprises a mutation in one amino acid residue selected from the group consisting of amino acid residues corresponding to E382R, H433R, H435R and H435A.

[0482] In addition to the specified mutations, the variant may have any of the characteristics described for the parent antibody. Specifically, it may be a human antibody. In addition to the mutations, the variant may further be of any IgG1 subtype.

[0483] When bound to its antigen on the surface of an antigen-expressing cell, a cell membrane, a virion or another particle, or when the antigen is associated with a virion, optionally wherein the antigen is included in the protein capsid or lipid envelope of the virion, such antibody variants may have an increase in at least one of the following 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) to (v). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, the variant may also or alternatively have retained or improved other effector functions, such as 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 cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-regulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0484] Without being limited to any particular theory, the effect caused by replacing an amino acid at a specified position with an amino acid residue of Table 1 can be, for example, to cause its own effect, to participate in a direct contact with the Fc domain of another molecule, or to be mutated to directly interact with another Fc domain or to indirectly affect an intermolecular Fc: Fc interaction. Therefore, without being limited to theory, substitutions are believed to directly or indirectly enhance the binding strength between antibody molecules in oligomeric form, enhance the stability of the oligomeric structure (e.g., hexamer, pentamer, tetramer, trimer or dimer structure). For example, amino acid substitutions can be amino acid substitutions that promote or enhance the formation of new intermolecular Fc: Fc bonds (e.g., but not limited to, van der Waals interactions, hydrogen bonds, charge-charge interactions), or amino acid substitutions that promote entropy increase by releasing water molecules under Fc: Fc interactions. With respect to Table 1, "exemplary substitutions" can be selected based on the size and physicochemical properties of participating in or promoting intermolecular Fc: Fc interactions or intramolecular interactions (isomeric mutations). "Exemplary substitutions" can be chosen based on size and physicochemical properties that are best suited to participate in or stimulate intermolecular Fc:Fc interactions or intramolecular interactions (isomeric mutations).

[0485] "Exemplary substitutions" of the amino acids listed in Table 1 include replacement of R residues with E residues, and replacement of R residues with H residues. Each "exemplary substitution" of amino acids in each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment of the present invention. In addition, each "preferred substitution" of amino acids in each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment of the present invention.

[0486] In another aspect, the present invention relates to a variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant comprises a mutation in at least two amino acid residues selected from the group consisting of:

[0487] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0488] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0489] (c) amino acid residues within the N-terminal CH3 helix,

[0490] (d) an amino acid residue within the C-terminal CH3 beta 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

[0491] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0492] At least two of the amino acid mutations are different.

[0493] In one embodiment, the parent polypeptide and variants thereof may be antibodies.

[0494] Therefore, the present invention also relates to a variant of a parent antibody, comprising an antigen binding region and an Fc domain, wherein the variant comprises a mutation in at least two amino acid residues selected from the group consisting of:

[0495] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0496] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0497] (c) amino acid residues within the N-terminal CH3 helix,

[0498] (d) an amino acid residue within the C-terminal CH3 beta 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

[0499] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0500] At least two of the amino acid mutations are different.

[0501] Thus, variants of the above embodiments may include mutations in at least two, such as two, three, four, five or more amino acids in Table 1.

[0502] In any embodiment where the variant comprises at least two such mutations of an amino acid, it may be present in each heavy chain of the variant, or one of the two may be included in one heavy chain and the other in the respective other heavy chain, or vice versa.

[0503] In one embodiment, the variant comprises mutations in at least two amino acid residues selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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 of S440 is S440Y or S440W. Optionally, the variant further comprises mutations in at least one residue selected from the group consisting of: H310, G385, H433, N434, Q438 and K439.

[0504] In one embodiment, the variant comprises mutations in at least two amino acid residues selected from the group corresponding to the following amino acid residues: 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, e.g., a naturally occurring amino acid.

[0505] For example, the antibody variant may include at least one mutation of E345, E430, S440 and Q386, optionally E382 and H433, such as two or all of E345, E430, S440 and Q386, optionally E382 and H433, optionally further including mutations of one or more other amino acids listed in Table 1. Thus, in a further embodiment, the variant includes mutations of at least two amino acid residues selected from the group corresponding to the following: 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.

[0506] Exemplary combinations of mutations of at least two amino acid residues are E345X / E430X, E345X / S440Y or W, E345X / Q386K, E430X / S440Y or W and E430X / Q386K.

[0507] In one embodiment, the mutation of at least two amino acid residues is a deletion, an insertion or a substitution. Such substitution of amino acids can be a substitution with any naturally occurring or artificial amino acid.

[0508] In a specific embodiment, the mutation of at least two amino acid residues can be an amino acid substitution corresponding to any of the following: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D / E356G, D / E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E 430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.

[0509] In a preferred embodiment, the variant comprises mutations in at least two amino acid residues selected from the group consisting of amino acid substitutions 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.

[0510] Optional further mutations are selected from mutations corresponding to: I253E,N,Q,S,T; H310N,Q,W,Y; Q311E,R; E382D,H,K,R,N,Q,S,T,W,Y; G385E,H,K,N,Q,R,S,T,W,Y; H433R; N434D,E,H,K,Q,R,S,T,W,Y; Y436,A,E,F,H,I,K,L,M,N,Q,R,S,T,V; Q438A,E,G,H,K,N,Q,R,S,T,W,Y; K439D,H,Q,R,W,Y; and S440D,E,H,F,N,Q.

[0511] In a preferred embodiment, the mutation of at least two amino acid residues is selected from the amino acid substitutions corresponding to the following amino acid substitutions: E345R / Q / N / K, E430T / S / G, S440Y / W and Q386K in the Fc region of a human IgG1 heavy chain. Optional further mutations are selected from the mutations corresponding to the following: I253N,Q; H310Q; Q311E,R; E382D,Q,K,R; G385D,E,K,R; H433R; N434H,K,Q,R; Y436N,Q,S,T; Q438N,S,T; K439Q; and S440D,E,Q.

[0512] Exemplary specific combinations of at least two amino acid residue mutations are E345R / E430T, E345R / S440Y, E345R / S440W, E345R / Q386K, E345R / E430G, E345Q / E430T, E345Q / S440Y, E345Q / S440W, E430T / S440Y, E430T / S440W, E430T / Q386K, and S440Y / Q386K.

[0513] In a specific embodiment, the mutation is not at the amino acid residue corresponding to I253, N434 or Q311. In additional or alternative embodiments, the mutation is not at H433, or the amino acid substitution is not H433A.

[0514] In one embodiment, the present invention relates to a variant comprising mutations in at least three amino acid residues selected from the group consisting of:

[0515] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0516] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0517] (c) amino acid residues within the N-terminal CH3 helix,

[0518] (d) an amino acid residue within the C-terminal CH3 beta strand, provided that it does not include a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, and

[0519] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0520] At least three of the amino acid mutations are different.

[0521] In a specific embodiment, the variant comprises mutations in amino acid residues corresponding to amino acid substitutions E345R, Q396K and E430G, which may be located in one or both heavy chains of the variant.

[0522] The mutations of at least three amino acid residues may be individually selected from the substitutions listed in Table 1. Non-limiting examples of variants comprising at least three mutations are: E345R / E430G / S440Y, E345R / E430G / S440W, E345K / E430G / S440Y, E345K / E430G / S440W, E345Q / E430G / S440Y, E345Q / E430G / S440W, E345N / E430G / S440Y, E345 5N / E430G / S440W,E345R / E430T / S440Y,E345R / E430T / S440W,E345K / E430T / S440Y,E345K / E430T / S440W,E345Q / E430T / S440Y,E345Q / E430T / S440W,E345N / E430T / S440Y,E345N / E4 30T / S440W,E345R / E430S / S440Y,E345R / E430S / S440W,E345K / E430S / S440Y,E345K / E430 S / S440W,E345Q / E430S / S440Y,E345Q / E430S / S440W,E345N / E430S / S440Y,E345N / E430S / S 440W, E345R / E430F / S440Y, E345R / E430F / S440W, E345K / E430F / S440Y, E345K / E430F / S440W, E345Q / E430F / S440Y, E345Q / E430F / S440W, E345N / E430F / S440Y, and E345N / E430F / S440W.

[0523] In addition to mutations in one or more amino acids in Tables 1 or 2A and B, the IgG heavy chain may include additional mutations known in the art, e.g., mutations that further improve effector function. Such additional mutations include known mutations that enhance CDC, Fc-receptor binding or FcRn binding and / or improve Fc-γ receptor-mediated effector function.

[0524] In one embodiment, the variants of the invention further comprise known CDC enhancing modifications, e.g., segment replacement between IgG isotypes to generate 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 in or near the C1q binding site of the CH2 domain 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 invention further comprise a combination of any one of the amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E, providing enhanced effector effects by CDC or ADCC (Moore et al., 2010 mAbs 2(2), 181-189). Other Fc mutations that affect binding to Fc receptors (described in WO 2006 / 105062, WO 00 / 42072, U.S. Pat. No. 6,737,056 and U.S. Pat. No. 7,083,784) or antibody physical properties (described in WO 2007 / 005612 A1) can also be used in the variants of the invention.

[0525] In one embodiment, the variants of the invention also include modifications that enhance Fc-γ receptor binding and / or Fc-γ receptor-mediated effector functions. Such modifications include (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 antibody hinge or CH2 region (protein engineering) (Lazar GA, et al., Proc Natl Acad Sci USA 2006; 103: 4005-10).

[0526] In one embodiment, the variants of the invention are further engineered at the FcRn binding site, for example, to extend the half-life (t1 / 2) of IgG antibodies. 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 to alanine residues to improve FcRn binding (Shields RL, et al. J. Biol. Chem. 2001; 276: 6591); and (iii) amino acid substitutions or combinations of amino acid substitutions of IgG1, the combination selected from: M252Y / S254T / T256E, M252W, M252Y, M252Y / T256Q, M252F / T256D, V308T / L309P / Q311S, G385D / Q386P / N389S, G385R / Q386T / P387R / N389P, H433K / N434F / Y436H, N434F / Y436H, H433R / N434Y / Y436H, M252Y / S254T / T256E-H433K / N434F / Y436H or M252Y / S254T / T256E-G385R / Q386T / P387R / N389P, increase affinity for FcRn (Dall'Acqua et al., supra).

[0527] “ Double mutants

[0528] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0529] As described above and below, the present invention also relates to a "double mutant" aspect, wherein the two mutations individually reduce the effector function but together restore the effector function to the level of the parent antibody. When used together, the specificity of the variant is increased. The antibody variant according to the "double mutant" aspect comprises two mutations, typically amino acid substitutions of the specific amino acid residue interaction pair K439 and S440.

[0530] Thus, in one aspect, the present invention relates to a variant of a polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant comprises a mutation at the following position:

[0531] (i) at least one amino acid residue selected from the group consisting of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, for example wherein the mutation in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E, and / or the mutation in the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / H / R;

[0532] (ii) at least one amino acid residue corresponding to K447D / E or to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or

[0533] (iii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H, 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

[0534] In one embodiment, the parent polypeptide and variants thereof may be antibodies.

[0535] Therefore, in one aspect, the present invention relates to a variant of an antibody comprising an antigen binding region and an Fc domain of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation of S440 is not S440Y or S440W, for example, wherein the mutation corresponding to the position K439 in the Fc region of a human IgG1 heavy chain is K439D / E, and / or the mutation corresponding to the position K440 in the Fc region of a human IgG1 heavy chain is S440K / H / R. Tables 2A and B show "exemplary" and "preferred substitutions" in terms of "double mutants" (Table A) and "mixed mutants" (Table 2B).

[0536] Table 2A Example mutation sites and amino acid substitutions in the “double mutant” aspect

[0537]

[0538] Table 2B: Example mutation sites and amino acid substitutions in the “mixed mutant” aspect (Antibody 1 + Antibody 2)

[0539]

[0540] In one variant embodiment, wherein the mutation is at a position other than S440 and K447 and wherein the variant further comprises a mutation at the following position:

[0541] (i) 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;

[0542] (ii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or

[0543] (iii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

[0544] Therefore, the present invention provides an antibody variant comprising a first mutation of a residue in the human IgG1 heavy chain CH2 and / or CH3 region corresponding to K439, and a second mutation of a residue in the human IgG1 heavy chain CH2 and / or CH3 region corresponding to S440.

[0545] The invention contemplates that variants may also comprise only one amino acid residue substitution, such as K439E or S440K, for example, the variant comprises a mutation at K439, and optionally no mutation at S440.

[0546] In one embodiment, the invention relates to variants, wherein the mutation of K439 is an amino acid substitution to an amino acid selected from E and D, such as K439E.

[0547] In another embodiment, the variant comprises a mutation in S440, optionally without a mutation in K439.

[0548] In one embodiment, the invention relates to variants, wherein the mutation of S440 is an amino acid substitution to an amino acid selected from E, R and H, such as S440K.

[0549] In one embodiment, the variant comprises mutations at both K439 and S440.

[0550] In another embodiment, the mutation of K439 is selected from K439 to D, E or R and the mutation of S440 is selected from S440 to D, E, K, H and R.

[0551] In another embodiment, the mutation of K439 is selected from K439D and K439E, and the mutation of S440 is selected from S440K, S440R and S440H.

[0552] In another embodiment, the variant comprises K439E and S440K mutations.

[0553] As described in Examples 4-6, antibody variants comprising only one of the K439E and S440K mutations had a K of 1.5 fold increase in C1q. DThe drastic increase reflects reduced complement activation and / or CDC capacity. Surprisingly, antibody variants of HuMAb 7D8 or 005 comprising two mutations were found to have restored or increased C1q binding or CDC. Without being bound by any particular theory, the underlying mechanism may be explained by the corresponding mutations spatially compensating for each other, such as Figure 4 and 5 shown.

[0554] Any "double mutant" described herein can also be used in combination with mutations that themselves can increase effector function. Thus, the "double mutant" aspect can be combined with the "single mutant" aspect, for example, the variant can further include a mutation at any amino acid position listed in Table 1 or any other embodiment described for the "single mutant" aspect above. Thus, in one embodiment, the mutation is located at a position other than S440, and wherein the variant further includes a mutation of 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 of S440 is not S440W or S440Y.

[0555] In one aspect, the present invention relates to a variant of a polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from the group consisting of:

[0556] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0557] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0558] (c) amino acid residues within the N-terminal CH3 helix,

[0559] (d) an amino acid residue within the C-terminal CH3 beta strand, provided that it does not include a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, and

[0560] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0561] wherein the variant comprises a further mutation

[0562] (i) 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;

[0563] (ii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or

[0564] (iii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

[0565] In one embodiment, the parent polypeptide and variants thereof may be antibodies.

[0566] Therefore, in one aspect, the present invention relates to a variant of a parent antibody, which comprises an antigen binding region of an immunoglobulin and an Fc domain, wherein the variant comprises a mutation of at least one amino acid residue selected from the group consisting of:

[0567] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0568] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0569] (c) amino acid residues within the N-terminal CH3 helix,

[0570] (d) an amino acid residue within the C-terminal CH3 beta strand, provided that it does not include a mutation corresponding to S440 in the Fc region of a human IgG1 heavy chain, and

[0571] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0572] Wherein the variant comprises a further mutation at the following position

[0573] (i) 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;

[0574] (ii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or

[0575] (iii) at least one amino acid residue corresponding to K447D / E or corresponding to K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

[0576] In one embodiment, the variant comprises a mutation in at least one amino acid residue selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgGl 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 IgGl heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.

[0577] In a further embodiment, the variant comprises a mutation of at least one amino acid residue selected from the group consisting of amino acid substitutions corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, e.g., a naturally occurring amino acid, and the variant comprises a further mutation of 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 of S440 is not S440W or S440Y.

[0578] In one embodiment, the variant comprises amino acid mutations at two positions corresponding to K439 and S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.

[0579] In a further embodiment, 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.

[0580] In a further embodiment, the first mutation is at an amino acid residue selected from E345, E430, Q386 and S440 corresponding to the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W; and the second and third mutations are amino acid substitutions at positions K439E or S440K.

[0581] In one embodiment, the first mutation is a deletion, insertion or substitution. Such substitution can be any naturally occurring or non-natural amino acid.

[0582] In a further embodiment, the first mutation is selected from 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 position K439E or S440K.

[0583] In preferred embodiments, one mutation is selected from E345R,Q,N,K,Y; E430T,S,G,F,H; S440W,Y; and Q386K.

[0584] As another example, in one embodiment of the present invention, the variant comprises E345R, K439E and S440K mutations, thereby providing an increased and more specifically mediated CDC response.

[0585] In one embodiment, the variant comprises a mutation in at least two amino acid residues selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgGl 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 IgGl heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.

[0586] In a further embodiment, the variant comprises a mutation of at least two amino acid residues selected from the group consisting of amino acid substitutions corresponding to E345X, E430X, S440W / Y, Q386K in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, e.g., a naturally occurring amino acid, and the variant comprises a further mutation of at least one human 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.

[0587] In one embodiment, the variant comprises amino acid mutations at two positions corresponding to K439 and S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.

[0588] In a further embodiment, 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.

[0589] In a further embodiment, the first and second mutations are in amino acid residues selected from the group consisting of amino acid residues corresponding to E345, E430, Q386 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W; and the third and fourth mutations are amino acid substitutions at positions K439E or S440K.

[0590] In another embodiment, the variant comprising mutations at positions K439 and S440 as described herein has an increase in Fc-mediated effector function selected from the group consisting of 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 cellular 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 the parent antibody or the antibody variant comprising only one of mutations at positions K439 and S440.

[0591] The present invention also provides for use of K439E and S440K mutations in an antibody to restore one or more of the following effects 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): (i) C1q binding affinity, (ii) antibody-mediated complement activation, (iii) antibody-mediated CDC, (iv) oligomer formation, (v) oligomer stability, or any combination of (i) to (v). In one embodiment of (iv) or (v), the oligomer is a hexamer.

[0592] In one embodiment, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0593] Mixed mutants

[0594] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0595] As mentioned above, the inventors of the present invention have also found that there are mutations that reduce effector function by themselves but restore effector function when used together, such as mutations in positions K439 and S440 of the Fc region of the human IgG1 heavy chain. Therefore, by introducing K439 in one antibody and S440 in another antibody, this principle can also be used to ensure the pairing of two different antibodies. Therefore, the antibody variants according to the "mixed mutant" aspect contain mutations, but generally one mutation that leads to a reduction or a large reduction in Fc: Fc interactions between the same Fc molecules. However, the antibody variants of the present invention as "mixed mutants" are able to pair with each other; providing, for example, restoration or even increased C1q binding, complement activation, CDC, oligomer formation, and / or oligomer stability of specific antibody variant pairs compared to each individual mutant or a mixture of parent antibodies. In one embodiment of the present invention, the oligomer is a hexamer. In one embodiment, the antibody variants may also or alternatively have retained or improved other effector functions, such as Clq 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 cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing polypeptides, target down-regulation, ADC uptake, apoptosis induction, cell death, cell cycle arrest, and any combination thereof. This aspect of the invention provides a large number of applications in which not only the strength of Clq binding, complement activation, CDC or other effector functions but also their selectivity can be modulated.

[0596] Exemplary mutation sites for each antibody variant in the "mixed mutant" pair are shown in Table 2. Specifically, the present invention provides an antibody variant comprising an immunoglobulin antigen binding region and an Fc domain, the variant comprising a mutation in a residue in the Fc region of a human IgG1 heavy chain corresponding to one of K439 and S440. In one embodiment, the mutation is located at K439 and is an amino acid substitution to an amino acid selected from E or D, such as K439E. In one embodiment, the mutation is located at S440 and is an amino acid substitution to an amino acid selected from K, R or H, such as S440K.

[0597] Therefore, in one embodiment, the present invention also relates to a variant comprising a mutation of at least one amino acid residue selected from:

[0598] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0599] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0600] (c) amino acid residues within the N-terminal CH3 helix,

[0601] (d) an amino acid residue within the C-terminal CH3 beta 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

[0602] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain;

[0603] and an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain.

[0604] In another embodiment, the present invention also relates to a variant comprising a mutation of at least one amino acid residue selected from:

[0605] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0606] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0607] (c) amino acid residues within the N-terminal CH3 helix,

[0608] (d) an amino acid residue within the C-terminal CH3 beta 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

[0609] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain;

[0610] and an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain.

[0611] In one embodiment, the above two embodiments can be combined into a "mixed mutant" pair of the present invention.

[0612] Each variant of the "mixed mutant" pair may further include the amino acid mutations listed in Table 1.

[0613] In one embodiment of the invention, a “mixed mutant” pair 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 of an immunoglobulin and an antigen binding region, wherein the first variant comprises (i) a first mutation of at least one amino acid residue selected from the following group of amino acid residues except for a K439 mutation:

[0614] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0615] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0616] (c) amino acid residues within the N-terminal CH3 helix,

[0617] (d) an amino acid residue within the C-terminal CH3 beta 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

[0618] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0619] (ii) a second mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and wherein the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue selected from the group consisting of the following amino acid residues except for the S440 mutation:

[0620] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0621] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0622] (c) amino acid residues within the N-terminal CH3 helix,

[0623] (d) amino acid residues within the C-terminal CH3β strand, and

[0624] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0625] and (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0626] Other exemplary "mixed mutant" pairs may further include, and are not limited to, any one of the following pairs: a first variant comprising mutation K447E and a second variant comprising mutations K447 / P448; a first variant comprising mutation K447E and a second variant comprising mutations K447 / K448 / P449.

[0627] In one embodiment, the first mutation is a deletion, insertion or substitution. This substitution of amino acids can be a substitution with any naturally occurring 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.

[0628] In a specific embodiment, the first variant and / or the second variant comprises a mutation in at least one amino acid residue selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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.

[0629] In a specific embodiment, the first variant and / or the second variant comprises a mutation of at least one amino acid residue, which mutation may correspond to any of the following amino acid substitutions: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D / E356G, D / E356R, T3 59R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W, and the first variant includes a second mutation corresponding to the position K439 in the Fc region of a human IgG1 heavy chain; and the second variant includes a second mutation corresponding to the position S440 in the Fc region of a human IgG1 heavy chain.

[0630] For example, in one embodiment, one variant of a "mixed mutant" pair includes E345R and K439E mutations, while the other variant includes E345R and S440K mutations, thereby providing increased and more specific C1q binding affinity, complement activation, CDC, oligomer formation, oligomer stability, and / or other effector-related functions such as FcRn binding, ADCC, Fc-γ receptor binding, protein A binding, protein G binding, ADCP, CDCC, complement-enhanced cytotoxicity, antibody-mediated phagocytosis, internalization, apoptosis, binding to complement receptors opsonized with antibodies, and / or combinations thereof.

[0631] The "mixed mutant" aspect may also include two variants, each variant including more than one mutation listed in Table 1 in the Fc region of the human IgG1 heavy chain, for example, the first variant comprises the mutations S440K / K447E, and the second variant comprises the mutations K439E / K447 / P448; for example, the first variant comprises the mutations K439E / K447E, and the second variant comprises the mutations S440K / K447 / P448.

[0632] The variants in the "mixed mutants" pairs described in the present invention can be derived from the same or different parent antibodies. In addition, the "mixed mutants" aspect can also be used for bispecific or asymmetric antibodies. In addition, the first, second and third antibodies can bind to different epitopes on the same or different targets.

[0633] In addition, the "mixed mutant" aspect provides a more specific CDC or other effector response against tumor cells expressing two specific tumor antigens by utilizing a first antibody with a K439E mutation against a first antigen and a second antibody with a S440K or S440R mutation against a second antigen. By utilizing a "mixed mutant" aspect comprising three variants, optionally a bispecific antibody, a more specific CDC or other effector response against tumor cells expressing at least two, e.g., two, three, four, five, or six specific tumor antigens can be provided.

[0634] In any of the embodiments of the "single mutant", "double mutant" and "mixed mutant" aspects, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0635] In any embodiment of the "mixed mutant" aspect, the first, second and / or third variant may comprise the same or different mutations of any amino acid substitution listed in Table 1.

[0636] Multispecific Antibodies

[0637] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0638] It will be appreciated that any of the embodiments of the "single mutant", "double mutant" and "mixed mutant" aspects described herein may be employed in the multispecific antibody aspects described below.

[0639] In one main aspect, the invention relates to a variant which 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 wherein

[0640] The first and second CH2-CH3 regions each include a first mutation of at least one amino acid residue selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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 of S440 is S440Y or S440W.

[0641] In one embodiment, the first mutation is a deletion, insertion or substitution. Such substitution of an amino acid may be a substitution with any naturally occurring or non-natural amino acid.

[0642] The bispecific antibody of the present invention is not limited to a specific form, it can be any one described above and herein.

[0643] In one embodiment of the invention, the first and second polypeptides comprise a first mutation of an amino acid residue selected from the group consisting of E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

[0644] In a specific embodiment, the mutation of at least one amino acid residue may be an amino acid substitution corresponding to any of the following: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W ,E345Y,D / E356G,D / E356R,T359R,E382L,E382V,Q386K,E430A,E430C,E430D,E430F,E430G,E430H,E430I, E430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.

[0645] In a specific 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 a further amino acid residue mutation selected from the amino acid residues 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 a further amino acid residue mutation selected from the amino acid residues corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, and wherein the further mutation of the first polypeptide is different from the further mutation of the second polypeptide.

[0646] In a specific embodiment, the bispecific antibody has a first polypeptide (including a further mutation of the amino acid residue corresponding to K409 of the Fc region of a human IgG1 heavy chain) and a second polypeptide (including a further mutation of the amino acid residue corresponding to F405 of the Fc region of a human IgG1 heavy chain). Such bispecific antibodies of the present invention can be produced as described in Example 22. In addition, the assay used in Example 23 can be used to detect the effect of the produced heterodimeric protein on CDC killing.

[0647] In a specific embodiment, the bispecific antibody comprises a first and a second polypeptide, wherein the first polypeptide comprises a mutation in an amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain; the second polypeptide comprises a mutation in an amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain; and the first and / or second polypeptide comprises a further mutation in an amino acid residue corresponding to amino acid substitution E345R in the Fc region of a human IgG1 heavy chain.

[0648] In a specific embodiment, the bispecific antibody comprises a first and a second polypeptide, wherein the first polypeptide comprises a mutation of an amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain; the second polypeptide comprises a mutation of an amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain; and the first and / or second polypeptide each comprises a further mutation of an amino acid residue corresponding to the amino acid substitutions E345R and Q386K in the Fc region of a human IgG1 heavy chain. The further mutation may be located in both the first and second polypeptides, or E345R may be located in the first polypeptide and Q386K in the second polypeptide; or vice versa.

[0649] In a specific embodiment, the bispecific antibody comprises a first and a second polypeptide, wherein the first polypeptide comprises a mutation of an amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain; the second polypeptide comprises a mutation of an amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain; and the first and / or second polypeptides each comprise further mutations of amino acid residues corresponding to amino acid substitutions E345R, Q386K and E430G in the Fc region of a human IgG1 heavy chain. The mutations may be located in both the first and second polypeptides, or the first polypeptide may comprise mutations E345R and E430G, and the second polypeptide may comprise mutations Q386K; or vice versa.

[0650] The bispecific antibody may, for example, include an antigen binding region of a CD20 antibody and an antigen binding region of a CD38 antibody, and amino acid substitutions of one or more of the amino acids listed in Tables 1 and / or 2. Exemplary CD20 binding regions include the CD20 binding regions of ofatumumab (2F2), 7D8 and 11B described in WO2004 / 035607 (which is hereby incorporated by reference in its entirety), and rituximab (WO2005 / 103081). Exemplary CD38 binding regions include those of 003 and daratumumab (005) described in WO2006 / 099875 (which is hereby incorporated by reference in its entirety).

[0651] In one embodiment, the bispecific antibody binds different epitopes on the same or different targets.

[0652] In another embodiment, the first mutation in the first and second polypeptides may be the same or different.

[0653] 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 full-length human antibody, such as a full-length human IgG1 antibody.

[0654] In any of the "single mutant", "double mutant", "mixed mutant" aspects and multispecific antibody aspects, the C1q binding of the antibody is determined according to the assay described in Example 4, the CDC is determined according to the assay described in Examples 5, 6 or 10, the mutation is not located in an amino acid residue directly involved in C1q binding, optionally determined by comparing the C1q binding according to the ELISA assay according to Example 3 with the C1q binding according to the cell-based assay according to Example 4, and the ADCC is determined according to the assay described in Example 12.

[0655] In addition, the present invention provides preparations of variants of any of the "single mutants", "double mutants", "mixed mutants" and multispecific antibody aspects or embodiments as described above. The present invention also provides compositions, e.g., pharmaceutical compositions, comprising variants of any of the "double mutant" aspects and embodiments as described above. The present invention also provides the use of any such variant, preparation or composition as a medicament.

[0656] The above-mentioned "single mutant", "double mutant", "mixed mutant" and multispecific antibody aspects of the present invention are particularly applicable to human antibody molecules having an IgG1 heavy chain, wherein the IgG1 heavy chain includes the relevant fragment P247 to K447 corresponding to the underlined residues 130 to 330 of the human IgG1 heavy chain constant region (UniProt accession number P01857; SEQ ID NO: 1):

[0657]

[0658] The present invention can also be used for 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)

[0659]

[0660] The present invention can also be used for 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 number P01860; SEQ ID NO: 3), which are underlined below:

[0661]

[0662] The present invention can also be used for 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)

[0663]

[0664] The present invention can also be used for antibodies having a heavy chain portion of the human IgG1m(f) allotype. Amino acid sequence of the IgG1m(f) allotype (CH3 sequence is underlined) - SEQ ID NO: 5

[0665]

[0666] The alignment of the corresponding fragments of the constant regions of IgG1, IgG2, IgG3, IgG4 and IgG1m(f) is shown in Figure 2 Therefore, any mutation of the amino acids described in Table 1 or Tables 2A and B can be introduced into the equivalent positions of IgG2, IgG3, IgG4 and / or IgG1m(f) determined by alignment to obtain the variants of the present invention.

[0667] In one embodiment, the invention provides variants of full-length IgG1, IgG2, IgG3 or IgG4 antibodies comprising one or more amino acid substitutions as described above.

[0668] In any of the “single mutant,” “double mutant,” “mixed mutant” aspects and multispecific antibodies, the Fc region of the IgG1 heavy chain may include the following sequence: residues 130 to 330 of SEQ ID NO:1, residues 126 to 326 of SEQ ID NO:2, residues 177 to 377 of SEQ ID NO:3, or residues 127 to 327 of SEQ ID NO:4.

[0669] In one embodiment, the parent antibody comprises a sequence selected from SEQ ID No.: 1-5, such as SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4 or SEQ ID No.: 5.

[0670] In one embodiment, the Fc region of an IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO:1.

[0671] The parent antibody may be any parent antibody described herein. Parent antibody in this context is also intended to be a first parent and a second parent antibody.

[0672] In one embodiment the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD or IgE antibody.

[0673] In one embodiment, the parent antibody is a full length human antibody, such as a full length human IgG1 antibody.

[0674] In one embodiment, the parent antibody, the first parent antibody and the 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.

[0675] In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO:2.

[0676] In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO:3.

[0677] In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO:4.

[0678] In a specific embodiment 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, except for the mutations introduced according to the present invention.

[0679] Thus, the variant may comprise the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, in addition to any mutations defined herein.

[0680] Any of the above-mentioned "single mutant", "double mutant", "mixed mutant" and multispecific aspects according to the present invention are to be understood to include the following embodiments.

[0681] In one embodiment, the first and / or second parent antibody is an antibody fragment, optionally selected from a monovalent antibody, a heavy chain antibody, a chain exchange engineering domain (SEED), a triomab, a dual variable domain immunoglobulin (DVD-Ig), a knob-into-holes antibody, a small antibody, a dual affinity redirecting molecule (Fc-DART or Ig-DART); 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 Fc domain, and dual scFv fragment fused to Fc domain.

[0682] In a further embodiment, both the first and the second parent antibody bind to an antigen expressed on the surface of a human tumor cell.

[0683] In a further embodiment, the antigens for the first and second parent antibodies are individually selected from erbBl (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.

[0684] In a further embodiment, the first and second parent antibodies are fully human.

[0685] In a further embodiment, the antigens directed against the first and second parent antibodies are, in any order, selected from CD20 and CD38, optionally wherein the first and second parent antibodies are, in any order, selected from 7D8 and 005.

[0686] In further embodiments, both the first antibody and the second antibody bind to an antigen expressed on the surface of a bacterial cell or a viral particle.

[0687] In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.

[0688] In a further embodiment, the first and second parent antibodies bind the same antigen.

[0689] In another embodiment, the first and second parent antibodies are the same antibody.

[0690] In another embodiment, the parent antibody is selected from 7D8 and 005. Composition

[0691] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0692] The present invention also relates to compositions comprising variants, and the parent antibody can be any variant and parent antibody described in the present invention. Specific aspects and embodiments will be described below. In addition, this variant can be obtained according to any method described in the present invention.

[0693] In one aspect, the invention relates to 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 of an immunoglobulin and a binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and a binding region, and wherein

[0694] (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 not S440Y or S440W,

[0695] (ii) the first variant comprises a mutation corresponding to the K447D / E position in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation corresponding to the K447K / R / H and 448P positions in the Fc region of a human IgG1 heavy chain, or

[0696] (iii) the first variant comprises mutations at positions 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.

[0697] In one embodiment, the first or both variants of the parent polypeptide and the second variant of the parent polypeptide may be antibodies.

[0698] Thus in one aspect the invention relates to 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 of an immunoglobulin and an antigen binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, and wherein

[0699] (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 not S440Y or S440W,

[0700] (ii) the first variant comprises a mutation corresponding to the K447D / E position in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation corresponding to the K447K / R / H and 448P positions in the Fc region of a human IgG1 heavy chain, or

[0701] (iii) the first variant comprises mutations at positions 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.

[0702] 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 of an immunoglobulin and an antigen binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, and wherein the first variant comprises a mutation at a position corresponding to K439 of 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 in S440 is not S440Y or S440W.

[0703] 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 of an immunoglobulin and an antigen binding region, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0704] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0705] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0706] (c) amino acid residues within the N-terminal CH3 helix,

[0707] (d) an amino acid residue within the C-terminal CH3 beta 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

[0708] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0709] (ii) a second mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and

[0710] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0711] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0712] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0713] (c) amino acid residues within the N-terminal CH3 helix,

[0714] (d) amino acid residues within the C-terminal CH3β strand, and

[0715] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0716] (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0717] In another embodiment, the composition comprises a first variant of an antibody and a second variant of a parent antibody, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0718] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0719] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0720] (c) amino acid residues within the N-terminal CH3 helix,

[0721] (d) an amino acid residue within the C-terminal CH3 beta 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

[0722] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0723] (ii) a second mutation at position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and

[0724] Wherein the second variant comprises a mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain, which is not S440Y or S440W.

[0725] In another 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 mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and

[0726] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0727] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0728] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0729] (c) amino acid residues within the N-terminal CH3 helix,

[0730] (d) amino acid residues within the C-terminal CH3β strand, and

[0731] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0732] and (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0733] In one embodiment, 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.

[0734] In another aspect, the present invention relates to a composition comprising a first variant of a parent polypeptide comprising an Fc domain of an immunoglobulin and a binding region and a second variant of a parent polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein

[0735] The first variant comprises a first Fc domain and an antigen binding region of an immunoglobulin, wherein the first variant comprises a first mutation of at least one amino acid residue selected from the group consisting of:

[0736] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0737] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0738] (c) amino acid residues within the N-terminal CH3 helix,

[0739] (d) an amino acid residue within the C-terminal CH3 beta 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

[0740] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and wherein

[0741] The second variant does not include an amino acid residue mutation of an amino acid residue selected from:

[0742] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0743] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0744] (c) amino acid residues within the N-terminal CH3 helix,

[0745] (d) an amino acid residue within the C-terminal CH3 beta 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

[0746] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0747] In one embodiment, the first and / or second parent polypeptide may be an antibody.

[0748] The present invention also relates to an embodiment of a composition, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0749] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0750] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0751] (c) amino acid residues within the N-terminal CH3 helix,

[0752] (d) an amino acid residue within the C-terminal CH3 beta 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

[0753] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0754] (ii) a second mutation at position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and

[0755] Wherein the second variant comprises a mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain, which is not S440Y or S440W.

[0756] The present invention also relates to an embodiment of the 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

[0757] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0758] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0759] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0760] (c) amino acid residues within the N-terminal CH3 helix,

[0761] (d) amino acid residues within the C-terminal CH3β strand, and

[0762] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0763] (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0764] In another aspect, the present invention relates to 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 of an immunoglobulin and a first antigen binding region, wherein the first variant comprises a first mutation of at least one amino acid residue selected from the group consisting of:

[0765] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0766] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0767] (c) amino acid residues within the N-terminal CH3 helix,

[0768] (d) an amino acid residue within the C-terminal CH3 beta 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

[0769] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0770] The second variant comprises a second Fc domain of an immunoglobulin and a second antigen binding region, wherein the second variant does not comprise a mutation of an amino acid residue selected from the group consisting of:

[0771] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0772] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0773] (c) amino acid residues within the N-terminal CH3 helix,

[0774] (d) an amino acid residue within the C-terminal CH3 beta 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

[0775] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0776] In embodiments where the second variant does not include any of the mutations listed herein, such second variant may include any of the examples of suitable second antibodies listed above in connection with effector methods.

[0777] In one embodiment, the first variant and the second variant comprise a first mutation selected from at least one amino acid residue corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436, and K447 in the Fc region of a human IgG1 heavy chain.

[0778] In one embodiment, the first variant comprises a mutation in at least one amino acid residue selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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.

[0779] In a specific embodiment, the first variant comprises mutations in the 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.

[0780] In a specific embodiment, the first variant comprises mutations in the amino acid residues corresponding to E345R, Q386K and E430G in the Fc region of a human IgGl heavy chain and the second variant does not comprise such mutations.

[0781] In one embodiment, at least one first mutation in the first and second variants is different.

[0782] 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 full human antibody, such as each a full human IgG1 antibody.

[0783] In one embodiment, the first variant and the second variant are selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0784] In further embodiments, the first and second variants bind to different epitopes on the same antigen or different antigens. Thus, in embodiments where the first and second antibodies are bispecific antibodies, they can each bind to two different epitopes. The at least two bispecific antibodies can be the same or different. If the bispecific antibodies are different, the composition includes up to 4 different epitopes on the same or different targets.

[0785] In further embodiments, one or both of the first variant and the second variant are conjugated to a drug, a toxin or a radiolabel, for example wherein one or both of the first variant and the second variant are conjugated to the toxin via a linker.

[0786] In further embodiments, one or both of the first variant and the second variant are part of a fusion protein.

[0787] In another aspect, the present invention relates to a composition comprising any variant, any bispecific antibody or any composition described in the present invention and a pharmaceutically acceptable carrier.

[0788] It is contemplated that any embodiment according to the "mixed mutant" aspect is also encompassed by any composition embodiment.

[0789] In one embodiment the variants of the first and second parent antibodies bind antigens expressed on the same cell.

[0790] In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 to an amino acid selected from E and D.

[0791] In another embodiment the amino acid substitution of the variant of the first parent antibody is K439E.

[0792] In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 to an amino acid selected from the group consisting of K, R and H.

[0793] In another embodiment the amino acid substitution of the variant of the second parent antibody is S440K.

[0794] In alternative embodiments, the variant of the first and / or second antibody further comprises a mutation of a residue selected from the group consisting of H310, G385, H433, N434 and Q438.

[0795] In further alternative embodiments, 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.

[0796] In a further embodiment, the variant of the first and second parent antibodies further comprises a mutation selected from E345R, E382R and H433R, such as E345R.

[0797] In another aspect, the invention relates to a pharmaceutical composition comprising a variant of the first parent antibody and a variant of the second parent antibody according to any one of the embodiments listed above.

[0798] Pharmaceutical compositions can be formulated according to conventional techniques, such as disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention can, for example, include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, isotonic agents, antioxidants, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions. Examples of suitable aqueous and non-aqueous carriers that can be used for pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, glucose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol).

[0799] The pharmaceutical composition can be administered by any suitable route and mode. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. The term "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0800] Set

[0801] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0802] The present invention also relates to a kit comprising a variant and a parent antibody for simultaneous, separate or sequential use in treatment, wherein any variant and parent antibody may be as described herein. Specific aspects and embodiments will be described below. In addition, such variants may be obtained according to any method described herein.

[0803] The present invention also relates to a kit comprising a variant and a parent antibody for simultaneous, separate or sequential use in treatment, which may be any variant and parent antibody as described herein. Specific aspects and embodiments will be described below. In addition, such variants may be obtained according to any method described herein.

[0804] In one aspect, the invention relates to a kit comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide for simultaneous, separate or sequential use in therapy, wherein the first variant comprises a first Fc domain of an immunoglobulin and a binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and a binding region, and wherein

[0805] (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 not S440Y or S440W,

[0806] (ii) the first variant comprises a mutation corresponding to the K447D / E position in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation corresponding to the K447K / R / H and 448P positions in the Fc region of a human IgG1 heavy chain, or

[0807] (iii) the first variant comprises mutations at positions 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.

[0808] In one embodiment, the first or both variants of the parent polypeptide and the second variant of the parent polypeptide may be antibodies.

[0809] Thus, in one aspect, the invention relates to a kit comprising a first variant of a parent antibody and a second variant of a parent antibody for simultaneous, separate or sequential use in therapy, wherein the first variant comprises a first Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, and wherein

[0810] (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 not S440Y or S440W,

[0811] (ii) the first variant comprises a mutation corresponding to the K447D / E position in the Fc region of a human IgG1 heavy chain; and the second variant comprises a mutation corresponding to the K447K / R / H and 448P positions in the Fc region of a human IgG1 heavy chain, or

[0812] (iii) the first variant comprises mutations at positions 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.

[0813] In one embodiment, a kit for simultaneous, separate or sequential use in therapy 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 of an immunoglobulin and an antigen binding region, wherein the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, and wherein the 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 in S440 is not S440Y or S440W.

[0814] In one embodiment, the kit for simultaneous, separate or sequential use in therapy comprises a first variant of a parent antibody and a second variant of a parent antibody,

[0815] The first variant comprises a first Fc domain and an antigen binding region of an immunoglobulin, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0816] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0817] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0818] (c) amino acid residues within the N-terminal CH3 helix,

[0819] (d) an amino acid residue within the C-terminal CH3 beta 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

[0820] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0821] (ii) a second mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and

[0822] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0823] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0824] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0825] (c) amino acid residues within the N-terminal CH3 helix,

[0826] (d) amino acid residues within the C-terminal CH3β strand, and

[0827] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0828] (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0829] In another embodiment, the kit for simultaneous, separate or sequential use in therapy comprises a first variant of an antibody and a second variant of a parent antibody, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0830] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0831] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0832] (c) amino acid residues within the N-terminal CH3 helix,

[0833] (d) an amino acid residue within the C-terminal CH3 beta 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

[0834] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0835] (ii) a second mutation at position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and

[0836] Wherein the second variant comprises a mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain, which is not S440Y or S440W.

[0837] In another embodiment, a kit for simultaneous, separate or sequential use in therapy comprises a first variant of a parent antibody and a second variant of a parent antibody,

[0838] wherein the first variant comprises a mutation in a position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and

[0839] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0840] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0841] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0842] (c) amino acid residues within the N-terminal CH3 helix,

[0843] (d) amino acid residues within the C-terminal CH3β strand, and

[0844] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0845] and (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0846] In one embodiment, 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.

[0847] In another aspect, the invention relates to a kit comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide for simultaneous, separate or sequential use in therapy, wherein the first variant comprises an Fc domain of an immunoglobulin and a binding region, and the second variant comprises an Fc domain of an immunoglobulin and a binding region, wherein

[0848] The first variant comprises a first Fc domain and an antigen binding region of an immunoglobulin, wherein the first variant comprises a first mutation of at least one amino acid residue selected from the group consisting of:

[0849] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0850] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0851] (c) amino acid residues within the N-terminal CH3 helix,

[0852] (d) an amino acid residue within the C-terminal CH3 beta 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

[0853] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and wherein

[0854] The second variant does not include a mutation in an amino acid residue group selected from:

[0855] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0856] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0857] (c) amino acid residues within the N-terminal CH3 helix,

[0858] (d) an amino acid residue within the C-terminal CH3 beta 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

[0859] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0860] In one embodiment, the first and / or second parent polypeptide may be an antibody.

[0861] The present invention also relates to an embodiment of a kit for simultaneous, separate or sequential use in therapy, wherein the first variant comprises (i) a first mutation of at least one amino acid residue other than a K439 mutation selected from the group consisting of:

[0862] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0863] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0864] (c) amino acid residues within the N-terminal CH3 helix,

[0865] (d) an amino acid residue within the C-terminal CH3 beta 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

[0866] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0867] (ii) a second mutation at position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and

[0868] Wherein the second variant comprises a mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain, which is not S440Y or S440W.

[0869] The invention also relates to an embodiment of a kit for simultaneous, separate or sequential use in therapy, wherein the first variant comprises a mutation corresponding to the position K439 of the Fc region of a human IgG1 heavy chain; and

[0870] The second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein the second variant comprises (i) a first mutation of at least one amino acid residue other than the S440 mutation selected from the group consisting of:

[0871] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0872] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0873] (c) amino acid residues within the N-terminal CH3 helix,

[0874] (d) amino acid residues within the C-terminal CH3β strand, and

[0875] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain,

[0876] and (ii) a second mutation at a position corresponding to S440 in the Fc region of an IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0877] In another aspect, the invention relates to a kit for simultaneous, separate or sequential use in therapy, comprising a first variant of an antibody and a second variant of a parent antibody, wherein

[0878] The first variant comprises a first Fc domain and an antigen binding region of an immunoglobulin, wherein the first variant comprises a first mutation of at least one amino acid residue selected from the group consisting of:

[0879] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0880] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0881] (c) amino acid residues within the N-terminal CH3 helix,

[0882] (d) an amino acid residue within the C-terminal CH3 beta 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

[0883] (e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain, and

[0884] The second variant comprises a second Fc domain of an immunoglobulin and a second antigen binding region, wherein the second variant does not comprise a mutation of an amino acid residue selected from the group consisting of:

[0885] (a) providing an isomeric mutated amino acid residue in the CH2-CH3 region,

[0886] (b) amino acid residues within the hydrophobic knob of the CH2-CH3 region,

[0887] (c) amino acid residues within the N-terminal CH3 helix,

[0888] (d) an amino acid residue within the C-terminal CH3 beta 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

[0889] (e) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

[0890] In embodiments where the second variant does not comprise any of the mutations listed herein, such second variant may comprise any of the examples of suitable second antibodies listed above in connection with effector methods.

[0891] In one embodiment, the first variant and the second variant comprise a first mutation selected from at least one amino acid residue corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, S254, Q311, D / E356, T359, E382, Y436 and K447 in the Fc region of a human IgG1 heavy chain.

[0892] In one embodiment, the first variant comprises a mutation in at least one amino acid residue selected from the group corresponding to the following amino acid residues: E345, E430, S440, Q386, P247, 1253, 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.

[0893] In a specific embodiment, the first variant comprises mutations in the 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.

[0894] In a specific embodiment, the first variant comprises mutations in the amino acid residues corresponding to E345R, Q386K and E430G in the Fc region of a human IgGl heavy chain and the second variant does not comprise such mutations.

[0895] In one embodiment, at least one first mutation in the first and second variants is different.

[0896] 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 full length human antibody, for example each a full length human IgG1 antibody.

[0897] In one embodiment, the first variant and the second variant are selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0898] In further embodiments, the first and second variants bind to different epitopes on the same antigen or different antigens. Thus, in embodiments in which the first and second antibodies are bispecific antibodies, they may each bind to two different epitopes. The at least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the kit for simultaneous, separate or sequential use for treatment includes targeting up to 4 different epitopes on the same or different targets.

[0899] In further embodiments, one or both of the first variant and the second variant are conjugated to a drug, a toxin or a radiolabel, for example wherein one or both of the first variant and the second variant are conjugated to the toxin via a linker.

[0900] In further embodiments, one or both of the first variant and the second variant are part of a fusion protein.

[0901] It is contemplated that any embodiment according to the "mixed mutant" aspect may be included in any kit embodiment for simultaneous, separate or sequential use in therapy.

[0902] In one embodiment the variants of the first and second parent antibodies bind antigens expressed on the same cell.

[0903] In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 to an amino acid selected from E and D.

[0904] In another embodiment the amino acid substitution of the variant of the first parent antibody is K439E.

[0905] In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 to an amino acid selected from the group consisting of K, R and H.

[0906] In another embodiment the amino acid substitution of the variant of the second parent antibody is S440K.

[0907] In alternative embodiments, the variant of the first and / or second antibody further comprises a mutation of a residue selected from the group consisting of H310, G385, H433, N434 and Q438.

[0908] In further alternative embodiments, 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.

[0909] In a further embodiment, the variant of the first and second parent antibodies further comprises a mutation selected from E345R, E382R and H433R, such as E345R.

[0910] In another aspect, the invention relates to a pharmaceutical kit for simultaneous, separate or sequential use in therapy, comprising a variant of the first parent antibody and a variant of the second parent antibody according to any of the embodiments listed above.

[0911] The pharmaceutical set for simultaneous, separate or sequential use in treatment can be administered by any suitable route and mode. In one embodiment, the pharmaceutical set of the present invention for simultaneous, separate or sequential use in treatment is administered parenterally. The term "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0912] combination

[0913] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0914] In addition, the present invention provides a formulation of a variant of any "single mutant" aspect or embodiment as described above, that is, a formulation comprising multiple copy variants. The present invention also provides a composition comprising any "single mutant" aspect and embodiment as described above, for example, a pharmaceutical composition. The present invention also provides the use of any such "single mutant" variant, preparation or composition as a drug.

[0915] The present invention also provides a combination of variants, wherein one variant comprises at least one mutation independently selected from those listed in Table 1, and one variant comprises at least one other mutation independently selected from those listed in Table 1, as well as products and pharmaceutical compositions of such a combination of variants and their use as a medicament. Preferably, the two variants bind to the same antigen or different antigens that are usually expressed on the surface of the same cell, cell membrane, virus particle and / or other particle.

[0916] Conjugate

[0917] It is to be understood that all embodiments of the present invention described with reference to a parent antibody, a first parent antibody or a second parent antibody should also be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region.

[0918] In one aspect, the invention relates to a variant, wherein the variant is conjugated to a drug, a toxin or a radiolabel, for example wherein the variant is conjugated to the toxin via a linker.

[0919] In one embodiment, the variant is part of a fusion protein.

[0920] In another aspect, the variants of the present invention are not conjugated to another molecule (e.g., a toxin or a label) at the C-terminus. In one embodiment, the variant is conjugated to another molecule at another site (usually at a site that does not interfere with oligomer formation). For example, an antibody variant can be connected to a compound selected from a toxin (including a radioisotope), a prodrug, or a drug at another site. Such a compound can make the killing of target cells more effective, for example, in cancer treatment. The variant thus obtained is an immunoconjugate.

[0921] Thus, in a further aspect, the invention provides an antibody linked or conjugated to one or more therapeutic moieties (e.g., cytotoxins, chemotherapeutic drugs, cytokines, immunosuppressants and / or radioisotopes). Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins".

[0922] Cytotoxins or cytotoxic agents include any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for use in forming immunoconjugates of the invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or its analogs or derivatives, enediyene antitumor antibiotics including neocarcinomatidylcholine, calicheamicin, esperamicin, anthracycline antibiotics, lidamycin, kedar cidin or its analogs or derivatives, anthracyclin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., nitrogen mustard, thioepa, chlorambucil, melphalan, bismuth nitrosamine (BSNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin; and duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin), or an analog or derivative of CC-1065, dolastatin, pyrrolo[ 2,1-c][1,4]benzodiazepins (PDBs) or their analogs, antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunorubicin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), antimitotic agents (e.g., microtubule inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; histone deacetylase inhibitors such as hydroxamic acid trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat as well as benzamide, entinostat, CI994, moxistat and aliphatic acid compounds such as phenyl butyrate and valproic acid, proteasome inhibitors such as Danoprevir, bortezomib, amantin peptides such as α-amantin, diphtheria toxin and related molecules (e.g., diphtheria A chain and active fragments and hybrid molecules thereof);Ricin toxin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, American pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curculin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxin. Other suitable conjugated molecules include antimicrobial / lytic peptides such as CLIP, magainin 2, melittin, cecropin, and P18; ribonucleases (RNases), DNase I, Staphylococcal enterotoxin-A species, American pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with the antibodies of the invention described elsewhere herein (such as, for example, anticancer cytokines or chemokines) can also be candidates for therapeutic moieties that can be conjugated to the antibodies of the invention. ;

[0923] In one embodiment, the drug conjugate of the present invention comprises an antibody conjugated to auristatins or auristatin peptide analogs and derivatives disclosed herein (US5635483; US5780588). Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and have anticancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). The auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (terminus) of the peptide drug moiety using a linker.

[0924] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Vol. 45, Abstract No. 623, published March 28, 2004 and described in US 2005 / 0238649.

[0925] An exemplary auristatin embodiment is MMAE (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).

[0926] In one embodiment, the antibodies of the invention include conjugated nucleic acids or nucleic acid binding molecules. In this embodiment, the conjugated nucleic acids are cytotoxic ribonucleases, antisense nucleic acids, inhibitory RNA molecules (e.g., siRNAs molecules) or immunostimulatory nucleic acids (e.g., DNA molecules containing immunostimulatory CpG motifs). In another embodiment, the antibodies of the invention are conjugated to aptamers or ribozymes.

[0927] In one embodiment, antibodies are provided that include one or more radiolabeled amino acids. Radiolabeled variants can be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581, U.S. Pat. No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S. RE 35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902. For example, radioactive isotopes can be conjugated by the chloramine T method.

[0928] In one embodiment, the variant of the present invention is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the variant can be conjugated to a chelating linker such as DOTA, DTPA or tiuxetan, which allows the antibody to form a complex with the radioisotope. The variant can also or optionally include one or more radiolabeled amino acids or other radiolabeled molecules, or conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled variants can be used for both diagnostic and therapeutic purposes. In one embodiment, the variant of the present invention is conjugated to an alpha emitter. Non-limiting examples of radioisotopes include 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 125 I, 111 In, 131 I, 186 Re, 213 Bs, 225 Ac and 227 Th.

[0929] In one embodiment, the variant of the present invention can be conjugated to a cytokine selected from the following group: IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, anacetum and TNFα.

[0930] Variants of the invention can also be chemically modified by covalent conjugation to polymers, for example to increase their circulation half-life. Exemplary polymers, and methods of attaching them to peptides, are shown in, for example, US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Other polymers include polyoxyethylene polyols and polyethylene glycols (PEG) (e.g., PEGs having a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000).

[0931] Any method known in the art for conjugating the variant of the present invention to the conjugated molecule, such as those described above, including the methods described below: Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such variants can be produced by chemically conjugating other parts to the N-terminal side or C-terminal side (e.g., antibody H or L chain) of the variant or its fragment (see, e.g., Antibody Engineering Handbook, Osamu Kanemitsu edited, Chijin Shokan published (1994)). In appropriate cases, such conjugated variant derivatives can also be produced by conjugating to internal residues or sugars.

[0932] The agent can be directly or indirectly conjugated to the variant of the present invention. The example of indirect conjugation of the second agent is to be conjugated to the cysteine ​​or lysine residues of the bispecific antibody through a spacer or a linker moiety. In one embodiment, the variant is conjugated to a prodrug molecule through a spacer or a linker, and the prodrug molecule can be activated as a therapeutic drug in vivo. In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint releases the drug unit from the antibody in the intracellular environment. In some embodiments, the joint is cut by a cleavable agent, and the cleavable agent is present in the intracellular environment (for example, in a lysosome or endosome or cave). For example, a spacer or a joint is cut by a tumor cell-related enzyme or other tumor-specific conditions, thereby forming an active drug. Examples of such prodrug techniques and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 of Syntarga BV et al. Suitable antibody prodrug techniques and multicarmycin analogs can also be found in U.S. Pat. No. 6,989,452 (Medarex), which is incorporated herein by reference. The linker can also or alternatively be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. The cleavage agent can include cathepsins B and D and plasmin, which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs in target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, e.g., US6214345, which describes the synthesis of doxorubicin with a Val-Cit linker and different examples of Phe-Lys linkers). Examples of Val-Cit and Phe-Lys linker structures include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA described below, wherein MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for Val-Cit, PAB is p-aminobenzylcarbamic acid tert-butyl ester and GABA is an abbreviation for gamma-aminobutyric acid. The advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is generally less toxic when conjugated and the serum stability of the conjugate is generally higher.

[0933] Still in another embodiment, the joint unit is not cleavable, and the drug is released by antibody degradation (see US2005 / 0238649). Generally, this joint is not substantially sensitive to the extracellular environment. In the context of joints, "not substantially sensitive to the extracellular environment" as used in the present invention means that when the variant antibody drug conjugate compound is present in the extracellular environment (such as blood plasma), no more than 20% in the sample of the variant antibody drug conjugate compound, usually no more than about 15%, more usually no more than about 10%, and even more usually no more than about 5%, no more than about 3%, or no more than about 1% of the joint is cut. By incubating the variant antibody drug conjugate compound with blood plasma for a predetermined time period (such as 2,4,8,16 or 24 hours) and then quantifying the amount of free drug present in blood plasma, it can be determined whether the joint is substantially sensitive to the extracellular environment. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures (wherein Ab represents an antibody, and p represents the drug loading (or the average number of cytostatic or cytotoxic drugs per antibody molecule) is 1 to about 8, for example, p can be 4-6, such as 3-5, or p can be 1, 2, 3, 4, 5, 6, 7 or 8).

[0934] Examples of cleavable linkers combined with auristatins include MC-vc-PAB-MMAF (also known as vcMMAF) and MC-vc-PAB-MMAF (also known as vcMMAE), wherein MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for a Val-Cit (valine-citrulline)-based linker, and PAB is an abbreviation for tert-butyl p-aminobenzylcarbamate.

[0935] Other examples include auristatins combined with non-cleavable linkers, such as mcMMAF (mc(MC is the same as mc in this context) is the abbreviation for maleimidocaproyl.

[0936] In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE drug linker moiety and conjugation methods are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US11 / 833,028 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the vcMMAE drug linker moiety is conjugated to the antibody at the cysteine ​​using a method similar to the method disclosed therein.

[0937] In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation methods are disclosed in US7498298, US11 / 833,954 and WO2005081711 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the mcMMAF drug linker moiety is conjugated to the variant at the cysteine ​​using methods similar to those disclosed therein.

[0938] In one embodiment, the variants of the invention are linked to a chelator linker, such as tiuxetan, which allows the bispecific antibody to be conjugated to a radioisotope.

[0939] In one embodiment, each arm (or Fab-arm) of the variant is coupled directly or indirectly to the same therapeutic moiety or moieties.

[0940] In one embodiment, only one arm of the variant is conjugated directly or indirectly to one or more therapeutic moieties.

[0941] In one embodiment, each arm of the variant is directly or indirectly coupled to a different therapeutic moiety. For example, in embodiments where the variant is a bispecific antibody and is prepared by controlled Fab-arm exchange of two different monospecific antibodies (e.g., the first and second antibodies described herein), such a bispecific antibody can be obtained by using monospecific antibodies conjugated or bound to different therapeutic moieties.

[0942] Further uses

[0943] It should be understood that all embodiments of the present invention are described with reference to parent antibodies, and that the first parent antibody or the second parent antibody should also be understood as embodiments of the parent, first parent or second parent polypeptide comprising an immunoglobulin and a binding region. In a further aspect, the present invention relates to the use of a variant of the present invention as described above as a medicament, in particular as a medicament for treating a disease or condition, wherein CDC-mediated killing of a target cell (e.g., a tumor, a bacterial or fungal cell) or a target organism (e.g., a virus) or a bacterial or virally infected cell is desired. Examples of such diseases and conditions include, but are not limited to, cancer and bacterial, viral or fungal infections.

[0944] In another aspect, the invention relates to the variants, bispecific antibodies, compositions and kits of the invention for use in treating a disease, such as cancer.

[0945] In another aspect, the invention relates to a method of treating a human comprising administering a variant, composition or kit as described herein.

[0946] In another aspect, the invention relates to a method of treating cancer in a human comprising administering a variant, composition or kit as described herein.

[0947] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of alleviating, ameliorating, arresting or eliminating (curing) symptoms or disease states.

[0948] An "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody will vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the antibody or antibody portion outweigh any toxic or adverse effects.

[0949] Without being limited by theory, when any of the "single mutant" aspects or embodiments of the invention are introduced into such a therapeutically active compound, the effective amount of the therapeutically active compound may be reduced.

[0950] Suitable antigens for cancer antibodies can be the same as those described herein. Examples 15 to 18 describe specific applications that provide enhanced and / or more sp...

Claims

1. A method for improving the effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, the method comprising introducing 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 in the parent polypeptide, with the proviso that the mutation in S440 is S440Y or S440W.

2. A method for reducing the effector function of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, the method comprising introducing a mutation in an amino acid residue selected from those corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain in the parent polypeptide, with the proviso that the mutation in S440 is not S440Y or S440W, such as 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 / H / R.

3. A method for increasing the effector function of a combination of at least a first and a second parent polypeptide, wherein each of the at least first and second parent polypeptides comprises an Fc domain and a binding region of an immunoglobulin, wherein the method comprises (i) introducing a mutation into at least the first and / or second parent polypeptide in one or more amino acid residues selected from the group consisting of: (a) amino acid residues in the CH2-CH3 region that provide 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) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

4. A method for inducing an effector response against a cell, cell membrane or virus particle, wherein the cell, cell membrane or virus particle expresses a target bound by a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, the method comprising (i) providing a parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide which has been mutated according to any one of claims 1 to 3; and (ii) contacting the mutant parent polypeptide of step (i) or the mutant combination of at least the first parent polypeptide and the second parent polypeptide of step (i) with a cell, cell membrane or virus particle expressing the antigen in the presence of human complement or effector cells.

5. A method for improving the specificity of a combination of at least a first and a second parent polypeptide, wherein the first and second parent polypeptides comprise an Fc domain and a binding region of an immunoglobulin, the method comprising A) (i) introducing a mutation in the amino acid residue at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain of the first parent polypeptide, and (ii) introducing a mutation in the amino acid residue at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain of the second parent polypeptide, provided that the mutation in S440 is not S440Y or S440W, B) (i) introducing a mutation in the amino acid residue at the position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain of the first parent polypeptide, and (ii) introducing mutations in the amino acid residues corresponding to the positions K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain of the second parent polypeptide; or C) (i) introducing a mutation in the amino acid residue at the position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain of the first parent polypeptide, and (ii) introducing mutations in the amino acid residues at positions corresponding to K447K / R / H, 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain of the second parent polypeptide.

6. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein the variant comprises a mutation in at least one of those amino acid residues corresponding to E345, E430, S440, Q386, P247, 1253, 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.

7. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein the variant comprises mutations in at least two amino acid residues selected from the group consisting of: (a) amino acid residues in the CH2-CH3 region that provide 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.

8. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein the variant comprises a mutation in at least one amino acid residue selected from the group consisting of: (a) amino acid residues in the CH2-CH3 region that provide 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, provided that it does not contain a mutation corresponding to S440 in the Fc region of a 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 Wherein the variant comprises additional mutations in the following amino acid residues: (i) 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) in at least one of the amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) in at least one of the amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

9. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein the variant comprises a mutation in the following amino acid residues: (i) 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 not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain is K439D / E, and / or the mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain is S440K / H / R; (ii) in at least one of the amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of a human IgG1 heavy chain; or (iii) in at least one of the amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

10. 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 a binding region of an immunoglobulin, wherein the second variant comprises a second Fc domain and a binding region of an immunoglobulin, and wherein (i) said first variant comprises a mutation in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain, wherein said second variant comprises a mutation in the position corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, (ii) the first variant comprises a mutation in a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; wherein the second variant comprises a mutation in 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 in a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; wherein the second variant comprises mutations in positions corresponding to K447K / R / H, 448K / R / H and 449P in the Fc region of a human IgG1 heavy chain.

11. 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, wherein the first variant comprises a first Fc domain of an immunoglobulin and a first antigen binding region, wherein the first variant comprises a first mutation in at least one amino acid residue selected from the group consisting of: (a) amino acid residues in the CH2-CH3 region that provide 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 second variant does not comprise a mutation in an amino acid residue selected from the group consisting of: (a) amino acid residues in the CH2-CH3 region that provide 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) The amino acid residue corresponding to E345, E382 or Q386 in the Fc region of a human IgG1 heavy chain.

12. A composition comprising a variant according to any one of claims 6 to 9 or a composition according to any one of claims 10 to 11 and a pharmaceutically acceptable carrier.

13. A kit for simultaneous, separate or sequential use in therapy comprising a first variant and a second variant as defined in any one of claims 6 to 9.

14. A method of treating a human comprising administering a variant, composition or kit according to any one of claims 6 to 13.

15. A method of treating cancer in a human comprising administering a variant, composition or kit according to any one of claims 6 to 13.

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