Human igg1 fc region variants and uses thereof

Abstract

Described herein are polypeptides comprising variant Fc domains and related antibodies. The variant Fc domains provide stable Fc:Fc interactions when the polypeptides, one or more antibodies, bind to their target(s), one or more antigens, on the surface of a cell, thus providing improved complement-dependent cytotoxicity (CDC).

Description

[0001] This application is a divisional application of Chinese invention patent application filed on January 10, 2013, application number 201380073913.X, entitled "Human IgG1 FC region variant and its use". Technical Field

[0002] This invention relates to polypeptides (such as antibodies) containing an Fc domain, said polypeptides having enhanced complement-dependent cytotoxicity (CDC), and may also have other modified effector functions resulting from modifications of one or more amino acids in the Fc domain. Background Technology

[0003] Effector functions mediated by the antibody Fc domain allow for the destruction of foreign entities, such as killing pathogens and clearing and degrading antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by the binding of the Fc domain to cells with Fc receptors (FcRs), while complement-dependent cytotoxicity (CDC) is initiated by the binding of the Fc domain to C1q, which initiates the classical pathway of complement activation.

[0004] Each IgG antibody contains two C1q binding sites, one in each heavy chain constant (Fc) region. However, due to the very weak affinity of monomeric IgG for C1q (K... d ~10 -4 In addition, single IgG molecules in solution do 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 IgM (M) and complement activation (Burton et al., 1990, Mol. Immunol. 22, 161-206). In contrast, IgM exists naturally as covalently linked pentameric or hexameric forms, and upon binding to cell-expressed or fixed antigens, IgM pentameric and hexameric forms can effectively induce CDC. Antigen binding is a requirement for inducing conformational changes in IgM to expose the C1q binding site (Feinstein et al., 1986, Immunology Today, 169-174).

[0005] It has been proposed that IgG can also perform complement activation through the interaction of the CH2 / CH3 domains in the Fc region to form a hexameric ring structure (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.), pp. 17.1-17.5. Blackwell, Edinburgh; Pinteric et al., 1971 Immunochem. 8, 1041-5) and three-dimensional crystals, as well as in solutions of IgG1, IgG2a, IgG4, and human Fc (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115). The formation of the hexamer ring was also observed in the crystal structure of the b12 human IgG1κ antibody against HIV-1gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug 10; 293(5532), 1155-9). In the b12 hexamer ring, six easily accessible C1q binding sites are located on the hexamer surface, one from each of the six antibodies, while the other six binding sites face downwards.

[0006] C1q resembles a six-headed tulip cluster, 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). It was found that C1q assembles with the b12 hexamer in the 1HZH crystal structure, such that each of the six bulb 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's topic, the Scripps Research Institute, La Jolla, California, November 2000). Mutations in selected amino acids at the observed Fc interface between symmetry-related b12 antibodies in the crystal structure were found to reduce C1q binding affinity, indicating the contribution of these amino acids to the intermolecular Fc:Fc interactions.

[0007] WO 2006 / 104989 describes the altered antibody Fc region and its uses.

[0008] WO2005 / 047327 describes neonatal Fc receptor (FcRn) binding peptide variants, dimer Fc binding proteins, and related methods.

[0009] WO 2010 / 106180 describes an Fc variant that has increased binding to the neonatal Fc receptor (FcRn).

[0010] WO2005 / 070963 describes the Fc region variants of the peptide and their uses.

[0011] WO2006 / 053301 describes an Fc variant with altered binding to FcRn.

[0012] US 2011 / 0123440 describes a modified antibody Fc region and its uses. The modified Fc region has one or more amino acid substitutions.

[0013] US 2008 / 0089892 describes Fc region variants of peptides and compositions comprising these Fc region variants.

[0014] US 2010 / 0184959 describes a method for providing Fc peptide variants with altered Fc ligand recognition and / or effector functions.

[0015] US 2010 / 015133 describes a method for producing peptides by modulating peptide associations.

[0016] US 2010 / 105873 describes a comprehensive method for generating multi-domain protein therapeutics.

[0017] US 6,737,056 describes peptide variants with altered effector functions.

[0018] Previous efforts have been made to identify antibody Fc variants with enhanced effector functions or other modified properties. Such studies have focused on amino acid substitutions such as exchanging fragments between IgG isotypes to produce chimeric IgG molecules (Natsume et al., 2008 CancerRes 68(10), 3863-72), or in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) or at or near the C1q binding site in the CH2 domain, surrounding 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 enhanced effector function by CDC or ADCC in detecting various combinations of S267E, H268F, S324T, S239D, I332E, G236A, and I332E. Other Fc mutations affecting binding to Fc receptors (WO 2006 / 105062, WO 00 / 42072, U.S. Patent 6,737,056 and U.S. Patent 7,083,784) or antibody physical properties (WO 2007 / 005612 A1) have also been proposed.

[0019] Despite these and other advances in the field, there remains a need for new and improved antibody-based therapies. Summary of the Invention

[0020] This invention provides peptide and antibody variants that, compared to their parent peptides / antibodies, exhibit enhanced complement-dependent cytotoxicity (CDC) and other enhanced effector functions. Not limited to theory, it is conceivable that the variants can achieve a more stable binding interaction between the Fc regions of the two peptide / antibody molecules, thereby providing a more avid surface that can lead to enhanced effector functions, such as improved or more specific CDC responses. Specific variants are also characterized by improved ADCC responses, ADCP responses, and / or other enhanced effector functions. As described in this invention, this subtle mechanism of peptide / antibody engineering can be applied, for example, to improve the potency or specificity of antibody-based therapeutics.

[0021] Therefore, in one aspect, the present invention relates to a method for enhancing complement-dependent cytotoxicity (CDC) of a parent polypeptide comprising the Fc domain and binding region of an immunoglobulin, the method comprising introducing a mutation in one or more amino acid residues into the parent polypeptide, said mutation being selected from the group consisting of E430X, E345X, and S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0022] On the other hand, the present invention relates to variants of parental polypeptides comprising the Fc domain and binding region of an immunoglobulin, wherein the variant contains one or more mutations selected from the group consisting of E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W corresponding to the Fc region of the human IgG1 heavy chain, provided that the variant does not contain any other mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor (FcRn).

[0023] The present invention also provides the use of one or more such mutations to enhance complement-dependent cytotoxicity (CDC) mediated by peptides or antibodies when bound to, for example, antigens on the surface of antigen-expressing cells, cell membranes, or viral particles.

[0024] In one respect, variants referred to in this paper as “single mutants” have increased CDC compared to the parental peptide or antibody and may also have other enhanced effector functions.

[0025] In one respect, variants referred to herein as “double mutants” contain at least two mutations in the fragment and have improved CDC compared to variants containing only one of the two mutations, and may also have other improved effector functions.

[0026] In one aspect, a variant referred to herein as a “hybrid mutant” when used in combination with a second variant of the same or different polypeptide or antibody that contains mutations at different amino acid residues in the said segment provides enhanced CDC compared to the individual variant, the second variant, and one or more of the parent polypeptide or antibody, and optionally may also have other enhanced effector functions.

[0027] Typically, mutations are amino acid substitutions, such as those described below, which exchange parental amino acid residues for amino acids of different sizes and / or physicochemical properties that promote the formation of new intermolecular Fc:Fc bonds or enhance the strength of existing pair interactions. Exemplary amino acid residues of mutations according to the invention, together with exemplary amino acid substitutions, are shown in Tables 1 and 2A and B. Non-limiting aspects of different aspects of the invention are shown in Figure 1 Provided by China.

[0028] These and other aspects of the invention, particularly the various uses and therapeutic applications of peptide and antibody variants, are described in more detail below. Attached Figure Description

[0029] Figure 1 (A) Schematic diagram of IgG molecules in hexamer formation. The dashed circle shows two close Fc:Fc interaction pairs between two adjacent IgG molecules. The arrows in the box indicate the direction of viewing the illustrations in B, C, and D: the two adjacent Fc molecules rotated 90° (in the plane of the figure) and viewed from the Fab arm in the CH3 domain direction. (B) Observed effect of oligomer-enhancing mutations on CDC. Schematic diagram showing Fc:Fc interaction pairs with enhancing efficacy according to the single mutant and double mutant aspects of the present invention. (C) Observed effect of oligomer-repressive mutations on CDC. Schematic diagram according to the double mutant and mixed mutant aspects of the present invention, showing how at least two mutually compensating oligomer-repressive mutations can combine into one molecule (double mutant aspect) or remain separate on two molecules (mixed mutant aspect) to restore or enhance Fc:Fc interactions. The mixed mutant achieves specific effector functions dependent on the binding of two antibodies that can recognize different targets. (D) Theoretical effect of C1q binding repressive mutations on CDC. A schematic diagram of the Fc:C1q interaction shows that because C1q cannot compensate for the defects introduced by the antibody, if the mutation inhibits C1q binding, they cannot combine or mix to restore CDC activity.

[0030] Figure 2Sequence alignments of human IgG1, IgG1f, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM Fc regions corresponding to residues P247 to K447 in the IgG1 heavy chain were performed using Clustel 2.1 software, according to the EU index numbers 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 allotypes of IgG1m(f); 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:2; UniProt accession number P01860); and residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO:1). NO:4; UniProt accession number P01861); and residues 225-428 of the IgE constant region (UniProt accession number P01854); and residues 133-353 of the IgA1 constant region (UniProt accession number P01876); and residues 120-340 of the IgA2 constant region (UniProt accession number P01877); and residues 230-452 of the IgM antibody constant region (UniProt accession number P01871); and residues 176-384 of the IgD constant region (UniProt accession number P01880).

[0031] Figure 3A and 3B Sequence alignment of anti-EGFr antibody 2F8 in the backbones of IgG1 (SEQ ID NO:3), IgG4 (SEQ ID NO:5), and (partially) IgG3 (SEQ ID NO:6). Amino acid numbers are described according to Kabat and according to the EU index (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0032] Figure 4 : Detailed view of the K439 / S440 interaction between neighboring molecules (Fc and Fc', respectively) in a multimer (such as a hexamer) arrangement, showing the interaction between wild-type, unmodified Fc and Fc' molecules.

[0033] Figure 5: A detailed view of the K439 / S440 interaction between Fc molecules of neighboring molecules (Fc and Fc', respectively) in a multimer (such as a hexamer) arrangement, showing the interaction between variant Fc and Fc' molecules containing K439E and S440K mutations.

[0034] Figure 6 ELISA using C1q binding of the 7D8 Fc:Fc mutant. Serial concentrations of antibody as shown were coated onto the wells of a microtiter plate and incubated with a fixed concentration of C1q. For all coated variants except I253D, the binding efficiency with C1q was comparable to that of wild-type 7D8. Representatives from at least three experiments are shown.

[0035] Figure 7 : 7D8 variant-mediated CDC in CD20-positive Raji cells. Raji cells were incubated with 7D8 variants (K439E, S440K, K439E / S440K double mutant, K439E+S440K mixture) and serial concentrations of C1q to test CDC potency by measuring cell lysis. A schematic diagram of the replicate experiment is shown.

[0036] Figure 8 :7D8 mutants (7D8-WT, K439E, S440K, K439E / S440K double mutant, K439E+S440K mixture) mediated CDC in CD20-positive Daudi cells. The potency of 7D8 mutants in inducing CDC was tested at a series of concentrations.

[0037] Figures 9A-9D : CD38 antibody HuMAb005 mutant-mediated CDC on CD38-positive cells. (A) CDC efficacy of HuMAb005 mutant series concentrations on Daudi cells. (B) CDC efficacy of HuMAb005 mutant series concentrations on Raji cells. (C) CDC efficacy of HuMAb005 E345R mutant with 20% or 50% NHS on Wien133 cells. (D) CDC efficacy of HuMAb005 and E345R mutant with 7D8 with 20% or 50% NHS on Raji cells.

[0038] The test was performed on unpurified antibody samples isolated from transient transfection. The supernatant from mock-transfected cells was used as a negative control.

[0039] Figure 10: CDC of wild-type and E345R mutants of CD38 antibody HuMAb 005 (A) and CD20 antibody HuMAb 7D8 (B) in a competition assay for Fc-binding peptide. Cell lysis was measured after incubation of antibody-conditioned Daudi cells with CDC containing serial concentrations of Fc-bound DCAWHLGELVWCT peptide (SEQ ID NO:7). Unpurified antibody samples isolated from transient transfection were used. Supernatant from simulated transfected cells was used as a negative control.

[0040] Figure 11 ADCC of CD38-expressing Daudi cells with wild-type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R. ADCC of a donor PBMC is shown, depicted as % cleavage.

[0041] Figures 12A-12C At pH 6, wild-type IgG1-7D8 and mutant IgG1-7D8-E345R bind to human, cynomolgus, and mouse FcRN, as determined by ELISA.

[0042] Figure 13 Plasma concentrations of wild-type IgG1-7D8 and -E354R, -S440K and K322A variants after intravenous injection in SCID mice.

[0043] Figure 14A , 14B 14C and 14D: CDC in CD20 and CD38 positive Wien133 cells.

[0044] Figure 15A and 15B Evaluation of the in vivo potency of IgG1-7D8-E345R in a subcutaneous xenograft model with Raji-luc#2D1 cells.

[0045] Figure 16A and 16B Evaluation of the in vivo potency of IgG1-005-E345R in a subcutaneous xenograft model with Raji-luc#2D1 cells.

[0046] Figure 17 : CD38 / EGFR bispecific antibody with E345R mutation against CD38-positive and EGFR-negative Wien133 cells in CDC.

[0047] Figure 18A and 18BCDCs with or without E345R mutations and CD20 / CD38 bispecific antibodies against CD20-positive, CD38-negative Wien133 or Raji cells.

[0048] Figure 19 EGFR antibody 2F8 with E345R mutation against CDC in EGFR-positive A431 cells.

[0049] Figure 20 A and 20B: E345R mutant antibody-mediated CDC.

[0050] Figure 21 Colocalization analysis of TF antibody (FITC) with lysosomal labeling LAMP1 (APC).

[0051] Figure 22A -D: Introduction of E345R resulted in enhanced CDC-mediated killing compared to wild-type rituximab tested in different B cell lines.

[0052] Figure 22E The introduction of E345R resulted in a maximum CDC-mediated killing effect compared to wild-type rituximab, independent of the expression levels of complement regulatory proteins CD46(A), CD55(B), or CD59(C) in different B cell lines with comparable CD20 expression levels.

[0053] Figure 23 CDC kinetics. Compared to wild-type antibodies, the E345R antibody resulted in more rapid and greater amounts of CDC-induced target cell lysis.

[0054] Figure 24 CDC kinetics. Introducing the E345R mutation into the bispecific CD38xCD20 antibody resulted in more rapid and greater CDC-mediated target cell lysis.

[0055] Figure 25 CDC kinetics. Introducing the E345R mutation into the bispecific antibodies CD38xEGFR and CD20xEGFR, which bind monovalently to EGFR-negative Raji cells, resulted in more rapid and greater CDC-mediated target cell lysis.

[0056] Figure 26 A-26F: Wild-type antibody combined with mutant antibodies containing (AC)E345R and Q386K or (DF)E345R, E430G and Q386K for CDC testing of Wien133 cells. The IgG1-b12 mutant does not bind to Wien133 cells and was used as a negative control antibody.

[0057] Figure 27CDC efficacy of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing the E345R mutation.

[0058] Figure 28 The introduction of the Fc-Fc stable E345R mutation into wild-type CD38 antibody 005 resulted in enhanced killing of primary CLL cells in the in vitro CDC assay (mean ± standard deviation of mean).

[0059] Figure 29 As determined by ELISA, wild-type IgG1-005 and IgG1-005 mutants bind to FcRn in humans, mice, and macaques at pH 6.0.

[0060] Figure 30 The CDC efficacy of various rituximab mutants, wild-type rituximab, and incoherent negative control antibody IgG1-B12 in 20% normal human serum in Ramos and SU-DHL-4 cell lines.

[0061] Figure 31 The production of wild-type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S, and IgG1-005-S440Y, and thermally aggregated IgG (HAG) (positive control) in normal human serum was determined by Micro Vue C4d fragment ELISA.

[0062] Figure 32A and Figure 32B : By total human IgG ELISA ( Figure 32A ) and human CD38 specific ELISA ( Figure 32B The plasma clearance rates of administered wild-type IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005-E430F, IgG1-005-E430G, IgG1-005-E430S, IgG1-005-E430T and IgG1-005-S440Y in SCID mice were determined. Detailed Implementation

[0063] As described in this invention, surprisingly, mutations in amino acids not directly involved in Fc:C1q binding can enhance antibody CDC and 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 subsequently bound by C1q. Furthermore, although some mutations have been found to reduce CDC induction, certain combinations of such mutations in the same or different antibody molecules lead to a restoration of CDC induction and show further specificity for antibody oligomers, thereby promoting more specific CDC induction. As shown in the examples, specific mutational features that enhance CDC response also include improved ADCC response, increased affinity, improved internalization, and in vivo potency in mouse tumor model systems. These findings allow for new antibody-based therapeutics with enhanced CDC induction capacity, more selective CDC induction, and / or other improved effector functions.

[0064] The polypeptide variants (including antibody variants) of the present invention fully encompass the immunoglobulin-binding region and the full-length or partial Fc domain, and contain at least one or more mutations in the region corresponding to amino acid residues E345 to S440 of IgG1. This is not limited to theory, but is based on… Figure 1 The diagram illustrates and is referred to in this invention as “single mutant,” “double mutant,” and “hybrid mutant,” three different principles that suggest that the identified mutations lead to more effective and / or more specific CDC induction.

[0065] The improved C1q and / or CDC effects of the variants of this invention are primarily detected only in assays that allow antibody oligomer formation, such as in cell-based assays where the antigen is not fixed but is present on a fluid membrane. Furthermore, according to Figure 1 The principle shown in Figure C is a validation of the fact that these effects are obtained from modifications of more stable antibody oligomers rather than from direct C1q binding sites.

[0066] definition

[0067] The term "single mutant" should be understood as a variant of the invention that has an increased CDC compared to the parental peptide or antibody and may also have other effector functions.

[0068] The term "double mutant" should be understood as a variant that contains at least two mutations in the fragment and has improved CDC compared to a variant that contains only one of the at least two mutations, and may also have other enhanced effector functions.

[0069] The term “hybrid mutant” should be understood as a variant that, when used in combination with a second variant of the same or different polypeptide or antibody containing mutations in different amino acid residues of the segment, provides enhanced CDC and optionally other enhanced effector functions compared to one or more of the individual variants, the second variant, and the individual parent polypeptide or antibody.

[0070] The term "polypeptide comprising an immunoglobulin Fc domain and a binding region" in the context of this invention refers to a polypeptide comprising an immunoglobulin Fc domain and a binding region capable of binding to any molecule, such as a polypeptide, present on, for example, cells, bacteria, or viral particles. The Fc domain of an immunoglobulin is defined as an antibody fragment (known to those skilled in the art) typically generated after digestion of an antibody with papain, comprising two CH2-CH3 regions of the immunoglobulin and a linker region, such as a hinge region. The antibody heavy chain constant region defines the antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc domain mediates the effector function of the antibody with cell surface receptors and complement system proteins, known as Fc receptors. The binding region can be a polypeptide sequence capable of binding to cells, bacteria, or viral particles, such as a protein, protein ligand, receptor, antigen-binding region, or ligand-binding region. If the binding region is, for example, a receptor, "polypeptide comprising an immunoglobulin Fc domain and a binding region" can be prepared as a fusion protein of the immunoglobulin Fc domain and said binding region. If the binding region is an antigen-binding region, a "peptide comprising an immunoglobulin Fc domain and a binding region" can be an antibody, such as a chimeric antibody, a humanized antibody, or a human antibody, or an antibody containing only the heavy chain, or an ScFv-Fc fusion. A peptide comprising an immunoglobulin Fc domain and a binding region may typically include a linker region, such as a hinge region, and two CH2-CH3 regions of the immunoglobulin heavy chain; therefore, a "peptide comprising an immunoglobulin Fc domain and a binding region" can be a "peptide comprising at least an immunoglobulin Fc domain and a binding region." In the context of this invention, the term "immunoglobulin Fc domain" means the presence of a linker region (e.g., a hinge) depending on the antibody subtype, as well as immunoglobulin CH2 and CH3 regions, such as human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE. The peptide is not limited to human origin but can be of any origin, such as mouse or macaque origin.

[0071] The term "CH2 region" or "CH2 domain" as used in this 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 can also be any other subtype described in this invention.

[0072] The term "CH3 region" or "CH3 domain" as used in this invention refers to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system. However, the CH3 region can also be any other subtype described in this invention.

[0073] The term "immunoglobulin" refers to a class of structurally related glycoproteins 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 potentially linked by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated VH in this invention) and a heavy chain constant region. The heavy chain constant region typically consists of three domains (CH1, CH2, and CH3). Heavy chains are linked by disulfide bonds in so-called "hinge regions." Each light chain typically consists of a light chain variable region (abbreviated VL in this invention) and a light chain constant region. The light chain constant region typically consists of one domain, CL. VH and VL regions can be further subdivided into highly variable regions (or highly variable regions that can be highly variable in sequence and / or structure-defined loop forms), also known as complement-determining regions (CDRs), which are interspersed with more conserved regions, known as framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by the context, the amino acids in the constant region sequences of this invention are based on EU-index numbers (described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)).

[0074] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, an immunoglobulin molecule fragment, or a derivative thereof, which has the ability to specifically bind an antigen under typical physiological conditions for a considerably long period of time (e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or longer, about 48 hours or longer, about 3, 4, 5, 6, 7 or more days, etc.), or any other relevant functionally defined time period (e.g., sufficient to induce, promote, enhance, and / or regulate physiological responses associated with antibody-antigen binding, and / or sufficient to recruit effector activity). The antibodies of this invention comprise an immunoglobulin Fc domain and a binding region. Antibodies typically contain two CH2-CH3 regions and a linker region, such as a hinge region, such as at least an Fc domain. Therefore, the antibodies of this invention can comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. An antibody's constant or "Fc" domain mediates the binding of immunoglobulins to host tissues or factors, encompassing 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 complement activation pathway. Antibodies can also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically 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 cells or different cells or cell types. As stated above, unless otherwise stated or clearly contradicted by the context, in this invention, the term antibody includes an antibody fragment that contains at least a portion of the Fc region and retains the ability to specifically bind antigens. Such fragments can be provided by any known technology, such as enzyme digestion, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments covered in the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, which consist of only two heavy chains and are naturally occurring, for example, in camels (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), chain exchange engineered domains (SEED or Seed bodies), which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219));FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-FV (Xencor, WO2011 / 028952), dual variable domain immunoglobulin (Abbott, DVD-Ig, US Patent 7,612,181); dual domain biheaded antibody (Unilever; Sanofi Aventis, WO20100226923), bispecific antibody (ImClone / EliLilly), knock-in-hole antibody form (Genentech, WO9850431); DuoBody (Genmab, WO2011 / 131746); electrostatically controlled antibody form (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-domain antibodies (GSK / Domantis), two-in-one antibodies recognizing two targets (Genentech, Novlmmune), cross-linked MAbs (Karmanos Cancer Center), CovX bodies (CovX / Pfizer), IgG-like bispecific antibodies (ImClone / Eli Lilly, Shen, J., et al. J Immunol) Methods, 2007, 318(1-2):p.65-74), and DIG and PIG bodies (Pharmabcine), as well as biphilic retargeting molecules (Macrogenics' Fc-DART or Ig-DART, WO / 2008 / 157379, WO / 2010 / 080538), Zybodies (Zyngenia), conventional light chains (Crucell / Merus, US7262028) or conventional heavy chains (Novlmmune's κλ body), and methods involving peptide sequence fusion proteins containing antibody fragments fused to Fc domains, such as scFv fusions, such as ZymoGenetics / BMS's BsAb, BiogenIdec's HERCULES (US007951918), Emergent BioSolutions / Trubion's SCORPIONS, 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 (WO2012025525,WO2012025530), f-Star's mAb; 2 (WO2008 / 003116) and double scFv-fusions. It should also be understood that, unless otherwise specified, the term antibody also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies) generated, for example, using techniques developed by Sympogen and Merus (Oligoclonics), and antibody-like peptides such as chimeric antibodies and humanized antibodies. The resulting antibodies may potentially have any isotype.

[0075] The term "full-length antibody," as used in this invention, refers to an antibody (such as a parental or variant antibody) that contains all the constant and variable domains of the heavy and light chains corresponding to those domains typically found in wild-type antibodies of the same type.

[0076] The term "human antibody" as used in this invention is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" as used in this invention is not intended to include antibodies in which the CDR sequence derived from another mammalian species (e.g., mouse) has been transplanted into a human backbone sequence.

[0077] As used in this invention, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," and "mAb" refer to antibody molecular formulations with a single-molecule composition. A monoclonal antibody composition exhibits single-binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an Ab that exhibits single-binding specificity and possesses variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be generated from hybridomas comprising B cells obtained from transgenic or transchromosomal nonhuman animals (such as transgenic mice) and fused with immortalized cells, wherein the nonhuman animals comprise a complete set of human heavy chain transgenic and light chain transgenic cells rearranged to produce functional human antibodies.

[0078] In this invention, "isotype" refers to immunoglobulins encoded by heavy chain constant region genes (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotypes thereof such as IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype may be combined with a κ or λ light chain.

[0079] In the context of this invention, the term "monovalent antibody" means an antibody molecule that can bind to an antigen only with one of its antibody-binding domains, for example, having a monoantigen-antibody interaction and therefore not being able to cross-link with the antigen.

[0080] The term "target" as used in this invention should be understood in the context of this invention as a molecule that binds to the binding region of a polypeptide comprising an Fc domain and a binding region, and when used in the context of antibody binding, includes any antigen against which the antibody is prepared. The terms "antigen" and "target" are used interchangeably with respect to antibodies and have the same meaning or purpose for any aspect or embodiment of this invention.

[0081] The term "binding" as used in this invention, in the context of antibody binding to a predetermined antigen, generally refers to the use of the antigen as a ligand and the antibody as an analyte, measured in a BIAcore 3000 instrument by, for example, surface plasmon resonance (SPR) technology, corresponding to approximately 10 -6 M or smaller, such as 10 -7 M or smaller, such as 10 -8 M or smaller, such as 10 -9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 M or even smaller K D The affinity binding, and in accordance with the following K D The K has an affinity for binding to a predetermined antigen. D It is at least 10-fold lower than the specificity of its binding to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). This is equivalent to at least 100-fold lower, at least 1,000-fold lower, at least 10,000-fold lower, or at least 100,000-fold lower. The amount of lower affinity depends on the antibody's Ka. D Thus, when the antibody's K D At very low levels (i.e., when the antibody is highly specific), the amount of antigen affinity that is lower than that for nonspecific antigens can be at least 10,000 times. The term "K" as used in this invention... D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction."

[0082] The term "variant," "antibody variant," or "parental antibody variant" of this invention refers to an antibody molecule containing one or more mutations compared to a "parental antibody." Different terms are used interchangeably and have the same meaning or purpose for any aspect or embodiment of this invention. Exemplary forms of parental antibodies include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, or any combination thereof. Similarly, the term "variant," "variant of a polypeptide containing an immunoglobulin Fc domain and a binding region," or "variant of a parental polypeptide containing an immunoglobulin Fc domain and a binding region" of this invention refers to a "polypeptide containing an immunoglobulin Fc domain and a binding region" containing one or more mutations compared to a "parental polypeptide containing an immunoglobulin Fc domain and a binding region." Different terms are used interchangeably and have the same meaning or purpose for any aspect or embodiment of this invention. Exemplary mutations include the deletion, insertion, or substitution of amino acids in the parental amino acid sequence. Amino acid substitutions may replace a natural amino acid with another naturally occurring amino acid or a non-naturally occurring amino acid derivative. Amino acid substitutions may be conserved or non-conserved. In the context of this invention, conservative substitution is defined by substitutions between amino acid classes reflected in one or more of the following three tables:

[0083] Conservatively substituted amino acid residues

[0084] acid residues 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 residues 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)

[0085] Optional conserved amino acid residue substitution types

[0086] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0087] Alternative physical and functional classification of amino acid residues

[0088]

[0089]

[0090] In the context of this invention, substitution in the variants is represented as:

[0091] Original amino acid - position - substituted amino acid

[0092] Amino acid residues are represented using either a three-letter code or a single-letter code, containing the codes Xaa and X. Therefore, the symbol "E345R" or "Glu345Arg" signifies that the variant contains a glutamic acid substitution with arginine at position 345 of the corresponding parent antibody.

[0093] When the position itself is not present in the antibody, but the variant contains an amino acid insertion, for example:

[0094] Position-substituted amino acid; use symbols such as "448E".

[0095] Such symbols are particularly associated with modifications in homologous peptides or antibody series.

[0096] Similarly, when the identity of the substituted amino acid residue is not important:

[0097] Original amino acid - position; or "E345".

[0098] The original amino acid and / or substituted amino acid can include more than one, but not all, amino acid modifications. For example, glutamic acid at position 345 is replaced with arginine, lysine, or tryptophan.

[0099] "Glu345Arg, Lys, Trp" or "E345R, K, W" or "E345R / K / W", or "E345 to R, K or W" may be used interchangeably in the context of this invention.

[0100] Furthermore, 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, 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 designation 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. This similarly applies to each position involved in the present invention, which specifically includes any of such substitutions.

[0101] An amino acid or fragment in a sequence that “corresponds” to an amino acid or fragment in another sequence is (i) aligned with another amino acid or fragment using standard sequence alignment programs such as ALIGN, ClustalW, etc., typically with default settings, and (ii) has an amino acid or fragment that is at least 50%, at least 80%, at least 90%, or at least 95% sequence identical to SEQ ID NO:1. For example, it can be used... Figure 2 The sequence was compared with that shown in Figure 3 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.

[0102] This invention relates to variants, namely parental polypeptides and parental antibodies, and / or variant polypeptides and variant antibodies, which have a certain degree of identity with amino acids P247 to K447 of SEQ ID No: 1, 2, 3, 4 and 5, such parental and / or variant antibodies are referred to below as "homological antibodies".

[0103] For the purposes of this invention, the degree of identity between two amino acid sequences and between two nucleotide sequences is determined using the Needleman-Wunsch alignment (i.e., global alignment) procedure. This procedure is used for the alignment of peptide and nucleotide sequences. The default score matrix BLOSUM50 is used for peptide alignment, while the default identity matrix is ​​used for nucleotide sequence alignment. The penalty for the first residue of the gap is -12 (for peptides) and -16 (for nucleotides). The penalties for the other residues in the gap are -2 (for peptides) and -4 (for nucleotides).

[0104] "Alignment" is part of FASTA package version v20u6 (see WRPearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and WRPearson (1990), "Rapid and Sensitive Sequence Comparaison with FASTP and FASTA", Methods in Enzymology 183:63-98). FASTA protein alignment uses the Smith-Waterman algorithm, which has no limit on the gap size (see "Smith-Waterman algorithm", TFSmith and MSWaterman (1981) J. Mol. Biolo. 147:195-197).

[0105] As used in this invention, the term "effective cell" refers to immune cells that participate in the effector phase of an immune response relative to the cognitive and activation phases. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, and 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 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 the presentation of antigens to other components of the immune system, or bind to antigen-presenting cells. In some embodiments, antibody-driven classical complement activation can further enhance ADCC, leading to the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands of complement receptors (CRs) expressed on myeloid cells, such as CR3. Complement fragment recognition of CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, the expression of FcγRI has been found to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxicity of cells carrying FcγRI to the target. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. These C3 cleavage products can promote phagocytosis either directly by effector cells or indirectly by enhancing antibody-mediated phagocytosis.

[0106] As used in this invention, the term "vector" refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid fragment ligated into the vector. One type of vector is a "plasmid," which is a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid fragment can be ligated into a viral genome. Some vectors can replicate autonomously in the host cell to which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episome mammalian vectors). Other vectors (such as non-epithelial mammalian vectors) can be integrated into the host cell's genome upon introduction and thereby replicate along with the host genome. Furthermore, some vectors can guide the expression of genes operatively ligated to them. Such vectors are referred to in this invention as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technology are typically in plasmid form. In this specification, since plasmids are the most commonly used vector form, "plasmid" and "vector" are used interchangeably. However, this invention intends to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0107] The term "recombinant host cell" (or simply "host cell") as used in this invention refers to a cell in which an expression vector has been introduced. It should be understood that this term refers not only to the specific target cell but also to the progeny of such cells. Because mutations or environmental influences can lead to modifications in the offspring, such progeny may not actually be identical to the parent cells, but are still included within the scope of the term "host cell" as used in this invention. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli, and other eukaryotic hosts such as plant cells and fungi.

[0108] The term "transfected tumor" as used in this invention includes recombinant host cells expressing antibodies or target antigens, such as CHO cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi, including yeast cells.

[0109] The term "formulation" refers to formulations of antibody variants and mixtures of different antibody variants that, when interacting with cells (e.g., antigens expressed on the cell surface), cell membranes, viral particles, or other structure-associated antigens, may have an enhanced ability to form oligomers, thereby resulting in enhanced 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, for example, C1q binding affinity (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25 and 35); ADCC (Example 12), in vivo potency (Examples 20, 21), plasma clearance (Example 37), FcRn binding (Example 34), and target-independent fluid-phase complement activation (Example 36). Variations of the present invention, referred to as “single mutants,” “double mutants,” and “hybrid mutants,” and their exemplary preparation processes and methods of use are described in more detail below.

[0110] The term “affinity” as used in this invention refers to the binding strength of a molecule (e.g., an antibody) to another (e.g., a target or antigen) at a single site (e.g., the individual antigen-binding site of an antibody and the antigen monovalently).

[0111] The term "avidity" as used in this invention refers to the binding strength between multiple binding sites of two structures (such as between multiple antigen-binding sites of an antibody that simultaneously interacts with a target, or, for example, between an antibody and C1q). When more than one binding interaction is present, the two structures will dissociate only when all binding sites dissociate, and therefore the dissociation rate will be slower than that of a single binding site, thus providing a greater effective total binding strength (affinity) compared to the strength (affinity) of binding at a single binding site.

[0112] As used in this invention, the term "oligomer" refers to a molecule composed of more than one but a finite number of monomer units (such as antibodies), compared to a polymer, which is at least in principle composed 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 monomer units in an oligomer, for example, a tetramer consisting of four units and a hexamer consisting of six units.

[0113] The term "oligomerization" as used in this invention refers to the process of converting monomers into polymers to a limited extent. In this invention, Fc domain oligomerization is observed to occur after target binding of Fc domain-containing peptides (such as antibodies, preferably but not limited to those on the cell surface). Antibody oligomerization can be evaluated, for example, using cell surface C1q binding assays (as described in Examples 4 and 9), C1q potency assays (as described in Example 5), and complement-dependent cytotoxicity assays as described in Examples 6, 10, and 19.

[0114] The term "C1q binding" as used herein refers to C1q binding in the context of an antibody that binds to its antigen. An antibody that binds to its antigen should be understood to occur both in vivo and in vitro in the context described herein. C1q binding can be evaluated, for example, by using antibodies immobilized on an artificial surface (e.g., the plastic in an ELISA plate as described in Example 3), or by using binding to a predetermined antigen on a cell or viral surface (as described in Examples 4 and 9). Binding of C1q to antibody oligomers should be understood herein as a multivalent interaction leading to high-affinity binding.

[0115] As used in this invention, the term "complement activation" refers to the activation of the classical complement pathway, triggered by the binding of complement component C1q to an antibody that binds to its antigen. C1q is the first protein in the early events of the classical complement cascade, which involves a series of cleavage reactions culminating in the formation of an enzyme 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 triggers the late events of complement activation, in which the final complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade leads to pore formation, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be evaluated using C1q potency (as described in Example 5), CDC kinetics (as described in Examples 28, 29, and 30), CDC detection (as described in Examples 6, 10, 19, 25, 27, 33, and 35), or by C3b and C4b cell deposition methods as described by Beurskens et al., April 1, 2012, vol. 188 no. 7 3532-3541.

[0116] As used in this invention, the term "complement-dependent cytotoxicity" ("CDC") refers to an antibody-mediated complement activation process resulting from the cleavage of antibodies bound to antigens on cells or viral particles due to membrane pores created by MAC assembly. CDC can be evaluated by in vitro assays (such as CDC assays, where normal human serum is used as the complement source, as described in Examples 6, 10, 19, 25, 27, 33, and 35) or by the C1q potency assay described in Example 5 (where normal human serum has been limited in C1q).

[0117] As used in this invention, the term "antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to the mechanism by which cells expressing an Fc receptor that recognizes and binds to the constant region of an antibody kill antibody-coated target cells or viral particles. ADCC can be determined using methods such as the ADCC assay described in Example 12.

[0118] As used in this invention, the term "antibody-dependent phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells or viral particles are eliminated through internalization by phagocytes. The internalized antibody-coated target cells or viral particles are contained in vesicles called phagosomes, which subsequently fuse with one or more lysosomes to form phagolysosomes. ADCP can be evaluated using macrophages as effector cells and in vitro cytotoxicity assays performed by visual microscopy, as described in van Bij et al., Journal of Hepatology, Volume 53, Issue 4, October 2010, pp. 677-685. Alternatively, regarding, for example, phagocytosis by Staphylococcus aureus via PMN, as described in Example 14.

[0119] As used in this invention, the term "complement-dependent cytotoxicity" ("CDCC") refers to a mechanism by which cells expressing complement receptors that recognize complement 3 (C3) cleavage products covalently bound to target cells or viral particles kill target cells or viral particles due to antibody-mediated complement activation. CDCC can be evaluated in a manner similar to that described for ADCC.

[0120] The term "plasma half-life" as used herein refers to the time it takes for the concentration of a peptide in plasma to decrease to half of its initial concentration during the elimination phase (after the distribution phase). For antibodies, the distribution phase is typically 1–3 days, during which time the plasma concentration decreases by approximately 50% due to redistribution between plasma and tissue. Plasma half-life can be measured using methods known in the art.

[0121] As used herein, the term "plasma clearance" is a quantitative measurement of the rate at which a peptide is removed from the blood following administration of the peptide to a living organism. Plasma clearance can be calculated as dose / AUC (mL / day / kg), where the AUC value (area under the curve) is determined from the concentration-time curve according to Example 37.

[0122] As used in this invention, the term "downregulation" refers to a process, for example, by binding an antibody to a receptor, to reduce the number of molecules (such as antigens or receptors) on the cell surface.

[0123] As used in this invention, the term "internalization" refers to any mechanism by which an antibody or Fc-containing polypeptide is internalized from the cell surface and / or from the surrounding medium, for example, through endocytosis, into a target-expressing cell. Antibody internalization can be evaluated using direct detection methods that measure the amount of internalized antibody (such as, for example, the lysosomal colocalization assay described in Example 26).

[0124] As used in this invention, the term "antibody-drug conjugate" refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker to which the drug is attached, for example, to an antibody. The linker may be cleavable or non-cleavable in the presence of malignant cells; wherein the antibody-drug conjugate kills malignant cells.

[0125] As used in this invention, “antibody-drug conjugate uptake” refers to the process by which an antibody-drug conjugate is attached to a target on a cell, subsequently taken up / engulfed by the cell membrane, and thus 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 ADCs in in vitro killing assays.”

[0126] As used in this invention, the term "apoptosis" refers to programmed cell death (PCD) that can occur in cells. Biochemical events lead to characteristic cellular alterations (morphological changes) and death. These alterations include vacuolization, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Antibodies binding to certain receptors can induce apoptosis.

[0127] Fc receptor binding can be measured indirectly as described in Example 12.

[0128] The term "FcRn" as used in this invention refers to the neonatal Fc receptor as an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk through the intestinal epithelial cells of newborn rodents into the newborn bloodstream. Further research revealed a similar receptor in humans. However, in humans, it has been found in the placenta to help facilitate the transport of maternal IgG to the growing fetus and has also been shown to play a role in detecting IgG turnover. FcRn binds IgG at an acidic pH of 6.0–6.5, but not at neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal lumen (the interior of the intestine) at slightly acidic pH and ensure efficient unidirectional transport to the basal side (inner side of the body) at pH neutral to alkaline (pH 7.0–7.5). This receptor functions in adult IgG rescue through its occurrence in the endocytic pathway within endothelial cells. FcRn receptors in acidic endosomes bind to IgG internalized via endocytosis, thereby recycling it to the cell surface and releasing it at the alkaline pH of the blood, thus preventing it from undergoing lysosomal degradation. This mechanism also explains the longer half-life of IgG in the blood compared to other isotypes. Examples 13 and 34 describe the detection of IgG binding to FcRn at pH 6.0 in an ELISA.

[0129] The term "protein A" as used in this invention refers to the 56 kDa MSCRAMM surface protein originally discovered on the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cellular osmotic pressure, and a two-component system called ArlS-ArlR. Due to its ability to bind immunoglobulins, it has also been found to be used in biochemical research. It consists of five homologous Ig-binding domains folded into a triple helix bundle. Each domain can bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain Fc region of most immunoglobulins (overlapping with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind to bacteria via their Fc region, rather than simply via their Fab region, thereby disrupting opsonization, complement activation, and phagocytosis.

[0130] The term "protein G" as used in this invention refers to an immunoglobulin-binding protein expressed in group C and G streptococci, which is very similar to protein A but has different specificities. It is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) cell surface protein, which is used in antibody purification through its binding to the Fc region.

[0131] Methods affecting peptide CDC

[0132] It should be understood that all embodiments of the parental antibody, first parental antibody, or second parental antibody described in this invention are also applicable to other parents, first parents, or second polypeptides that include the immunoglobulin Fc domain and binding region.

[0133] On one hand, the present invention relates to a method for improving complement-dependent cytotoxicity (CDC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding domain, the method comprising introducing a mutation in one or more amino acid residues of the parent polypeptide selected from the group corresponding to E430X, E345X and S440W in the Fc region of the human IgG1 heavy chain.

[0134] In one embodiment, the parental polypeptide may be a parental antibody comprising the Fc domain and antigen-binding region of an immunoglobulin.

[0135] Introducing mutations into a parent polypeptide using the methods or uses of the present invention yields a variant polypeptide (which may also be referred to herein as a "variant"). Therefore, the methods of the present invention can be performed to obtain any variant or variant polypeptide described herein.

[0136] The variant peptides obtained from the methods or uses of this invention have increased CDC compared to the parent peptides. Typically, the effect of a peptide on effector function can be determined by the EC50 value, which is the peptide concentration required to obtain half of the maximum cleavage value.

[0137] Maximum cleavage is the cleavage obtained when using a saturated amount of peptide, where saturation means the amount of peptide when all targets of the peptide are bound by the peptide.

[0138] The terms “enhanced CDC,” “improved CDC,” or “enhanced effector function,” or “improved effector function,” in the context of this invention, refer to a decrease in the EC50 value of the variant peptide compared to the parent peptide. The decrease in the EC50 value can be, for example, at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “enhanced CDC,” “improved CDC,” or “enhanced effector function,” or “improved effector function” means that, under the condition that the parent peptide cleavage is less than 100% of all cells, the maximum number of lysed cells (where the total number of cells is set to 100%) is increased, 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] The enhanced or improved effector function of a variant can be detected by cloning the IgG1-005 or IgG1-7D8 heavy chain variable domain into the variant and testing its potency in a CDC assay, as described for Daudi (Example 6) and Wien (Example 10). Using the IgG1-7D8 HC variable domain and Daudi cells, enhancement was defined as an EC50 value more than 2-fold lower than the EC50 of IgG1-7D8 under study conditions, such as approximately 2-fold, approximately 3-fold, approximately 5-fold, approximately 10-fold, or more than 10-fold lower EC50 values ​​(at which the maximum cleavage half-concentration was observed). Using the IgG1-005 HC variable domain and Daudi cells, enhancement was defined as an EC50 value more than 2-fold lower than the EC50 of IgG-005 under study conditions, such as approximately 2-fold, approximately 3-fold, approximately 5-fold, approximately 10-fold, or more than 10-fold lower EC50 values ​​(at which the maximum cleavage half-concentration was observed). Using IgG1-7D8 HC variable domain and Wien133 cells, enhancement was defined as an EC50 value more than 2-fold lower than that of IgG1-7D8 under study conditions, such as approximately 2-fold, 3-fold, 5-fold, 10-fold, or more than 10-fold lower EC50 values ​​(at the concentration where the maximum cleavage half was observed). Using IgG1-005 HC variable domain and Wien133 cells, enhancement was defined as an enhancement ranging from 10% to 100% of the maximum cleavage value across all cells, such as approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, and approximately 100%. An improvement in CDC potency can also be defined as an EC50 greater than 2 times that of IgG-005 under the study conditions, such as 2 times lower, approximately 3 times lower, approximately 5 times lower, approximately 10 times lower, or more than 10 times lower (the concentration at which the maximum lysis half was observed under conditions where lysis of Wien133 cells could be detected).

[0140] The inventors of this invention have surprisingly discovered that mutations at these specific positions have an improving effect on variant antibodies CDC, which are obtained by introducing one or more mutations into the parent antibody using the method according to the invention (e.g., as shown in Example 19). Not limited to theory, it is believed that substitution of one or more amino acids from the above-mentioned positions will induce oligomerization. The antibody binds with higher affinity (Example 2 illustrates this; direct labeling of IgG-7D8-E345R yielded increased binding to Daudi cells compared to IgG-7D8-WT), which allows the antibody to bind to cells for a longer period and thereby enable different effector functions, such as enhanced C1q binding, C1q potency CDC, ADCC, internalization, ADCP, and / or in vivo potency. These effects have been illustrated by Examples 4 (C1q binding on cells), Example 5 (C1q potency in CDC assay), Examples 6, 7, 27, 28 and 29 (CDC assay), Example 12 (ADCC), Example 26 (internalization), Examples 21 and 22 (in vivo potency), plasma clearance (Example 37), FcRn binding (Example 34), and target-independent fluid-phase complement activation (Example 36).

[0141] Therefore, mutations of amino acid residues selected from those corresponding to E430X, such as E430G, E430S, E430F or E430T, E345X, such as E345K, E345Q, E345R or E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain can also be referred to as “single mutation” aspects or “CDC enhancing mutations” in the context of this invention.

[0142] Therefore, in one embodiment, in the method of improving CDC, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of the human IgG1 heavy chain.

[0143] In a preferred embodiment, in the method of improving CDC, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0144] In one embodiment, the parental polypeptide is a parental antibody comprising an immunoglobulin Fc domain and an antigen-binding region.

[0145] In another aspect, the present invention also relates to a method for enhancing CDC and antibody-dependent cell-mediated cytotoxicity (ADCC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, the method comprising introducing a mutation in one or more amino acid residues of the parent polypeptide corresponding to E430X, E345X, and S440W in the Fc region of the human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid.

[0146] In one embodiment, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of the human IgG1 heavy chain.

[0147] In a preferred embodiment, the mutation in one or more amino acid residues is selected from the group corresponding to positions E345R, E430T, and E430F in the Fc region of the human IgG1 heavy chain.

[0148] In one embodiment, at least one other effector function of the antibody, such as C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc-γ receptor binding, protein A binding, protein G binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhancing cytotoxicity, antibody-mediated binding to complement receptors of opsonized antibodies, antibody-mediated phagocytosis (ADCP), internalization, apoptosis, and / or binding to complement receptors of opsonized antibodies is also enhanced, such as ADCC.

[0149] In one embodiment, the parental polypeptide is a parental antibody comprising an immunoglobulin Fc domain and an antigen-binding region.

[0150] In one implementation, the CDC of the parent antibody is enhanced when the parent antibody binds to its antigen on an antigen-expressing cell, cell membrane, or viral particle.

[0151] In one implementation, the parental antibody is a monospecific, bispecific, or multispecific antibody.

[0152] In another aspect, the present invention relates to a method for enhancing complement-dependent cytotoxicity (CDC) of a parent antibody, said parent antibody being a bispecific antibody comprising a first polypeptide and a second polypeptide, said first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, said second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of an immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and said method comprising introducing a mutation into one or more amino acid residues in the first and / or second CH2-CH3 region, said mutation being selected from the group consisting of E430X, E345X, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, and wherein

[0153] The first CH2-CH3 region contains additional amino acid mutations at positions selected from the group corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the human IgG1 heavy chain Fc region; and the second CH2-CH3 region contains additional amino acid mutations at positions selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the human IgG1 heavy chain Fc region, and the additional amino acid mutations in the first CH2-CH3 region are different from the additional amino acid mutations in the second CH2-CH3 region.

[0154] In one embodiment, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of the human IgG1 heavy chain.

[0155] In a preferred embodiment, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0156] In one embodiment, the method includes introducing a mutation into only one of the first or second polypeptides of the bispecific antibody.

[0157] In one embodiment, the method includes introducing a mutation into both the first and second peptides of a bispecific antibody.

[0158] In a preferred embodiment, other amino acid mutations in the first CH2-CH3 region occur at the position of K409, such as K409R, in the Fc region corresponding to the human IgG1 heavy chain; and other amino acid mutations in the second CH2-CH3 region occur at the position of F405, such as F405L, in the Fc region corresponding to the human IgG1 heavy chain.

[0159] The inventors of this invention have also discovered that introducing mutations into amino acid residues corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain reduces the effector function of the parental antibody (Examples 5, 6, 10).

[0160] As shown in Example 6, the amino acid substitution at position K439E or S440K as a "single mutant" reduces CDC compared to any of the first mutations according to the method of the present invention.

[0161] The variant antibodies obtained from the method described above, which reduces effector function, exhibit reduced effector function compared to the parent antibody. Typically, the effect of an antibody on effector function can be measured by the EC50 value, which is the antibody concentration required to obtain the maximum cleavage half-value.

[0162] Maximum lysis is the lysis obtained when using a saturated amount of antibody, where saturation means the amount of antibody that binds to all the antigens present in the antibody.

[0163] The term "reduced effector function" in the context of this invention refers to an increase in the EC50 value of the variant antibody compared to the parent antibody. This increase in EC50 value can be, for example, at least or about 2-fold, such as at least or about 3-fold, at least or about 5-fold, or at least or about 10-fold. Optionally, "reduced effector function" means that, under conditions where the parent antibody lyses less than 100% of all cells, the maximum number of lysed cells is reduced, for example, by 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%.

[0164] The reduced effector function of a variant can be detected by cloning the IgG1-005 or IgG1-7D8 heavy chain variable domain into the variant and testing its potency in a CDC assay, as described for Daudi cells (Example 6) and Wien 133 cells (Example 10). Using the IgG1-7D8 HC variable domain and Daudi cells, a reduction is defined as an EC50 value that is more than 2-fold lower than the EC50 of IgG1-7D8 under study conditions, such as approximately 2-fold, approximately 3-fold, approximately 5-fold, approximately 10-fold, or more than 10-fold lower EC50, where EC50 is the concentration at which the maximum cleavage half-maximum is observed. Using the IgG1-005 HC variable domain and Daudi cells, a reduction is defined as an EC50 value that is more than 2-fold lower than the EC50 of IgG-005 under study conditions, such as approximately 2-fold, approximately 3-fold, approximately 5-fold, approximately 10-fold, or more than 10-fold lower EC50, where EC50 is the concentration at which the maximum cleavage half-maximum is observed. Using IgG1-7D8 HC variable domain and Wien133 cells, a reduction will be defined as an EC50 value that is more than 2-fold lower than that of IgG1-7D8 under study conditions, such as approximately 2-fold, approximately 3-fold, approximately 5-fold, approximately 10-fold, or more than 10-fold lower EC50 values, where EC50 is the concentration at which the maximum lysis was observed to occur. Using IgG1-005HC variable domain and Wien133 cells, a reduction will be defined as a reduction in the maximum lysis range of 10% to 100% across all cells, such as reductions of approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, and approximately 100%. A decrease in CDC potency can also be defined as a decrease of more than 2 times the EC50 of IgG-005 under study conditions, such as approximately 2 times, approximately 3 times, approximately 5 times, approximately 10 times, or more than 10 times the EC50 value, where EC50 is the concentration at which the maximum lysis half is observed under conditions where lysis of Wien133 cells can be detected.

[0165] In another aspect, the present invention relates to a method disclosed according to the present invention and embodiments thereof, the method comprising introducing a mutation at one of a plurality of positions other than S440Y and S440W, and further introducing mutations in the following:

[0166] (i) In each of the amino acids corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation on S440 is not S440Y or S440W.

[0167] (ii) In each of the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, such as K447K / R / H and 448E / D in the Fc region of the human IgG1 heavy chain, preferably K447K and 448E in the Fc region of the human IgG1 heavy chain, or

[0168] (iii) In each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of the human IgG1 heavy chain, such as K447D / E, 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain, preferably K447E, 448K and 449P in the Fc region of the human IgG1 heavy chain.

[0169] For embodiments that introduce other mutations as described in steps (ii) or (iii) above, it should be noted that the lysine residue at position K447 is cleaved during antibody production in cells under normal circumstances. This can be prevented by adding one or more other amino acid residues (such as 448 or 448 / 449) to protect position K447. This is further described in WO2013 / 0048411 (Genmab A / S).

[0170] In one embodiment, the method includes introducing a mutation into one of a plurality of positions other than S440Y and S440W, and introducing the mutation into each of the amino acid residues corresponding to K439 and / or S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W.

[0171] In a preferred embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E, and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R.

[0172] In one embodiment, the parental polypeptide is a parental antibody comprising an immunoglobulin Fc domain and an antigen-binding region.

[0173] In one embodiment, the parental antibody is a monospecific, bispecific, or multispecific antibody. A bispecific antibody can be any of the embodiments described herein.

[0174] Furthermore, any mutations listed in Table 1 can be introduced into bispecific antibodies. Example 24 shows that introducing the E345R mutation into a bispecific CD20xEGFR antibody enhances CDC efficacy. Examples 23, 29, and 30 also describe several different bispecific antibodies containing mutations according to the present invention.

[0175] Introducing mutations into two amino acid residues in the parental antibody corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain (provided the mutation at S440 is not S440Y or S440W) is also referred to in this invention as a “double mutant.” As elsewhere described, S440Y or S440W mutations have been found to increase CDC when introduced into the parental peptide.

[0176] As described elsewhere, the inventors have discovered that introducing the identified mutations at amino acid residues corresponding to K439 or S440 in the Fc region of the human IgG1 heavy chain leads to a reduction in effector function (Examples 5, 6, 10). However, when repressive mutations are introduced at the 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 parental antibodies without mutations at K439 and S440. However, without being limited to any theory, it is considered that the presence of K439 and S440 mutations restricts effector function to oligomeric complexes that correspond only to antibodies containing both K439 and S440 mutations. Therefore, if a therapeutic antibody contains K439 and S440 mutations, it is not limited to any theory that when such a therapeutic antibody is administered to a patient, effector function induction is restricted to an oligomeric antibody complex containing a therapeutic antibody with K439 / S440 mutations but not the patient's autoantibody, which does not contain K439 and S440 mutations, thereby limiting any potential side effects caused by the interaction between the therapeutic antibody and the patient's autoantibody.

[0177] When the mutation at position K439 and / or S440 is combined with the first mutation, enhancement of CDC and increased specificity of CDC are obtained. In a similar manner, enhancement of CDC and increased specificity of CDC can be obtained by introducing the mutations disclosed in embodiments (ii) and (iii) above.

[0178] In another aspect, the present invention relates to a method for enhancing complement-dependent cytotoxicity (CDC) of at least a combination of first and second parental peptides, wherein each of the first and second parental peptides comprises an Fc domain and a binding region of an immunoglobulin, wherein the method comprises: introducing a mutation in one or more amino acid residues into at least the first and / or second parental peptide, said mutation being selected from the group consisting of E430X, E345X, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0179] In one embodiment, the method includes introducing a mutation in one or more amino acid residues into at least a first and / or a second parental polypeptide, said mutation being selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0180] In a preferred embodiment, the method includes introducing a mutation in one or more amino acid residues into at least a first and / or a second parental polypeptide, said mutation being selected from the group consisting of E430G, E430S, E345K, and E345Q corresponding to the Fc region of the human IgG1 heavy chain.

[0181] In one embodiment, the method includes introducing the same or different mutations into both the first and second parental polypeptides.

[0182] In other implementations, the method includes:

[0183] (i) Introducing a mutation into one or more amino acid residues of the first parent polypeptide, said mutation being selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0184] (ii) Provide a second parent polypeptide that does not contain a mutation in one or more amino acid residues selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region corresponding to the human IgG1 heavy chain.

[0185] In one embodiment, the method includes introducing a mutation in one or more amino acid residues into a first parent polypeptide, said mutation being selected from the group consisting of E430G, E430S, E345K, or E345Q corresponding to the Fc region of the human IgG1 heavy chain.

[0186] In other embodiments, the mutations at one or more locations are not S440Y and S440W, and the method further comprises the following steps

[0187] (i) Introducing a second mutation into the first parent polypeptide at the amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain; and

[0188] (ii) Introducing a second mutation into the second parent polypeptide at the amino acid residue corresponding to the S440 position in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W; wherein steps (i) and (ii) may alternatively be

[0189] (iii) Introduce a second mutation into the first parent polypeptide, which corresponds to the amino acid residue at position S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W.

[0190] (iv) Introduce a second mutation into the second parent polypeptide at the amino acid residue corresponding to the K439 position in the Fc region of the human IgG1 heavy chain.

[0191] The second parent polypeptide can be any parent polypeptide that does not provide a sufficient CDC response after binding to the target cell.

[0192] Therefore, without being bound by theory, it is believed that the method provides a first variant polypeptide containing a mutation in one or more amino acid residues as listed above, thus the variant polypeptide has an enhanced CDC response, and provides a second variant polypeptide that does not contain such a mutation, thereby obtaining a CDC response of the second parent polypeptide.

[0193] Combining a first antibody containing a mutation that enhances CDC with a second antibody not modified according to the invention can result in an enhanced CDC, as shown in Example 31. Therefore, in one embodiment, this method can be used to combine a therapeutic antibody that has been proven safe but not sufficiently effective (or for which enhanced efficacy is desired) as a second antibody with a first antibody containing the mutation, thereby obtaining an effective combination.

[0194] Examples of suitable secondary antibodies that do not contain mutations in amino acid residues include, but are not limited to, any of the following mutations selected from those corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of the human IgG1 heavy chain: (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; alefacept; alemtuzumab; Alirocumab; altumomab; Altumomabpentetate; alvircept sudotox; amatuximab; AMG714 / HuMax-IL15; anatumomab mafenatox); Anrukinzumab (=IMA-638); apolizumab; acitumomab; aesilizumab; atacicept; atinumab; Atlizumab (=tocilizumab); atorolimumab; baminercept; Bapineuzumab; basiliximab; bavituximab; bectumomab; beetacept; 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; cixutumab; Clazakizumab; clenoliximab; Clivatuzumab tetraxetan; conatumumab; conbercept; CR6261; crenezumab; dacetuzumab; daclizumab; dalantercept; dalotuzumab; daratumumab; Demcizumab; denosumab; detumomab; dorlimomab aritox; drozitumab; dulaglutide; ecrometasemab; eculizumab; edobacomab; edrecolomab; efalizumab; efungumab; elotuzumab; elsilimomab; enavatuzumab; enlimomab; pegolimomab pegol); enokizumab; ensituximab; epitumomab; epitumomab cituxetan; epratuzumab; erlizumab; ertumaxomab; etaracizumab; etrolizumab; exbivirumab; Fanolesomab; faralimomab; farletuzumab; fasinumab; FBTA05; felvizumab; Fezakinumab; ficlatuzumab; figitumumab; flanvolumab; fontolizumab; foralumab; foravirumab;fresolimumab; fulranumab; galiximab; ganitumab; gantenerumab; gavilimomab; gemtuzumab; gemtuzumab ozogamicin; geokizumab; girentuximab; glembatumumab; glembatumumab vedotin; 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-TAC antibody as disclosed in WO2010 / 066803 or WO Published in 2011 / 157741 are the following anti-tissue factor (TF) antibodies: ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, inclacumab, indatuximab ravtansine, infliximab, inolimomab, and inotuzumab. ozogamcin; intetumumab; iodine (I241) girentuximab; ipilimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; lemalesomab; lenercept; lerdelimumab; lexatumumab; libivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; lumiliximab; mapatumumab; maslimoma; matuzumab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minretumomab; mirococept; mitumomab; mogamulizumab; morolimumab;motavizumab; moxetumomab; pasudotox; muromonab-CD3; nacolomab tafenatox; namilumab; naptumomab estafenatox; narnatumab; natalizumab; nebacumab; necitumumab; nerelimomab; nimotuzumab; Nivolumab; Nofetumomab; merpentan; obinutuzumab; Ocaratuzumab; ocrelizumab; odulimomab; olaratumab; olokizumab; omalizumab; onartuzumab; onercept; oportuzumab monatox; oregovomab; otelixizumab; oxelumab; ozoralizumab; pagibazimab; palivizumab; panitumumab; panobacumab; pascolizumab; pateclizumab; patritumab; pegsunercept; pemtumomab; pertuzumab; pexelizumab; pintumomab; placulumab; ponezumab; priliximab; pritumumab; PRO 140; 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 / pendimethicone; secukinumab; sevirumab; sibrotuzumab; sifalimumab; siltuximab; siplizumab; sirukumab; solanezumab; solitomab; sonetuzumab; sonetuzumab; sotatercept; stamulumab; sulesomab; suvizumab; tabalumab; tabatuzumab tetraxetan; tadocizumab; talizumab; tanezumab; tapetumomab / aptox; tefibazumab; telimomab aritox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; ticilimumab (tremelimumab); tigatuzumab; TNX-650; atlizumab; toralizumab; torapsel; tositumomab; tralokinumab; trastuzumab emtansine; TRBS07; trebananib; tregalizumab; tremelimumab; tucotuzumab Celmoleukin; tuvirumab; ublituximab; urelumab; urtoxazumab; ustekinumab; vapaliximab; vaporizumab; vedolizumab; veltuzumab; vepalimomab; vesencumab; visilizumab; volociximab; Vorsetuzumab mafodotin; votumumab; zalutumumab; zanolimumab; ziralimumab; and zolimomab aritox.

[0195] As shown in Example 10, the first and second variant antibodies show a preference for oligomerization compared to any wild-type or naturally occurring antibody.

[0196] In one embodiment, the mutation at the position of K439 in the Fc region corresponding to the human IgG1 heavy chain is K439D / E, and / or the mutation at the position of S440 in the Fc region corresponding to the human IgG1 heavy chain is S440K / R.

[0197] Therefore, the improvement in specificity refers to "CDC induction". Thus, the method described in one embodiment is a method for improving the induction specificity of effector function through a combination of at least first and second parental antibodies.

[0198] By performing a method to enhance the specificity of the inducible effector function through the combination of at least the first and second parental peptides, a combination of the first and second variant peptides was obtained.

[0199] By introducing mutations into the parental peptides K439 or S440, the resulting variant peptides exhibit reduced effector function compared to the parental peptides. However, as described elsewhere in this invention, the mutations at K439 and S440 can compensate for each other to restore the effector function of the peptide containing both mutations. This compensatory ability of the mutations at K439 and S440 can be similarly applied to both peptides. Therefore, when a mutation at K439 is introduced into the first parental peptide and a mutation at S440 is introduced into the second parental peptide, or vice versa, no further reduction in effector function is observed due to the combined use of the first and second variant peptides. The terms "enhanced specificity" or "improved specificity" in this context refer to the fact that the effector response induced by the combination of the first variant peptide containing the K439 mutation and the second variant peptide containing the S440 mutation is higher than the effector response induced by either the first variant peptide containing the K439 mutation or the second variant peptide containing the S440 mutation.

[0200] The specificity of the oligomer is improved by introducing two amino acid substitutions at K439 and S440.

[0201] When the 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.

[0202] In one embodiment, at least the first and second parental peptides bind to the same binding site, or, in relation to the antibody, bind to the same epitope.

[0203] In one embodiment, at least the first and second parental polypeptides bind to different binding sites on the same target, or, in terms of antibodies, bind to different epitopes on the same antigen.

[0204] In one embodiment, at least the first and second parental peptides bind to different epitopes on different targets.

[0205] In one embodiment, the first and second parental polypeptides are first and second parental antibodies having the same or different VL and VH sequences.

[0206] In one embodiment, the combination of at least the first and second parental polypeptides comprises a first parental polypeptide and a second parental polypeptide.

[0207] In one embodiment, specificity is enhanced when the combination of the first and second parental polypeptides binds to their binding sites or antigens on antigen-expressing cells, cell membranes, or viral particles.

[0208] Therefore, in another aspect, the present invention also relates to using mutations at two or more amino acid residues of a polypeptide to improve, for example, the specificity of CDC induced by the polypeptide when binding to its antigens on antigen-expressing cells, cell membranes, or viral particles, wherein,

[0209] The first mutation occurs at the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain;

[0210] The second mutation occurs at the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain.

[0211] In one embodiment, the first and second parental polypeptides are first and second parental antibodies, each comprising an immunoglobulin Fc domain and an antigen-binding region.

[0212] In one implementation, the first and second parental antibodies are monospecific, bispecific, or multispecific antibodies.

[0213] In one embodiment, the first and / or second parental antibody is a bispecific antibody comprising a first polypeptide containing a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, and a second polypeptide containing a second CH2-CH3 region 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 contains a further amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of the human IgG1 heavy chain; and wherein the second CH2-CH3 region contains a further amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of the human IgG1 heavy chain; and wherein the further amino acid mutation in the first CH2-CH3 region is different from the further amino acid mutation in the second CH2-CH3 region.

[0214] In a preferred embodiment, the first CH2-CH3 region contains a further amino acid mutation at the position corresponding to K409 in the human IgG1 heavy chain Fc region, such as K409R; and the second CH2-CH3 region contains a further amino acid mutation at the position corresponding to F405 in the human IgG1 heavy chain Fc region, such as F405L.

[0215] By performing this method, a combination of at least first and second variant antibodies was obtained. The at least first and second variant antibodies obtained by this method, when combined, have a higher CDC compared to a combination of first and second parental antibodies.

[0216] The term "enhanced CDC" should be understood as described in this invention.

[0217] The first and / or second parental antibodies can be any parental antibody described in this invention.

[0218] Specifically, methods for enhancing the CDC of the first and second antibody combination can be performed to obtain a first and / or second antibody having any of the characteristics of the variant antibodies described in this invention.

[0219] In one implementation, at least the first and second parental antibodies bind to the same epitope.

[0220] In one implementation, at least the first and second parental antibodies bind to different epitopes on the same antigen.

[0221] In one implementation, at least the first and second parental antibodies bind to different epitopes on different targets.

[0222] In one embodiment, at least the first and second parental antibodies have the same or different VL and VH sequences.

[0223] In one embodiment, the combination of at least the first and second parental antibodies comprises a first parental antibody and a second parental antibody.

[0224] In one embodiment, the combination of at least the first and second parental antibodies includes more parental antibodies, such as a third, fourth, or fifth parental antibody. In one embodiment, the first and second bispecific or multispecific parental 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. Therefore, in one embodiment, the at least first and second parental antibodies are bispecific or multispecific antibodies that bind to the same antigen or different epitopes on different antigens.

[0225] In one embodiment of the method and / or use of the present invention, the parental antibody (whether it is a parental antibody, a first parental antibody, or a second parental antibody) contains other mutations besides those of the present invention that have been found to affect effector function. Such mutations may be introduced simultaneously with the present invention mutations affecting effector function, or they may be introduced sequentially; the method or use of the present invention is not limited to the simultaneous or sequential introduction of mutations. The bispecific antibody can be any bispecific antibody, and the method and use of the present invention are not limited to any particular bispecific form, as different forms are anticipated to be usable.

[0226] In one implementation, the method does not alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the parental peptide or parental antibody.

[0227] In one implementation, as determined by the method disclosed in Example 34, the method does not alter the binding of the parental polypeptide or parental antibody to the neonatal Fc receptor (FcRn).

[0228] In one embodiment, as determined by the method disclosed in Example 34, by measuring the change in absorbance at OD405, the method does not increase or decrease the binding of the parental peptide or parental antibody to the neonatal Fc receptor (FcRn) by more than 30%, for example, more than 20%, 10%, or 5%.

[0229] In one embodiment, as determined by the method disclosed in Example 34, the method does not increase the apparent affinity of the parent peptide or parent antibody to the mouse neonatal Fc receptor (FcRn) by more than 0.5-fold, or decrease the apparent affinity of the parent peptide or parent antibody to the mouse neonatal Fc receptor (FcRn) by more than 2-fold.

[0230] In one implementation, as determined by the method disclosed in Example 37, the method does not alter the plasma clearance rate of the parental polypeptide or parental antibody.

[0231] In one embodiment, as determined by the method disclosed in Example 37, the method does not increase or decrease the plasma clearance rate of the parental peptide or parental antibody by more than 3.0 times, such as more than 2.5 times, 2.0 times, 1.5 times, or 1.2 times.

[0232] In one implementation, as determined by the method disclosed in Example 36, the method does not alter the target-independent fluid phase complement activation of the variant.

[0233] In one implementation, the method does not alter the plasma half-life of the parental peptide or parental antibody.

[0234] Any mutation or combination thereof described in this invention may be introduced using the method of this invention.

[0235] Mutations selected from exemplary or preferred amino acid substitutions can be tested using appropriate detection methods that allow for the formation of oligomers of antigen-bound antibodies and the detection of enhanced C1q binding, complement activation, CDC, ADCC, and / or internalization, as described in the examples. For example, C1q binding affinity can be determined using cells expressing antigens that express antibody variants, according to a detection method similar to that described in Example 4. Exemplary CDC detection methods are provided in Examples 5, 6, 10, 16, 19, 22, 23, 24, 25, or 35. Exemplary ADCC detection methods are provided in Example 12. Exemplary internalization detection methods are provided in Example 26. Finally, to distinguish between mutations in amino acid residues that directly participate in C1q binding and 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. Plasma clearance can be compared according to the assay described in Example 37, FcRn binding can be compared according to Example 34, and target-independent fluid phase complement activation can be evaluated according to the assay in Example 36.

[0236] In one implementation, the mutation at one or more amino acid residues can be an amino acid substitution, an amino acid deletion, or an amino acid insertion.

[0237] In one implementation, a mutation at one or more amino acid residues can be an amino acid deletion.

[0238] In one implementation, a mutation at one or more amino acid residues can be an amino acid insertion.

[0239] In one implementation, the mutation at one or more amino acid residues can be an amino acid substitution.

[0240] In one embodiment, the mutation at one or more amino acid residues may be selected from any amino acid substitution or deletion listed in Table 1.

[0241] Therefore, 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 a further preferred embodiment, E345X is E345K or E345Q.

[0242] In another further embodiment, E430X may be E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W, or Y; specifically, E430T, S, G, F, or H. In a further preferred embodiment, E430X is E430G or E430S. In another embodiment, optionally, the mutation is not located at an amino acid residue directly involved in C1q binding, 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.

[0243] In one implementation, one or more mutations are a single mutation, meaning that no more than one mutation is introduced into the parent antibody.

[0244] 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 in Table 1.

[0245] Any combination of mutations described in this invention can be introduced using the method of this invention.

[0246] In one embodiment, the method includes introducing more than one mutation, such as two, three, four, or five, specifically two or three mutations, into the parent polypeptide, of amino acid residues selected from the group corresponding to E345X, E430X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain. For example, at least one amino acid residue corresponding to E345X, E430X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain can be mutated, such as two or all of E345X, E430X, S440Y, and S440W, optionally in combination with mutations at one or more other amino acids listed in Table 1. At least two mutations can be any amino acid residue substitution at position E345 combined with any amino acid substitution at positions E430, S440Y, or S440W, or any amino acid residue substitution at position E430 combined with any amino acid substitution at positions S440Y or S440W. In a further embodiment, two or three mutations are introduced into the amino acid residues of the parent antibody, said mutations being selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0247] This combination of two mutations in amino acid residues, wherein the mutations are selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y and E430X / S440W in the Fc region of the human IgG1 heavy chain.

[0248] In the method or use according to the invention, when the antibody binds to its antigen, the CDC is increased.

[0249] Without being limited by any theory, it is believed that when an antibody binds to its antigen, it enhances the CDC, where the antigen is on the antigen-expressing cell, cell membrane, or viral particle. In one embodiment, the Fc region of the IgG1 heavy chain contains the sequence of residues 130-330 of SEQ ID NO:1.

[0250] The parental polypeptide or parental antibody can be any parental polypeptide or parental antibody described in this invention. In this context, parental polypeptide and parental antibody also refer to the first parent and the second parental polypeptide and the first parent and the second parental antibody.

[0251] In one embodiment, the parental antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

[0252] In one implementation, the parental antibody is a full-length human antibody, such as a full-length human IgG1 antibody.

[0253] In one embodiment, the parental antibody, the first parental antibody and the second parental antibody are human IgG1 antibodies, such as IgG1m(za) or IgG1m(f) allotypes, optionally comprising an Fc region containing SEQ ID NO:1 or 5.

[0254] In one embodiment, the parental antibody is a human IgG2 antibody, optionally comprising an Fc region containing SEQ ID NO:2.

[0255] In one embodiment, the parental antibody is a human IgG3 antibody, optionally comprising an Fc region containing SEQ ID NO:3.

[0256] In one embodiment, the parental antibody is a human IgG4 antibody, optionally comprising an Fc region containing SEQ ID NO:4.

[0257] In one implementation, the parental antibody is a bispecific antibody.

[0258] In one embodiment, the parental antibody is any antibody described in this invention, such as an antibody fragment containing at least a portion of the Fc region, a monovalent antibody (described by Genmab in WO2007059782); a heavy chain antibody consisting of only two heavy chains and naturally occurring, for example, in camels (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), a chain exchange modification 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-domain variable immunoglobulin (Abbott, DVD-Ig, US Patent 7,612,181); dual-domain biheaded antibody (Unilever; Sanofi Aventis, WO20100226923), bispecific antibody (ImClone / Eli Lilly), button-in-hole antibody form (Genentech, WO9850431); DuoBody (Genmab, WO2011 / 131746); electrostatically controlled antibody form (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-domain antibody (GSK / Domantis), Two-in-one antibody recognizing two targets (Genentech, Novlmmune), Cross-linked MAbs (Karmanos Cancer Center), CovX body (CovX / Pfizer), IgG-like bispecific antibody (ImClone / Eli Lilly, Shen, X, et al. J Immunol Methods, 2007, 318(1-2):p.65-74), and DIG and PIG bodies (Pharmabcine), as well as biaffinity retargeting molecules (Macrogenics' Fc-DART or Ig-DART, WO / 2008 / 157379, WO / 2010 / 080538), Zybodies (Zyngenia), using conventional light chains (Crucell / Merus, US7262028) or conventional heavy chains (Novlmmune's κλ body), and fusion proteins containing polypeptide sequences fused to an antibody fragment containing an Fc domain, such as scFv fusions, such as ZymoGenetics / BMS's BsAb, Biogen Idec's HERCULES (US007951918), Emergent BioSolutions / Trubion's SCORPIONS, Ts2Ab (Medlmmune / AZ Dimasi, N., et al. JMol 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 mAb2 (WO2008 / 003116) and bispecific scFv-fusions. It should also be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies), for example, antibody-like peptides generated by techniques developed by Sympogen and Merus (Oligoclonics), such as chimeric antibodies and humanized antibodies. The generated antibodies can potentially have any isotype.

[0259] In another implementation, the antigen is expressed on the cell surface.

[0260] In another implementation, the cells are human tumor cells.

[0261] In a further implementation, the antigen is selected from the following group: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3.

[0262] In another implementation, the antigen is associated with the cell membrane.

[0263] In another embodiment, the antigen is associated with the viral particle, optionally wherein the antigen is contained in the protein capsid or lipid envelope of the viral particle.

[0264] In another embodiment, the antibody is a human antibody, optionally binding to at least one antigen selected from CD20 and CD38.

[0265] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally including a variable weight and / or variable light chain region of at least one of 7D8 and 005.

[0266] In any use according to the disclosed invention, the antibody without any of the present invention mutations can be any parental antibody. Therefore, the use of the invention provides for any variant of such a parental antibody.

[0267] In one implementation, the effector function is Fc receptor binding, for example, including Fc-gamma receptor binding.

[0268] In one implementation, the effector function is the internalization of an Fc-containing polypeptide.

[0269] In one implementation, the effector function is a combination of complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0270] The term "C1q binding" as used in this invention, when used in the context of a variant of the parent antibody or the antibody itself, encompasses any mechanism by which the variant or antibody binds to a host tissue or factor containing various cells of the immune system, such as effector cells, mediated by the binding of the variant or antibody to the first component of the classical complement activation pathway. C1q binding of the antibody can be evaluated using an ELISA (e.g., the C1q binding ELISA used in Examples 3 and 4), or C1q potency can be evaluated using a CDC assay (e.g., the CDC assay used in Example 5). In a further embodiment, the C1q binding affinity of the antibody is determined according to the detection method described in Example 4.

[0271] In all the methods according to the disclosed invention, the antibody without any of the inventive mutations can be any parental antibody. Therefore, the methods of the present invention provide for any variant of such a parental antibody.

[0272] Parental antibodies, first parental antibodies, second parental antibodies, or variants thereof obtained by the methods and / or uses of the present invention can bind to any target described in the present invention.

[0273] Examples of antigens or targets that this invention can target include: 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; CD72; CD 74; 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; FORR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (Hepatitis B Virus); HCMV (Human Cytomegalovirus); Heat shock protein 90 homolog [Candida albicans]; Herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); Human respiratory syncytial virus; Glycoprotein F; ICAM-1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGE junction region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; Interleukin-2 receptor β 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.

[0274] In a key aspect, the present invention relates to a method for inducing CDC in cells, cell membranes, or viral particles, said cells, cell membranes, or viral particles expressing a target that binds to a parental polypeptide comprising an immunoglobulin Fc domain and a binding region, said method comprising

[0275] (i) providing a parental polypeptide that has been mutated according to any of the embodiments disclosed herein, or at least a combination of a first parental polypeptide and a second parental polypeptide; and

[0276] (ii) The formulation of the mutant parent polypeptide of step (i) or a mutant combination of at least the first parent polypeptide and the second parent polypeptide of step (i) is contacted with cells, cell membranes or viral particles expressing the antigen in the presence of human complement or effector cells.

[0277] In one implementation, any or all of the parental polypeptides, the first parental polypeptide and the second parental polypeptide, may be antibodies.

[0278] In another embodiment, the method adds a further effect response selected from: ADCC, Fc-γ receptor binding, protein A binding, protein G binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, via antibody-mediated binding to the complement receptor of the opsonized antibody, and any combination thereof.

[0279] In a further embodiment, the method also induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0280] In a further embodiment, the method also induces the internalization of Fc-containing peptides.

[0281] In one implementation, the cells are human tumor cells or bacterial cells.

[0282] In another implementation, the IgG1 parent antibody is a human IgG1 antibody.

[0283] In another embodiment, the first and second antigens are selected from erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), EpCAM, and MRP3, respectively.

[0284] In another embodiment, the first and second parental antibodies are fully human, optionally wherein the first and second parental antibodies bind antigens selected from CD20 and CD38, respectively.

[0285] In a further embodiment, the first and second parental antibodies are selected from 7D8 and 005, respectively.

[0286] In even more advanced implementations, the cells are bacterial cells.

[0287] In another embodiment, the bacterial cells are selected from Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.

[0288] In another embodiment, the first and / or second antigen is lipoteichoic acid (LTA), and optionally at least one of the first and second parental antibodies is pagibaximab.

[0289] In another implementation, the antigen is expressed on the viral particle.

[0290] In another implementation, the first and second antibodies bind to the same antigen.

[0291] In another embodiment, the first and second antibodies contain the same VH sequence, VL sequence, or both VH and VL sequences.

[0292] For the purposes of this invention, the target cell expressing 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, particularly human cells, such as human cancer cells; and single-celled organisms such as bacteria, protozoa; and single-celled fungi such as yeast cells. The cell membrane containing or otherwise binding to the antigen comprises a portion and / or a disrupted cell membrane derived from the antigen-expressing cell. Antigens associated with viral particles or viral grains may be contained in, or otherwise bound to, the protein coat and / or lipid envelope of the viral particles.

[0293] Target cells can be, for example, human tumor cells. Suitable tumor antigens include any of the targets or antigens described in this invention, 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-capsule protein, periosteal protein, Biggs3, 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 WO2004 / 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 WO 06 / 099875, and anti-EGFr antibody 2F8 disclosed in WO 02 / 100348. Examples of other specific antibodies are provided in this invention.

[0294] Optionally, the target cells can be bacterial cells, such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis. Exemplary antigens include lipoteichoic acid (LTA), and exemplary antibodies include paxisumab.

[0295] Optionally, the target may be present on the surface of viruses, fungal cells, or other particles, such as, for example, West Nile virus, dengue virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papillomavirus, Epstein-Barr virus, herpesvirus, poxvirus, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus, and Histoplasma.

[0296] In one implementation, the contact step (ii) occurs in vitro.

[0297] In one implementation, the contact step (ii) occurs in vivo.

[0298] In another implementation, step (ii) includes administering the variant to the subject.

[0299] In a further embodiment, the subject has cancer, a bacterial infection, or a viral infection. The contact step (ii) of the above embodiments can occur in vitro or in vivo. In the latter case, step (ii) may further include administering one or more formulations to the subject (optionally, the subject with cancer or a bacterial infection). Further details regarding therapeutic applications are provided below.

[0300] The first and second antibodies contain antigen-binding regions that can bind to the same or different epitopes. These epitopes can be on the same or different targets.

[0301] In one implementation, the first and second antibodies bind to different epitopes on different targets. These targets may be expressed on the same cell or cell type, or on different cell or cell types. In this type of implementation, the enhancement of effector function targets only 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.

[0302] Without any theoretical constraints, it is believed that the enhancement of CDC can be limited to target cells that simultaneously express two specific targets / antigens, provided that the first and second antibodies bind to epitopes found on the same cells, thereby utilizing the combined expression of targets to improve the selectivity of enhanced CDC-induced CDC.

[0303] In cases where the target is expressed on different cells or cell types, it is not theoretically constrained to assume that administration of the first and second antibodies in any order will improve CDC enhancement and possibly other effector functions by “recruiting” the second cell or cell type expressing the second target.

[0304] In embodiments in which a combination of first and second antibodies is used, step (ii) can be performed by contacting cells with the mutated first and second parental antibodies simultaneously, separately, or sequentially in the presence of human complement and / or effector cells.

[0305] The present invention also provides a method for inducing a CDC or other effector response, such as ADCC, against an antigen-associated target cell, cell membrane, viral particle, or other particle that binds to an IgG1 or IgG3 antibody, comprising the steps of: (i) providing an antibody variant comprising a K439 mutation (K439E) and an S440 mutation (S440K or S440R) in the Fc region of the antibody; and (ii) contacting the variant formulation with the cells in the presence of human complement and / or effector cells.

[0306] The present invention also provides a method for inducing a CDC or other effector response, such as ADCC, against a target cell, cell membrane, or viral particle, wherein the target cell, cell membrane, or viral particle expresses a first antigen that binds to a first IgG1 antibody and a second antigen that binds to a second antibody, the method comprising the steps of: (i) providing a first variant (which is a first antibody containing a K439E mutation) and a second variant (which is a second antibody containing an S440K or S440R mutation); and (ii) contacting the cells with the first and second variant formulations simultaneously, separately, or sequentially in the presence of human complement or effector cells.

[0307] In individual and specific embodiments, the first and second antibodies bind to (i) different antigens, (ii) different epitopes on the same antigen, (iii) the same epitope on the antigen, and (iv) the same epitope on the antigen and contain the same VH and / or VL sequences.

[0308] Other methods

[0309] In another key aspect, the present invention relates to a method for identifying antibody mutations that enhance the effector function of antibody binding to C1q, comprising the steps of:

[0310] (i) Prepare at least one antibody containing a mutation in one or more amino acids selected from the group consisting of E430X, E345X, S440Y and S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0311] (ii) When binding to the surface of antigen-expressing cells, the C1q activity of the antibody is evaluated compared to that of the parent antibody; and

[0312] (iii) Select mutations of any variant with increased C1q affinity.

[0313] In one embodiment, at least one antibody comprises one or more amino acid substitutions selected from E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, such as the group consisting of E430G, E430S, E345K, and E345Q.

[0314] In another key aspect, the present invention relates to a method for identifying parental antibody mutations that increase the ability of an antibody to induce a CDC response, comprising the steps of:

[0315] (i) Prepare at least one parental antibody variant comprising a mutation in one or more amino acids selected from the group consisting of E430X, E345X, S440Y or S440W corresponding to the Fc region of the human IgG1 heavy chain.

[0316] (ii) In the presence of effector cells or complement, assess the variant-induced CDC response compared to the parental antibody when binding to the surface of antigen-expressing cells; and

[0317] (iii) Select mutations of any variant that have increased C1q response.

[0318] In one embodiment, at least one antibody comprises one or more amino acid substitutions selected from E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E345K, and E345Q in the Fc region of the human IgG1 heavy chain.

[0319] The polypeptide of the present invention

[0320] Parental polypeptides

[0321] As described in this invention, the invention particularly relates to parental polypeptide variants containing one or more mutations in the CH3 region of immunoglobulins (e.g., in the heavy chain of antibodies). The “parental polypeptide” can be a “parental antibody.” The “parental” antibody (which may be a wild-type antibody) to be used as the starting material for this invention before modification can be produced, for example, by the hybridoma method first described in Kohler et al., Nature 256, 495 (1975), or by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in 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. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with an antigen of interest (e.g., in the form of cells expressing the antigen on their surface, or in the form of nucleic acids encoding the antigen of interest). Monoclonal antibodies can also be obtained from hybridomas of antibody-expressing cells derived from immune human or non-human mammals (e.g., rabbits, rats, dogs, primates, etc.).

[0322] Parental antibodies can be, for example, chimeric or humanized antibodies. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies can be generated using transgenic or transchromosomal mice (e.g., HuMAb mice, which carry a portion of the human immune system instead of the mouse system). HuMAb mice contain minilocus of human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856 859 (1994)). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to the immune response, the introduced human heavy and light chain transgenes undergo type switching and somatic mutations to produce high-affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), above; 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. NYAcad. Sci 764 536 546 (1995)). The preparation of HuMAb mice is detailed 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, hybridomas secreting human monoclonal antibodies can be generated using spleen cells from these transgenic mice.

[0323] Furthermore, the human antibodies of the present invention, or antibodies of the present invention derived from other species, can be identified by display techniques, including but not limited to phage display, retroviral display, ribosome display, mammalian display, yeast display, and other techniques known in the art. The resulting molecules can undergo additional maturation, such as affinity maturation, as these techniques are well-known in the art. The specific strategy described in Example 17 is applicable to any antibody used to prepare and obtain variants of the present invention using phage display.

[0324] Parental antibodies are not limited to antibodies having a natural, for example, human Fc domain, but can also be antibodies having mutations other than those of the present invention, such as mutations affecting glycosylation or enabling the antibody to become a bispecific antibody. The term "natural antibody" means any antibody that does not contain any genetically introduced mutations. Antibodies containing naturally occurring modifications (e.g., different allotypes) are therefore understood as "natural antibodies" for the purposes of this invention, and thus can be understood as parental antibodies. Such antibodies can serve as templates for one or more mutations according to the present invention, thereby providing variant antibodies of the present invention. An example of a parental antibody containing mutations other than those of the present invention is the bispecific antibody described in WO2011 / 131746 (Genmab), which utilizes reducing conditions to promote half-molecule substitution of two antibodies containing an IgG4-like CH3 region, thereby forming a bispecific antibody without the co-formation of aggregates. Other examples of parental antibodies include, but are not limited to, bispecific antibodies such as heterodimeric bispecific antibodies: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); button-in-hole (Genentech); electrostatic harnessing (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary forms of parental antibodies include, but are not limited to, wild-type antibodies, full-length antibodies or antibody fragments containing Fc, human antibodies, or any combination thereof.

[0325] The parental antibody can bind to any target. Examples of such targets or antigens in this invention include, but are not limited to, 5T4; ADAM-10; ADAM-12; ADAM. 17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA72-4; Cancer-associated antigen CTAA 16.88; 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; Endothelial angiotensin 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]; Herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gpl20 V3 ring; HLA-DRB (HLA-DRβ); Human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGE linker region; IL12B; IL13; IL15; IL17A; ILIA; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; Interleukin-2 receptor β subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; Lipid A, lipopolysaccharide LPS domain; LTA; MET; MMP14; MMpl5; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte antigen; conjugation 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.

[0326] The parental antibody can be any isotype of any human antibody, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, and IgD, optionally a full-length human antibody, such as a full-length human IgG1 antibody. The parental antibody may contain a sequence according to any one of SEQ ID NO: 1, 2, 3, 4, and 5.

[0327] Monoclonal antibodies used in this invention, such as parents and / or variants, can be produced by the hybridoma method first described in Kohler et al., Nature 256, 495 (1975), or by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in 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. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with an antigen of interest (e.g., in the form of cells expressing the antigen on their surface, or nucleic acids encoding the antigen of interest). Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals (e.g., rats, dogs, primates, etc.).

[0328] In one embodiment, the antibody is a human antibody. Human monoclonal antibodies against any antigen can be generated using transgenic or transchromosomal mice carrying a portion of the human immune system instead of the mouse system. Such transgenic and transchromosomal mice are respectively referred to in this invention as... In this invention, mice and KM mice are collectively referred to as "transgenic mice". The mice contain mini-loci of human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and κ immunoglobulin light chain sequences, as well as targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg, N. et al., Nature). 368 ,856-859(1994)). Thus, mice showed decreased expression of mouse IgM or κ, and in response to immunity, the introduced human heavy and light chain transgenes underwent type switching and somatic mutations to produce high-affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), above; 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. NYAcad. 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.

[0329] HCo7, Hco12, Hco17, and HCo20 mice exhibit JKD disruption in their endogenous light chain (κ) gene (described in Chen et al., EMBO J.12, 821-830 (1993)), CMD disruption in their endogenous heavy chain gene (described in Example 1 of WO 01 / 14424), and the KCo5 human κ light chain transgene (described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Furthermore, 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 mice obtained expressed human immunoglobulin heavy chain and κ light chain transgenes in a background of homozygosity in the disruption of endogenous mouse heavy chain and κ light chain loci.

[0330] In the KM mouse strain, as described by Chen et al., EMBO J.12, 811-820 (1993), the endogenous mouse κ light chain gene has been homozygous destroyed, and as described in Example 1 of WO 01 / 09187, the endogenous mouse heavy chain gene has been homozygous destroyed. As described by Fishwild et al., Nature Biotechnology 14, 845-851 (1996), this mouse strain carries the human κ light chain transgene KCo5. As described in WO 02 / 4347814, this mouse strain also carries a human heavy chain transchromosomal consisting of a segment of chromosome 14, hCF (SC20). As described in WO / 2009 / 097006, HCo12-Balb / C mice are generated by crossing HCo12 with KCo5[J / K](Balb). According to known techniques, hybridomas secreting human monoclonal antibodies can be generated using spleen cells from these transgenic mice.

[0331] Furthermore, arbitrary antigen-binding regions can be obtained from human antibodies or antibodies from other species identified by display techniques (including, but not limited to, phage display, retroviral display, ribosome display, and other techniques) using techniques known in the art, and the resulting molecules can undergo additional maturation, such as affinity maturation, because these techniques 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), ScottTIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russell et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and US 5, 733, 743. If the display technology is used to generate non-human antibodies, these antibodies can be humanized.

[0332] The mutations according to the present invention can be, but are not limited to, the deletion, insertion, or substitution of one or more amino acids. Such substitution of amino acids can be carried out using any naturally occurring or non-natural amino acid.

[0333] "Single mutant"

[0334] It should be understood that all embodiments described herein with reference to parental antibodies, first parental antibodies, or second parental antibodies may also be applied to other parental, first parental, or second parental polypeptides containing the Fc domain and binding region of an immunoglobulin.

[0335] The antibody or peptide variants of the present invention, in the "single mutant" aspect, comprise mutations in one or more amino acid residues as shown in Table 1, typically amino acid substitutions. Table 1 lists each amino acid residue numbered according to the EU index of the human IgG1 antibody, along with the corresponding amino acid in the parental IgG2, IgG3, and IgG4 antibodies, as well as "exemplary" and "preferred" amino acid substitutions. Figure 2The IgG2 segment corresponding to residues 126 to 326 of IgG1, the IgG3Fc segment corresponding to residues 177 to 377, and the IgG4 segment corresponding to residues 127 to 327 are shown.

[0336] Table 1. Example mutation sites and amino acid substitutions for "single mutants".

[0337]

[0338] As can be seen from Table 1, the amino acid substitutions that led to increased cell lysis in Wien133 cells in Example 19 are included as "preferred substitutions".

[0339] In one aspect, the present invention relates to a variant of a parental polypeptide comprising the Fc domain and binding region of an immunoglobulin, wherein said variant contains one or more mutations selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, provided that the variant does not contain any other mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor. (FcRn) can be determined by the method disclosed in Example 34.

[0340] In another aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein said variant comprises one or more mutations selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, provided that said variant does not contain any other mutations in the Fc domain that increase or decrease the binding of the variant to the neonatal Fc receptor (FcRn) by more than 30%, for example more than 20%, 10%, or 5%, as measured by changes in OD 405nm absorbance determined by the method disclosed in Example 34.

[0341] In another aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant contains one or more mutations selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, provided that the variant does not contain any other mutations in the Fc domain that increase the apparent affinity of the parental antibody to the mouse neonatal Fc receptor (FcRn) by more than 0.5-fold or decrease the apparent affinity of the parental polypeptide or parental antibody to the mouse FcRn by more than 2-fold, as determined by the method disclosed in Example 34.

[0342] In one embodiment, one or more mutations are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0343] In one implementation, the variant does not contain any other mutations that alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the variant in the Fc domain.

[0344] In one embodiment, the variant does not contain any other mutations that alter the plasma clearance rate of the variant in the Fc domain, as determined by the method disclosed in Example 37.

[0345] In another embodiment, the variant does not contain any other mutation in the Fc domain that increases or decreases the plasma clearance of the variant by more than 3.0 times, such as more than 2.5 times, 2.0 times, 1.5 times, or 1.2 times, as determined by the method disclosed in Example 37.

[0346] In one implementation, the variant does not contain any other mutations that alter the serum half-life of the variant in the Fc domain.

[0347] In one embodiment, the variant does not contain any other mutations that alter the variant’s target-independent fluid phase complement activation in the Fc domain, as determined by the method disclosed in Example 36.

[0348] In one implementation, the variant does not contain any other mutations in the Fc domain.

[0349] In one implementation, the variant contains only one mutation.

[0350] In one embodiment, the variant peptide may be a variant antibody comprising the Fc domain and antigen-binding region of an immunoglobulin.

[0351] In one specific implementation, the amino acid substitution is E345R.

[0352] As illustrated in the examples, variants of CD38 antibodies HuMab-005 and -003 (as described in WO2006 / 099875) and / or CD20 antibodies HuMab-7D8 and -11B8 (as described in WO 2004 / 035607) and rituximab and / or EGFR antibody HuMab-2F8 (as described in WO 2002 / 100348) containing one of these amino acid substitutions have higher C1q binding, complement activation, and / or CDC than wild-type HuMab 005 and 7D8, respectively.

[0353] It should be understood that the variants may also include one of the mutations of the “Exemplary Substitutions” listed in Table 1. The variants may also include more than one mutation, such as two, three, four, five or six of any of the mutations listed in Table 1.

[0354] In addition to the specified mutation, the variant can have any characteristics described for the parent antibody. Specifically, it can be a human antibody. Furthermore, besides the mutation, the variant can be any IgG1 subtype.

[0355] When binding to antigens on the surface of antigen-expressing cells, cell membranes, viral particles, or other particles, or when the antigen is associated with a viral particle, optionally wherein the antigen is contained within a protein capsid or lipid envelope of the viral particle, such antibody variants may have an increase in at least one of the following compared to the parent antibody: (i) antibody-mediated CDC, (ii) antibody-mediated complement activation, (iii) C1q binding, (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 have an increased ADCC compared to the parent peptide or parent antibody. In a further embodiment, the variant retains the same or similar plasma clearance rate compared to the parent peptide or parent antibody. In a further embodiment, the variant does not have a plasma clearance rate that is increased or decreased by more than 3.0 times compared to the parent peptide or parent antibody, such as more than 2.5 times, 2.0 times, 1.5 times or 1.2 times, as determined by the method disclosed in Example 37.

[0356] Not limited to any specific theory, the effect induced by substituting amino acid residues at designated positions in this invention can, for example, cause an effect on its own, participate in direct contact with the Fc domain of another molecule, or be mutated to directly interact with another Fc domain or indirectly affect intermolecular Fc:Fc interactions. Therefore, not limited to theory, substitutions are considered to directly or indirectly enhance the binding strength between oligomeric antibody molecules, and enhance the stability of oligomeric structures (e.g., hexamers, pentamers, tetramers, trimers, or dimers). 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 aromatic superposition interactions), or amino acid substitutions that promote entropy increase through water molecule release during Fc:Fc interactions. Furthermore, referring to Table 1, “exemplary substitutions” can be selected based on the magnitude and physicochemical properties of participation in or promotion of intermolecular Fc:Fc interactions or intramolecular interactions. “Exemplary substitutions” can be selected based on the magnitude and physicochemical properties most suitable for participation in or activation of intermolecular Fc:Fc interactions or intramolecular interactions.

[0357] In one implementation, the variant may include other mutations selected from Table 1.

[0358] In one embodiment, the variant comprises a combination of two mutations in amino acid residues selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y, and E430X / S440W.

[0359] In any embodiment of a variant containing such a mutation in at least two amino acids, it may be present in each heavy chain of the variant, or one of the two may be contained in one heavy chain while the other may be contained in another heavy chain, or vice versa.

[0360] In one embodiment, the mutation in at least two amino acid residues is a deletion, insertion, or substitution. Such substitution of amino acids can be made using any naturally occurring or artificially occurring amino acid.

[0361] The mutations according to the invention can be, but are not limited to, the deletion, insertion, or substitution of one or more amino acids. Such substitutions of amino acids can be made using any naturally occurring or non-naturally occurring amino acid.

[0362] Therefore, in one implementation, the mutation at at least one amino acid residue can be a deletion.

[0363] In another embodiment, the mutation at at least one amino acid residue can be an insertion.

[0364] In another implementation, the mutation at at least one amino acid residue can be a substitution.

[0365] Exemplary specific combinations of mutations in at least two amino acid residues are E345R / E430T, E345R / S440Y, E345R / S440W, E345R / E430G, E345Q / E430T, E345Q / S440Y, E345Q / S440W, E430T / S440Y, and E430T / S440W.

[0366] In addition to mutations in one or more amino acids according to embodiments of the invention, the IgG heavy chain may contain additional mutations known in the art, such as mutations that further improve effector function. These additional mutations include known mutations that enhance CDC, Fc-receptor binding, or FcRn binding and / or improve Fc-γ receptor-mediated effector function.

[0367] In one embodiment, the variant according to the invention further comprises known CDC-enhancing modifications, such as fragment exchange between IgG isotypes to produce 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 surrounding 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 according to the invention further comprise a combination of amino acid substitutions for any one of S267E, H268F, S324T, S239D, G236A, and I332E, providing enhanced effector function via CDC or ADCC (Moore et al., 2010 mAbs 2(2), 181-189). Other Fc mutations affecting binding to the Fc receptor (described in WO 2006 / 105062, WO 00 / 42072, US Patent 6,737,056, and US Patent 7,083,784) or antibody physical properties (described in WO 2007 / 005612 A1) may also be used in the variants of the invention.

[0368] In one embodiment, the variant according to the invention further comprises modifications that enhance Fc-γ receptor binding and / or Fc-γ receptor-mediated effector function. Such modifications include (i) reducing the amount of fucose in the glycosylation of CH2 attachment (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).

[0369] In one embodiment, the variant according to the invention is further modified at the FcRn binding site, for example, to prolong the half-life (t1 / 2) of the IgG antibody. Such modifications include (i) mutations in N434A and T307A / E380A / N434A (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 for alanine residues, thereby improving FcRn binding (Shields). RL, et al. J. Biol. Chem. 2001; 276:6591); and (iii) amino acid substitutions or combinations of amino acid substitutions selected from the group consisting of: M252Y / S254T / T256E, M252W, M252Y, M252Y / T256Q, M252F / T256D, V308T / L309P / Q311S, G385D / Q386P / N389S, G385R / Q386T / P 387R / N389P, H433K / N434F / Y436H, N434F / Y436H, H433R / N434Y / Y436H, M252Y / S254T / T256E-H433K / N434F / Y436H or M252Y / S254T / T256E-G385R / Q386T / P387R / N389P, thereby increasing affinity for FcRn (Dall'Acqua et al., above).

[0370] "Double mutant"

[0371] It should be understood that all embodiments described in this question involving parental antibodies, first parental antibodies, or second parental antibodies may also be applied to other parental, first parental, or second parental polypeptides that contain the Fc domain and binding region of an immunoglobulin.

[0372] As described above and further below, the present invention also relates to a “double mutant” aspect, wherein two individual mutations each reduce effector function but together restore effector function to the parent antibody level. When used together, the specificity of the variant is increased. Antibody variants according to the “double mutant” aspect contain two mutations, typically amino acid substitutions, that specifically interact with the amino acid residue pairs K439 and S440, K447 and 448, or K447, 448 and 449.

[0373] Therefore, 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 first mutation and a second mutation, the first mutation being selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to E430G, E430S, E345K, or E345Q in the Fc region of the human IgG1 heavy chain, and the second mutation being selected from the group consisting of the following amino acid residues:

[0374] (i) The amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W, and if the first mutation is S440Y or S440W, then the second mutation is in the amino acid residues corresponding to K439 in the Fc region of the human IgG1 heavy chain.

[0375] (ii) Mutant residues corresponding to K447D / E or K447K / R / H and 448P in the Fc region of the human IgG1 heavy chain; or

[0376] (iii) Amino acid residues corresponding to K447D / E or K447K / R / H, 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain. Tables 2A and 2B show exemplary and preferred substitutions for “double mutants” (Table A) and “mixed mutants” (Table 2B).

[0377] Table 2A lists example mutation sites and amino acid substitutions related to "double mutants".

[0378]

[0379] Table 2B: Example mutation sites and amino acid substitutions in the "mixed mutant" category (antibody 1 + antibody 2).

[0380]

[0381] In one embodiment, the variant comprises a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y in the Fc region of the human IgG1 heavy chain, and a second mutation in amino acid residues corresponding to K439 and S440, provided that the mutation in S440 is not S440Y or S440W.

[0382] The present invention envisions variants that may also contain only one of the amino acid residue substitutions, such as K439E or S440K, for example, variants containing a mutation in K439 and optionally no mutation in S440.

[0383] In one embodiment, the present invention relates to variants wherein the mutation in K439 is an amino acid substitution selected from E and D, such as K439E.

[0384] In another implementation, the variant contains the mutation in S440, and optionally has no mutation in K439.

[0385] In one embodiment, the present invention relates to a variant wherein the mutation of S440 is an amino acid substitution with an amino acid selected from K and R, such as S440K.

[0386] In one implementation, the variant contains mutations at both K439 and S440.

[0387] In another implementation, the mutation of K439 is selected from K439 to D, E, or R, such as K439D / E, and the mutation of S440 is selected from S440 to D, E, K, and R, such as S440K / R.

[0388] In another embodiment, the mutations in K439 are selected from K439D and K439E, and the mutations in S440 are selected from S440K and S440R.

[0389] In another implementation, the variant contains K439E and S440K mutations.

[0390] In one embodiment, the parental polypeptide is a parental antibody comprising an immunoglobulin Fc domain and an antigen-binding region.

[0391] As described in Examples 4-6, antibody variants containing only one of the K439E and S440K mutations against C1q K D A significant increase was observed, reflecting decreased complement activation and / or CDC capacity. Surprisingly, antibody variants of HuMAb 7D8 or 005 containing both mutations were found to have restored or increased C1q binding or CDC. Without being limited to any specific theory, the underlying mechanism may perhaps be explained by corresponding mutations that spatially complement each other, such as… Figure 4 and 5 As shown.

[0392] In one embodiment, the parental polypeptide and its variants may be a parental antibody comprising an immunoglobulin Fc and an antigen-binding region.

[0393] In another embodiment, compared to a parental antibody or an antibody variant containing only a mutation at one of K439 and S440, a variant as described in this invention containing a mutation at both K439 and S440 has an increased Fc-mediated effector function selected from complement-dependent cytotoxicity (CDC), C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, including Fc-γ receptor binding, protein A binding, protein G binding, antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing peptide internalization, target downregulation, ADC uptake, apoptosis induction, cell death, cell cycle arrest, and any combination thereof.

[0394] The present invention also provides the use of K439E and S440K mutations in antibodies in restoring 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 containing only one of the K439E or S440K mutations): (i) antibody-mediated CDC, (ii) antibody-mediated complement activation, (iii) C1q binding affinity, (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.

[0395] In one implementation, the variant is selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0396] Hybrid mutant

[0397] It should be understood that all embodiments described herein with reference to parental antibodies, first parental antibodies, or second parental antibodies are applicable to other parental, first parental, or second parental peptides containing immunoglobulin Fc domains and binding regions.

[0398] As described above, the inventors of this invention have also discovered mutations that reduce effector function on their own but restore it when used together, such as mutations at positions K439 and S440 in the Fc region of the human IgG1 heavy chain. Therefore, this concept, by introducing K439 into one antibody and S440 into another, can also be used to ensure pairing of two different antibodies. Thus, antibody variants according to the “hybrid mutant” aspect contain mutations, but a single mutation typically results in reduced or significantly reduced Fc:Fc interactions between the same Fc molecules. However, the antibody variants of this invention, as “hybrid mutants,” are able to pair with each other; compared to, for example, mixing each individual variant or parent antibody, this provides restored or even increased CDC, C1q binding, complement activation, oligomer formation, and / or oligomer stability for specific antibody variant pairs. In one embodiment of this invention, the oligomer is a hexamer. In one embodiment, the antibody variant may also optionally have other effector functions that are retained or enhanced, such as C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, Fc receptor binding, Fc-γ receptor binding, protein A binding, protein G binding, antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, internalization of Fc-containing peptides, target downregulation, ADC uptake, apoptosis induction, cell death, cell cycle arrest, and any combination thereof. This aspect of the invention provides numerous applications in which not only the intensity but also the selectivity of C1q binding, complement activation, CDC, or other effector functions can be modulated.

[0399] Exemplary mutation sites for each antibody variant in the “hybrid mutant” pair are shown in Table 2B. Specifically, the present invention provides an antibody variant comprising an immunoglobulin Fc domain and an antigen-binding region, the variant comprising a mutation in a residue corresponding to one of K439 and S440 in the Fc region of the human IgG1 heavy chain.

[0400] In one embodiment, the mutation is located at K439, and the amino acid is substituted with an amino acid selected from E or D, such as K439E. In another embodiment, the mutation is located at S440, and the amino acid is substituted with an amino acid selected from K or R, such as S440K.

[0401] In one implementation, the variant contains K439 in the Fc region corresponding only to the IgG1 heavy chain, rather than an amino acid mutation at the position corresponding to S440.

[0402] In one embodiment, the variant contains an amino acid mutation at a position corresponding only to S440, provided that the mutation at S440 is not S440Y or S440W, and the variant does not contain an amino acid mutation at a position corresponding to K439 in the Fc region of the IgG1 heavy chain.

[0403] Therefore, in one embodiment, the invention also relates to variants comprising a first mutation and a second mutation, wherein the first mutation is selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to the Fc region of the human IgG1 heavy chain, and the second mutation is in an amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain.

[0404] In another embodiment, the invention also relates to variants comprising a first mutation and a second mutation, wherein the first mutation is selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y corresponding to the Fc region of the human IgG1 heavy chain, and the second mutation is in an amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the second mutation is not S440Y or S440W.

[0405] In one implementation, the two implementations described above can be combined to form a "hybrid mutant" pair according to the present invention.

[0406] Each variant of a “hybrid mutant” pair may further include mutations in the amino acids listed in Table 1.

[0407] In one embodiment of the invention, the "hybrid mutant" comprises a first variant of a parental antibody and a second variant of a parental antibody, wherein 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 in one or more amino acid residues other than the mutation in K439 and a second mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain, the first mutation being selected from the group consisting of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W corresponding to E430X, E345X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain; and,

[0408] The second variant comprises a second Fc domain and an antigen-binding region of an immunoglobulin, wherein the second variant comprises (i) a first mutation in one or more amino acid residues other than the mutation in S440, the first mutation being selected from the group consisting of E430X and E345X corresponding to the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y.

[0409] (ii) a second mutation at the position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W.

[0410] Other exemplary "hybrid mutant" pairs may further include, but are not limited to, any of the following pairs: a first variant containing the K447E mutation and a second variant containing the K447 / P448 mutation; a first variant containing the K447E mutation and a second variant containing the K447 / K448 / P449 mutation.

[0411] In one implementation, the first mutation is a deletion, insertion, or substitution. This substitution of amino acids can be made using any naturally occurring or non-natural amino acid.

[0412] In one implementation, the mutation is a deletion.

[0413] In another implementation, the mutation is an insertion.

[0414] In another implementation, the mutation is the substitution of an amino acid.

[0415] In one specific embodiment, the first variant and / or the second variant contains a mutation in one or more amino acid residues selected from the group consisting of E430G, E430S, E345K, and E345Q in the Fc region of the human IgG1 heavy chain.

[0416] For example, in one embodiment, one variant of the “hybrid mutant” pair contains one of E430G, E430S, E345K, or E345Q and a K439E mutation, while the other variant contains one of E430G, E430S, E345K, or E345Q and an S440K mutation, thus providing increased and more specific C1q binding affinity, complement activation, CDC, oligomer formation, oligomer stability, and / or other effector-related functions such as 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 of opsonized antibodies, and / or combinations thereof.

[0417] The “hybrid mutant” aspect can also include two variants, each containing more than one mutation listed in Table 2A in the Fc region of the human IgG1 heavy chain, such as a first variant containing the mutation S440K / K447E and a second variant containing the mutation K439E / K447 / P448; or a first variant containing the mutation K439E / K447E and a second variant containing the mutation S440K / K447 / P448.

[0418] The variants in the "hybrid mutant" pairs described in this invention can be derived from the same or different parental antibodies. Furthermore, the "hybrid mutant" aspect can also be used for bispecific or asymmetric antibodies. Additionally, the first, second, and third antibodies can bind to different epitopes on the same or different targets.

[0419] Furthermore, the "hybrid mutant" aspect provides a more specific CDC or other effector response against tumor cells expressing two specific tumor antigens by utilizing a first antibody against the first antigen with a K439E mutation and a second antibody against the second antigen with an S440K or S440R mutation. By utilizing the "hybrid mutant" aspect containing three variants (optionally bispecific antibodies), a more specific CDC or other effector response can be provided against tumor cells expressing at least two, such as two, three, four, five, or six specific tumor antigens.

[0420] In any of the embodiments of "single mutant", "double mutant" and "mixed mutant", the variant is selected from monospecific antibody, bispecific antibody or multispecific antibody.

[0421] In any implementation of the “hybrid mutant”, the first, second and / or third variants may contain the same or different mutations of any amino acid substitutions listed in Table 1.

[0422] Multispecific antibodies

[0423] It should be understood that all embodiments described in this invention involving parental antibodies, first parental antibodies, or second parental antibodies are applicable to other parental, first parental, or second parental polypeptides containing immunoglobulin Fc domains and binding regions.

[0424] It should be understood that any of the embodiments described in this invention regarding "single mutant," "double mutant," and "mixed mutant" can be used in relation to multispecific antibodies as described below.

[0425] Therefore, in one embodiment, the variant is an antibody selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0426] In one specific implementation, the bispecific antibody has the form described in WO 2011 / 131746.

[0427] In one key aspect, the present invention relates to a variant of a parental antibody, said parental antibody being a bispecific antibody comprising a first polypeptide and a second polypeptide, said first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, said second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of an immunoglobulin, wherein said first and second antigen-binding regions bind to different epitopes on the same antigen or different antigens, and wherein said first and / or second CH2-CH3 regions comprise one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of the human IgG1 heavy chain, and wherein

[0428] The first polypeptide contains further mutations in amino acid residues selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of the human IgG1 heavy chain; and

[0429] The second polypeptide contains further mutations in amino acid residues selected from those corresponding to the Fc region of the human IgG1 heavy chain, namely F405, T366, L368, K370, D399, Y407, and K409, and wherein the further mutations in the first polypeptide are different from the further mutations in the second polypeptide.

[0430] In one implementation, the mutation is a deletion, insertion, or substitution. Such substitution of amino acids can be made using any naturally occurring or non-natural amino acid.

[0431] The bispecific antibody of the present invention is not limited to a specific form, and it can be any of the bispecific antibodies described above and herein.

[0432] In one specific embodiment of the invention, (i) the first polypeptide comprises a further mutation in an amino acid residue corresponding to K409, such as K409R, in the Fc region of the human IgG1 heavy chain; and

[0433] (ii) The second polypeptide contains a further mutation in an amino acid residue corresponding to F405, such as F405L, in the Fc region of the human IgG1 heavy chain; or alternatively,

[0434] (iii) The first polypeptide contains further mutations in amino acid residues corresponding to F405 in the Fc region of the human IgG1 heavy chain, such as F405L.

[0435] (iv) The second polypeptide contains further mutations in amino acid residues corresponding to the K409 region of the human IgG1 heavy chain Fc region, such as K409R.

[0436] In one specific implementation, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0437] The bispecific antibody according to the invention can be generated according to the description in Example 22. Furthermore, the effect of the generated heterodimeric protein on CDC killing can be detected using the assay method used in Example 23.

[0438] Bispecific antibodies may, for example, comprise an antigen-binding region of a CD20 antibody and an antigen-binding region of a CD38 antibody, with amino acid substitutions for one or more amino acids listed in Tables 1 and / or 2A / B. Exemplary CD20-binding regions comprise the CD20-binding regions of ofatumumab (2F2), 7D8, and 11B as described in WO2004 / 035607 (which is therefore fully incorporated herein by reference), and rituximab (WO 2005 / 103081). Exemplary CD38-binding regions comprise those CD38-binding regions of 003 and daratumumab (005) as described in WO2006 / 099875 (which is therefore fully incorporated herein by reference).

[0439] In one implementation, bispecific antibodies bind to different epitopes on the same or different targets.

[0440] In another embodiment, the first mutation in the first and second polypeptides can be the same or different.

[0441] In one embodiment of “single mutant,” “double mutant,” “mixed mutant,” and multispecific antibody, the variant is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, or IgE antibody, optionally a full-length human antibody, such as a full-length human IgG1 antibody.

[0442] In terms of any "single mutant", "double mutant", "mixed mutant" and multispecific antibodies, 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 on an amino acid residue directly involved in C1q binding, optionally determined by comparing the C1q binding in the ELISA assay according to Example 3 with the C1q binding in the cell-based assay according to Example 4, and the ADCC is determined according to the assay described in Example 12.

[0443] Furthermore, the present invention provides articles of manufacture comprising any of the "single mutant," "double mutant," "mixed mutant," and multispecific antibody aspects or embodiments as described above. The present invention also provides compositions comprising any of the "double mutant" aspects and embodiments as described above, such as pharmaceutical compositions. The present invention also provides the use of any such variants, articles, or compositions as pharmaceuticals.

[0444] The above-described aspects of the "single mutant," "double mutant," "mixed mutant," and multispecific antibodies of the present invention are particularly applicable to human antibody molecules having an IgG1 heavy chain, wherein the IgG1 heavy chain comprises the relevant fragments P247 to K447 corresponding to underscore residues 130 to 330 (UniProt accession number P01857; SEQ ID NO:1) of the constant region of the human IgG1 heavy chain.

[0445]

[0446] This invention can also be applied to antibody molecules having the human IgG2 heavy chain moiety. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to underlined residues 126 to 326 of the constant region of the IgG2 heavy chain (accession number P01859; SEQ ID NO:2).

[0447]

[0448] This invention can also be applied to antibody molecules having the human IgG3 heavy chain moiety. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to residues 177 to 377 of the constant region of the IgG3 heavy chain (UniProt accession number P01860; SEQ ID NO:3), which are underlined below:

[0449] 1 astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs wnsgaltsgv

[0450] 51 htfpavlqss glyslssvvt vpssslgtqt ytcnvnhkps ntkvdkrvel

[0451] 101 ktplgdttht cprcpepksc dtpppcprcp epkscdtppp cprcpepksc

[0452] 151 dtpppcprcp apellggpsv flfppk pkdt lmisrtpevt cvvvdvshed

[0453] 201 pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh qdwlngkeyk

[0454] 251 ckvsnkalpapiektisktkgqprepqvytlppsreemtknqvsltclvk

[0455] 301 gfypsdiave wessgqpenn ynttppmlds dgsfflyskl tvdksrwqqg

[0456] 351 nifscsvmhe alhnrftqks lslspgk

[0457] This invention can also be applied to antibody molecules having the human IgG4 heavy chain moiety. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to underlined residues 127 to 327 of the constant region of the IgG4 heavy chain (accession number P01859, SEQ ID NO:4).

[0458]

[0459] This invention can also be applied to antibodies containing the heavy chain portion of the human IgG1m(f) allotype. The amino acid sequence of the IgG1m(f) allotype (CH3 sequence is underlined) - SEQ ID NO:5

[0460]

[0461] The alignment of corresponding fragments from 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 introduce equivalent positions of IgG2, IgG3, IgG4 and / or IgG1m(f) determined by alignment to obtain variants according to the invention.

[0462] In one embodiment, the present invention provides a variant of a full-length IgG1, IgG2, IgG3, or IgG4 antibody comprising one or more amino acid substitutions according to any of the aspects described above.

[0463] In any “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody, the Fc region of the IgG1 heavy chain may contain the following sequences: 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.

[0464] In one embodiment, the parental 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.

[0465] In one embodiment, the Fc region of the IgG1 heavy chain contains the sequence of residues 130 to 330 of SEQ ID NO:1.

[0466] The parental antibody can be any parental antibody described in this invention. In this context, the parental antibody is also intended to be a first parent and a second parental antibody.

[0467] In one embodiment, the parental antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

[0468] In one implementation, the parental antibody is a full-length human antibody, such as a full-length human IgG1 antibody.

[0469] In one embodiment, the parental antibody, the first parental antibody, and the second parental antibody are human IgG1 antibodies, such as IgG1m(za) or IgG1m(f) allotypes, optionally comprising an Fc region containing SEQ ID NO:1 or 5.

[0470] In one embodiment, the parental antibody is a human IgG2 antibody, optionally comprising an Fc region containing SEQ ID NO:2.

[0471] In one embodiment, the parental antibody is a human IgG3 antibody, optionally comprising an Fc region containing SEQ ID NO:3.

[0472] In one embodiment, the parental antibody is a human IgG4 antibody, optionally comprising an Fc region containing SEQ ID NO:4.

[0473] In any specific embodiment of the “single mutant,” “double mutant,” “mixed mutant,” and multispecific antibody, the variant comprises an amino acid sequence having 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% identity with the amino acids P247 to K447 of SEQ ID NOs: 1, 2, 3, 4, and 5, except for the mutations introduced according to the invention.

[0474] Therefore, in addition to any mutations defined in this invention, variants may contain sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0475] Any of the above-described "single mutant", "double mutant", "mixed mutant" and multispecific aspects of the present invention should be understood to include the following embodiments.

[0476] In one embodiment, the first and / or second parental antibody is an antibody fragment, optionally selected from monovalent antibodies, heavy chain antibodies, chain exchange modified domain (SEED), triomab, dual variable domain immunoglobulin (DVD-Ig), button-in-pore antibodies, mini-antibodies, biaffinity redirecting molecules (Fc-DART or Ig-DART); LUZ-Y antibodies, Biclonic antibodies, dual-targeting (DT)-Ig antibodies, two-in-one antibodies, cross-linked mabs, mAbs. 2 CovXbody, IgG-like bispecific antibody, Ts2Ab, BsAb, HERCULES antibody, TvAb, ScFv / Fc fusion antibody, SCORPION, scFv fragment fused to the Fc domain, and dual scFv fragment fused to the Fc domain.

[0477] In a further embodiment, both the first and second parental antibodies bind to antigens expressed on the surface of human tumor cells.

[0478] In a further embodiment, the antigens targeting the first and second parental antibodies are individually selected from erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3.

[0479] In a further embodiment, the first and second parental antibodies are fully human.

[0480] In a further embodiment, the antigens against the first and second parental antibodies are selected from CD20 and CD38 in any order, optionally wherein the first and second parental antibodies are selected from 7D8 and 005 in any order.

[0481] In a further embodiment, both the first antibody and the second antibody bind to the antigen expressed on the surface of bacterial cells or viral particles.

[0482] In another embodiment, the bacterial cells are selected from Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.

[0483] In a further embodiment, the first and second parental antibodies bind to the same antigen.

[0484] In another implementation, the first and second parental antibodies are the same antibody.

[0485] In another embodiment, the parental antibody is selected from 7D8 and 005.

[0486] Composition

[0487] It should be understood that all embodiments described in this invention involving parental antibodies, first parental antibodies, or second parental antibodies are applicable to other parental, first parental, or second parental polypeptides containing immunoglobulin Fc domains and binding regions.

[0488] This invention also relates to compositions comprising variants, and the parental antibody can be any variant and parental antibody described in this invention. Specific aspects and embodiments are described below. Furthermore, such variants can be obtained by any method described in this invention.

[0489] In one aspect, the present invention relates to a composition comprising a first variant and a second variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the first variant and / or the second variant comprises one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain.

[0490] In one aspect, the first variant and / or the second variant contains one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain.

[0491] In a preferred embodiment, the first variant and / or the second variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[0492] In one implementation, the first variant and the second variant contain one or more mutations, which may be the same or different.

[0493] In another embodiment, the first variant comprises one or more mutations selected from the group corresponding to E430X, E345X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W, and wherein,

[0494] The second variant does not contain one or more mutations in amino acid residues selected from the group corresponding to E430X, E345X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W.

[0495] In one embodiment, the composition comprises at least one molecule containing at least one immunoglobulin CH2-CH3 domain and a variant according to the invention, wherein the molecule contains a mutation in one or more amino acid residues selected from the group consisting of E430X, E345X, S440Y and S440W, such as E430G, E430S, E345K and E345Q, corresponding to the Fc region of the human IgG1 heavy chain.

[0496] The molecule described in the implementation scheme may be referred to as an "Fc-only molecule" and may further include, for example, a hinge region. However, such a hinge region may not be included.

[0497] Compositions containing only Fc molecules and any variants according to the invention can be applied to imaging diagnostic methods or to modulate the affinity of variants when they bind to cell surfaces.

[0498] Only the Fc molecule may further include further mutations in the amino acid residues of K439 and / or S440 corresponding to the Fc region of the human IgG1 heavy chain, provided that the mutation of S440 is not S440Y or S440W, and if the first mutation is S440Y or S440W, then the further mutation is in the amino acid residue of K439 corresponding to the Fc region of the human IgG1 heavy chain.

[0499] In another embodiment, (i) the first variant further includes a mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and

[0500] (ii) The second variant further includes a mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W; or

[0501] Steps (i) and (ii) can be alternative locations.

[0502] (iii) The first variant further includes a mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W, and

[0503] (iv) The second variant further includes a mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain.

[0504] In one implementation, the mutation at position K439 in the Fc region of the human IgG1 heavy chain is K439D / E, and the mutation at position S440 in the Fc region of the human IgG1 heavy chain is S440K / R.

[0505] In a further embodiment, the present invention relates to a composition as defined herein, wherein

[0506] (i) The first variant further includes a prodrug, and

[0507] (ii) The second variant contains an activator for use with the prodrug in the first variant; or

[0508] Where (i) and (ii) are alternative sites, they can be

[0509] (iii) The second variant contains a prodrug, and

[0510] (iv) The first variant contains an activator for use as a prodrug in the second variant.

[0511] The term "prodrug" is understood according to the present invention as a relatively non-cytotoxic drug precursor that must undergo chemical transformation (e.g., through metabolic processes) before becoming an active pharmacological (anticancer) agent. Examples of prodrugs and methods for preparing prodrugs are well known in the art. One example is an antibody combination comprising an enzyme prodrug, wherein drug delivery is provided by binding an antibody conjugated to the prodrug and an antibody conjugated to an activator for said prodrug to its antigenic target present on the same cell. This brings the prodrug and its activator close to each other, thereby locally releasing the drug, which in turn can penetrate, for example, surrounding cells and kill those cells. (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31; 53(3):247-64, Senter, 1994 FASEB J. 1990 Feb 1; 4(2):188-93).

[0512] The term "activator of prodrug" is understood according to the present invention as a molecule capable of converting a prodrug into an active drug. Examples of activators of prodrugs and methods for preparing these are well known in the art. Examples of activators may be enzymes, which act as catalysts for converting prodrugs into active drugs (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31; 53(3):247-64, Senter, 1994 FASEB J. 1990 Feb 1; 4(2):188-93).

[0513] In one embodiment, the first and / or second parental polypeptides are first and second parental antibodies, each comprising the Fc domain and antigen-binding region of an immunoglobulin.

[0514] In one embodiment, the first and second antibodies are each of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or IgE antibodies, optionally each being a full-length human antibody, such as each being a full-length human IgG1 antibody.

[0515] In one embodiment, the first and second antibodies are each selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0516] In a further embodiment, the first and / or second parental antibodies are each bispecific antibodies comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, and the second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of an immunoglobulin, wherein the first and second antigen-binding regions bind to the same antigen or different epitopes on different antigens, wherein the first CH2-CH3 region comprises a further amino acid mutation at a position selected from those corresponding to the human IgG1 heavy chain Fc region K409, T366, L368, K370, D399, F405, and Y407; wherein the second CH2-CH3 region comprises a further amino acid mutation at a position selected from those corresponding to the human IgG1 heavy chain Fc region F405, T366, L368, K370, D399, Y407, and K409, and wherein the further amino acid mutation in the first CH2-CH3 region is different from the further amino acid mutation in the second CH2-CH3 region.

[0517] In a preferred embodiment, a further amino acid mutation in the first CH2-CH3 region occurs at a position corresponding to human IgG1 heavy chain Fc region K409, such as K409R; and a further amino acid mutation in the second CH2-CH3 region occurs at a position corresponding to human IgG1 heavy chain Fc region F405, such as F405L.

[0518] In one embodiment, the first and second variants of the composition bind to different epitopes on the same or different antigens.

[0519] In one embodiment, one or both of the first variant and the second variant are conjugated to a drug, a toxin, or a radiolabel, for example, where one or both of the first variant and the second variant are conjugated to a toxin via a connector.

[0520] In one implementation, one or both of the first and second variants are part of the fusion protein.

[0521] In one specific implementation, the first and second variants of the composition contain only one mutation.

[0522] In one embodiment, the second variant does not contain any of the mutations listed in this invention, and such second variant may contain any of the above-listed suitable second antibody instances relating to methods for improving CDC.

[0523] In one implementation, at least one first mutation is different in the first and second variants.

[0524] In one embodiment, the first variant and the second variant are each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each being a full-length human antibody, such as each being a full-length human IgG1 antibody.

[0525] In one implementation, the first variant and the second variant are each selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0526] In a further embodiment, the first and second variants bind to different epitopes on the same antigen or different antigens. Therefore, in embodiments where the first and second antibodies are bispecific antibodies, they may each bind to two different epitopes. At least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the composition comprises up to four different epitopes targeting the same or different targets.

[0527] In another aspect, the present invention relates to a composition comprising any variant, any bispecific antibody or any combination thereof described herein and a pharmaceutically acceptable carrier.

[0528] It is anticipated that any embodiment based on the "hybrid mutant" aspect can also be included in any composition embodiment.

[0529] In one implementation, variants of the first and second parent antibodies bind to antigens expressed on the same cells.

[0530] In another embodiment, a variant of the first parent antibody comprises an amino acid substitution of K439 to an amino acid selected from E and D.

[0531] In another embodiment, the amino acid substitution of the variant of the first parent antibody is K439E.

[0532] In another embodiment, a variant of the second parent antibody comprises an amino acid substitution from S440 to an amino acid selected from K and R.

[0533] In another embodiment, the amino acid substitution of the variant of the second parent antibody is S440K.

[0534] In another aspect, the present invention relates to a pharmaceutical composition comprising a variant of a first parent polypeptide or parent antibody and a variant of a second polypeptide or parent antibody according to any of the embodiments listed above.

[0535] The pharmaceutical compositions can be formulated using conventional techniques, for example, as disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutical compositions of the present invention may, for example, contain 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, isotropic agents, antioxidants, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextran, and polyols (e.g., glycerol, propylene glycol, polyethylene glycol).

[0536] The pharmaceutical composition can be administered via any suitable route and method. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, the term "parenteral administration" refers to a method of administration other than intestinal and surface administration, typically by injection, and includes injections and infusions via the epidermis, intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendonal, trachea, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal routes.

[0537] Kit-of-parts

[0538] It should be understood that the present invention relates to parental antibodies, and all embodiments described with first or second parental antibodies are also applicable to other parental, first or second parental polypeptides containing immunoglobulin Fc domains and binding regions.

[0539] The present invention also relates to kits for simultaneous, separate, or continuous use in treatment, comprising variants of the parental polypeptide and the parental antibody, wherein any variant of the parental polypeptide and the parental antibody may be as described in the present invention. Specific aspects and embodiments are described below. Furthermore, such variants may be obtained by any of the methods described in the present invention.

[0540] In one aspect, the present invention relates to a kit for simultaneous, separate, or sequential use in treatment, comprising a first variant of a parental polypeptide and a second variant of a parental polypeptide, wherein the first variant contains one or more mutations selected from the group corresponding to E430X, E345X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W, provided that the variant does not contain any other mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor (FcRn), and wherein...

[0541] (i) The first variant contains a mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain, and the second variant contains a mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W.

[0542] (ii) The first variant contains a mutation at the position corresponding to K447D / E in the Fc region of the human IgG1 heavy chain; and the second variant contains mutations at the positions corresponding to K447K / R / H and 448P in the Fc region of the human IgG1 heavy chain, or

[0543] (iii) The first variant contains a mutation at the position corresponding to K447D / E of the Fc region of the human IgG1 heavy chain; and the second variant contains mutations at the positions corresponding to K447K / R / H, 448K / R / H and 449P of the Fc region of the human IgG1 heavy chain.

[0544] In one embodiment, one or both of the first variant and the second variant of the parent polypeptide may be an antibody containing an immunoglobulin Fc domain and an antigen-binding region.

[0545] In one implementation, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E, and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R.

[0546] In another aspect, the present invention relates to a kit for simultaneous, separate, or continuous use in treatment, comprising a first variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin and a second variant of a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein

[0547] The variant contains one or more mutations selected from the group corresponding to E430X, E345X, S440Y, and S440W in the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W, provided that the variant does not contain any other mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor (FcRn), and wherein...

[0548] The second variant does not contain mutations in amino acid residues selected from the group consisting of E430X, E345X, S440Y, and S440W corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440Y in the Fc region of the human IgG1 heavy chain.

[0549] In one implementation, the second variant does not contain any of the mutations listed herein, and such second variant may contain any of the above-listed examples of suitable second antibodies that involve methods relating to effector function.

[0550] In one implementation, at least one first mutation in the first and second variants is different.

[0551] In one embodiment, the first variant and the second variant are each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each being a full-length human antibody, such as each being a full-length human IgG1 antibody.

[0552] In one implementation, the first variant and the second variant are each selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0553] In a further embodiment, the first and second variants bind to different epitopes on the same antigen or different antigens. Therefore, in embodiments where the first and second antibodies are bispecific antibodies, they may each bind to two different epitopes. At least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the treatment kit, used simultaneously, separately, or sequentially, contains up to four different epitopes targeting the same or different targets.

[0554] In a further embodiment, one or both of the first variant and the second variant are conjugated to a drug, a toxin, or a radiolabel, for example, where one or both of the first variant and the second variant are conjugated to a toxin via a connector.

[0555] In a further embodiment, one or both of the first and second variants are part of a fusion protein.

[0556] It is anticipated that any implementation scheme based on the "hybrid mutant" can be included in any set used simultaneously, separately, or continuously during treatment.

[0557] In one implementation, variants of the first and second parent antibodies bind to antigens expressed on the same cells.

[0558] In another embodiment, a variant of the first parent antibody comprises an amino acid substitution of K439 to an amino acid selected from E and D.

[0559] In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.

[0560] In another embodiment, a variant of the second parent antibody comprises an amino acid substitution from S440 to an amino acid selected from K and R.

[0561] In another embodiment, the amino acid substitution in the variant of the second parent antibody is S440K.

[0562] In another aspect, the present invention relates to a pharmaceutical kit for simultaneous, separate or continuous use in treatment, comprising a variant of a first parent polypeptide or parent antibody and a variant of a second parent polypeptide or parent antibody of any of the embodiments listed above.

[0563] The drug kits used concurrently, separately, or continuously during treatment can be administered via any suitable route and method. In one embodiment, the drug kits used concurrently, separately, or continuously during treatment according to the present invention are administered parenterally. The term "parentereal administration" as used in this invention refers to administration methods other than intestinal and surface administration, typically by injection, and includes injections and infusions via the epidermis, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendonal, trachea, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal.

[0564] combination

[0565] Furthermore, the present invention provides a formulation of any variant of the "single mutant" aspect or embodiment as described above, i.e., a formulation comprising a multi-copy variant. The present invention also provides compositions comprising any variant of the "single mutant" aspect and embodiment as described above, for example, pharmaceutical compositions. The present invention also provides the use of any such "single mutant" variant, article, or composition as a medicine.

[0566] The present invention also provides combinations of variants, wherein one variant comprises at least one mutation according to the invention, and another variant comprises at least one other mutation according to the invention, as well as articles and pharmaceutical compositions of such combinations of variants and their use as pharmaceuticals. Preferably, the two variants combine the same or different antigens that are normally expressed on the surface of the same cells, cell membranes, viral particles and / or other particles.

[0567] Conjugate

[0568] It should be understood that the present invention relates to parental antibodies, and all embodiments described with first or second parental antibodies are also applicable to other parental, first or second parental polypeptides containing immunoglobulin Fc domains and binding regions.

[0569] In one aspect, the present invention relates to a variant, wherein the variant is conjugated with a drug, toxin, or radioactive marker, for example, wherein the variant is conjugated with a toxin via a connector.

[0570] In one implementation, the variant is part of a fusion protein.

[0571] In another aspect, variants of the 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 a different site (typically at a site that does not interfere with oligomer formation). For example, antibody variants may be linked at a different site to a compound selected from toxins (containing radioisotopes), prodrugs, or drugs. Such compounds can make the killing of target cells more effective, for example, in cancer treatment. Thus, the resulting variants are immunoconjugates.

[0572] Therefore, in a further aspect, the present invention provides an antibody linked to or conjugated to one or more therapeutic components (e.g., cytotoxins, chemotherapeutic agents, cytokines, immunosuppressants, and / or radioisotopes). Such conjugates are referred to herein as “immunoconjugates” or “pharmaceutical conjugates.” Immunoconjugates containing one or more cytotoxins are referred to as “immunotoxins.”

[0573] Cytotoxins or cytotoxic agents comprise any agent that is harmful to cells (e.g., kills cells). Suitable therapeutic agents for forming the immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraxetine. Anthracindione, maytansine or its analogues or derivatives, enediyene (enediyne) antitumor antibiotics including neocarzinostatin, calicheamycins, esperamicins, anthracyclines, lidamycin, kedarcidin or their analogues or derivatives, anthracyclins, mitoxantrone, mithramycin, actinomycin D D), 1-Dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine). Itabine), cladribine, alkylating agents (e.g., nitrogen mustard, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 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 analogs or derivatives of CC-1065, dolastatin, pyrrolo[2,1-c][1,4]benzodiazepin (PDBs) or analogs thereof, anti- Antibiotics (e.g., dactinomycin, bleomycin, daunomycin, doxorubicin, idarubicin, sclerosingmycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), and antimitotic agents (e.g., tubulin inhibitors) such as monomethyl alistatin E) auristatin E, methylauristatin F, or other analogues or derivatives of dolastatin 10; histone deacetylase inhibitors such as hydroxamic acids trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat; and benzamides, entinostat, CI994, mocetinostat; and fatty acid compounds such as phenylbutyrate and valproic acid. Proteasome inhibitors such as Danoprevir, bortezomib, amatoxins such as α-amantin, diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules); ricin toxins (e.g., ricin A or deglycosylated ricin A chain toxins), 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 inhibitors, Pseudomonas exotoxin, alarin, saporin, modeccin, gelanin, absinthecin A chain, and modeccin A chain.α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxin. Other suitable conjugated molecules include antimicrobial / cleaving peptides such as CLIP, Xenopus magainin 2, melitin, bactericidal peptides, and P18; ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenbenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents (e.g., anticancer cell factors or chemokines) that can be administered in combination with the antibodies of the present invention described elsewhere in this invention may also be candidates for conjugating the therapeutic portion of the antibodies of the present invention.

[0574] In one embodiment, the pharmaceutical conjugate of the present invention comprises an antibody disclosed herein conjugated to alistatin or an alistatin peptide analog and derivative (US5635483; US5780588). Alistatin has 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 has anticancer (US5663149) and antifungal activities (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). The alistatin pharmaceutical moiety can be attached to the antibody via a linker, through the N (amino) terminus or C (terminus) of the peptide pharmaceutical moiety.

[0575] An exemplary aristatin implementation comprising N-terminally linked monomethylaristatin drug moieties DE and DF is disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, March 28, 2004, and described in US 2005 / 0238649.

[0576] An exemplary alistatin implementation is MMAE (methylalistatin E). Another exemplary alistatin implementation is MMAF (methylalistatin F).

[0577] In one embodiment, the antibody of the present invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In this embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., siRNA molecules), or an immunostimulatory nucleic acid (e.g., an immunostimulatory DNA molecule containing a CpG motif). In another embodiment, the antibody of the present invention is conjugated to an aptamer or ribozyme.

[0578] In one embodiment, an antibody comprising one or more radiolabeled amino acids is provided. Variations of the radiolabeled amino acids can be used for both diagnostic and therapeutic purposes (conjugation to a radiolabeled molecule is another possible feature). Non-limiting examples of labeling for peptides 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, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US4,681,581, US4,735,210, US5,101,827, US5,102,990 (US RE35,500), US5,648,471, and US5,697,902). For example, radioisotopes can be conjugated using the chloramine-T method.

[0579] In one embodiment, variants of the invention are conjugated to a radioisotope or a chelate containing a radioisotope. For example, variants may be conjugated to a chelating agent linker, such as DOTA, DTPA, or tiuxetan, which allows the antibody to form a complex with the radioisotope. Variants may also or optionally contain one or more radiolabeled amino acids or other radiolabeled molecules, or be 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, variants of the invention are 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.

[0580] In one embodiment, variants of the invention may be conjugated with cytokines selected from the group consisting of: 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, ancestim, and TNFα.

[0581] Variations of the invention can also be chemically modified by covalent conjugation to polymers to, for example, increase their cyclic half-life. Exemplary polymers, and methods of attaching them to peptides, are shown, for example, in US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546. Other polymers comprise polyoxyethylene polyols and polyethylene glycol (PEG) (e.g., PEG with a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000).

[0582] Any method known in the art for conjugating variants of the invention to conjugated molecules can be used, such as those described above, including those described in: 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 generated by chemically conjugating another portion to the N-terminal or C-terminal side of the variant or a fragment thereof (e.g., antibody H or L chain) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Where suitable, such conjugated variant derivatives can also be generated by conjugation at internal residues or sugars.

[0583] The agent can be directly or indirectly conjugated to variants of the invention. One example of indirect conjugation of the second agent is conjugation via a spacer or linker portion to a cysteine ​​or lysine residue in a bispecific antibody. In one embodiment, the variant is conjugated to a prodrug molecule via a spacer or linker, the prodrug molecule being activated in vivo as a therapeutic agent. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In some embodiments, the linker is cleavable by a cleavable agent present in the intracellular environment (e.g., within lysosomes, endosomes, or pits). For example, the spacer or linker is cleavable by tumor cell-associated enzymes or other tumor-specific conditions, thereby forming the active drug. Examples of such prodrug technology and adapters are described in Syntarga BV et al., WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171. Suitable antibody prodrug technologies and duocarmycin analogs are also found in U.S. Patent No. 6,989,452 (Medarex), which is incorporated herein by reference. The adapter may also be, for example, a peptide adapter, which is cleaved by an intracellular peptidase or protease, comprising, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptide adapter is at least two amino acids long or at least three amino acids long. The cleavage agent may comprise cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug into the target cell (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In one specific embodiment, the peptide linker that can be cleaved by the intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, for example, US6214345, which describes the synthesis of doxorubicin with a Val-Cit linker and various examples of Phe-Lys linkers). Examples of Val-Cit and Phe-Lys connector structures include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB, or MC-Phe-Lys-GABA as described below, where MC is an abbreviation for maleimido caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid. The advantage of using intracellular protein hydrolysis to release therapeutic agents is that the agent is generally weakened upon conjugation and the serum stability of the conjugate is generally high.

[0584] In another embodiment, the linker unit is non-cleavable, and the drug is released via antibody degradation (see US 2005 / 0238649). Typically, such linkers are substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" means that when the variant antibody-drug conjugate is present in the extracellular environment (e.g., plasma), no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker is cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined by incubating the variant antibody-drug conjugate with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structure (where Ab represents an antibody, and p represents a drug load (or the average number of cell-inhibiting or cytotoxic drugs per antibody molecule) which is 1 to about 8, for example p may be 4-6, such as 3-5, or p may be 1, 2, 3, 4, 5, 6, 7 or 8).

[0585] Examples of combinations of cuttable connectors with alistatin include MC-vc-PAB-MMAF (also known as vcMMAF) and MC-vc-PAB-MMAF (also known as vcMMAE), where MC is an abbreviation for maleimide hexanoyl, vc is an abbreviation for Val-Cit (valine-citrulline) connector, and PAB is an abbreviation for para-aminobenzylcarbamate.

[0586] Other examples include arithmetic amines combined with non-cuttable connectors, such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation for maleimide hexanoyl).

[0587] In one embodiment, the drug linker is vcMMAE. The vcMMAE drug linker and conjugation methods are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US 11 / 833,028 (SeattleGenetics, Inc.), (which are incorporated herein by reference), and the method of binding the vcMMAE drug linker to an antibody at a cysteine ​​residue using a method similar to that disclosed therein.

[0588] In one embodiment, the drug linker is mcMMAF. The mcMMAF drug linker and conjugation methods are disclosed in US7498298, US 11 / 833,954 and WO2005081711 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the method of binding the mcMMAF drug linker to the variant at the cysteine ​​residue using a method similar to that disclosed therein.

[0589] In one embodiment, a variant of the invention is attached to a chelating agent connector, such as tiuxetan, which allows bispecific antibodies to conjugate to a radioisotope.

[0590] In one implementation, each arm (or Fab-arm) of the variant is directly or indirectly coupled to the same one or more therapeutic components.

[0591] In one implementation, only one arm of the variant is directly or indirectly coupled to one or more therapeutic components.

[0592] In one embodiment, each arm of the variant is directly or indirectly coupled to a different therapeutic moiety. For example, in an embodiment 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 in this invention), such a bispecific antibody can be obtained by using monospecific antibodies conjugated to or bound to different therapeutic moieties.

[0593] Further uses

[0594] It should be understood that all embodiments described in this invention involving parental antibodies, first parental antibodies, or second parental antibodies can also be applied to other parental, first parental, or second parental polypeptides that include the immunoglobulin Fc domain and binding region.

[0595] In a further aspect, the present invention relates to variants of the invention as described above, used as a medicament, specifically as a medicament for treating diseases or conditions, wherein CDC-mediated killing of target cells (e.g., tumor, bacterial, or fungal cells) or target organisms (e.g., viruses) or bacterial or viral-infected cells is desired. Examples of such diseases and conditions include, but are not limited to, cancer and bacterial, viral, or fungal infections.

[0596] In another aspect, the present invention relates to variants of the invention, bispecific antibodies, compositions, and kits for treating diseases such as cancer.

[0597] In another aspect, the present invention relates to a method for treating a person, comprising administering the variants, compositions or kits described herein.

[0598] In another aspect, the present invention relates to a method of treating human cancer, comprising administering the variants, compositions or kits described herein.

[0599] "Treatment" refers to the application of an effective amount of the therapeutically active compound of this invention with the aim of alleviating, improving, blocking or eliminating (curing) symptoms or disease states.

[0600] "Effective dose" or "therapeutic effective dose" is the amount of antibody that is effective at achieving the desired therapeutic outcome at the necessary dosage and time period. Therapeutic effective doses of antibodies can vary depending on various factors, such as an individual's disease condition, age, sex, and weight, and the antibody's ability to elicit the desired response in the individual. Therapeutic effective dose is also the amount by which any toxicity or adverse effects of the antibody or antibody fraction exceed the beneficial effects of the treatment.

[0601] In another aspect, the present invention relates to the use of variations, compositions or kits of any of the embodiments described herein in diagnostic methods.

[0602] In another aspect, the present invention relates to a diagnostic method comprising applying a variant, composition, or kit of any embodiment described herein to at least a portion of the body of a human or other mammal.

[0603] In another aspect, the present invention relates to the use of variations, compositions, or kits of any of the embodiments described herein for imaging at least a portion of the body of a human or other mammal.

[0604] In another aspect, the present invention relates to a method for imaging at least a portion of the body of a human or other mammal, comprising applying a variant, composition or kit according to any embodiment described herein.

[0605] Without being limited by theory, when any “single mutant” aspect or embodiment of the invention is introduced into such a therapeutically active compound, the effective amount of the therapeutically active compound may be reduced.

[0606] The suitable antigens for cancer antibodies can be the same as those described in this invention. Examples 15 to 18 describe specific applications of providing enhanced and / or more specific complement activation or CDC for tumor cells. For example, antitumor antibodies containing, for example, the E345R mutation according to the "single mutant aspect" can provide enhanced CDC or ADCC, ADCP responses in tumor cells. Furthermore, in variations of this approach, mutations according to the "single mutant aspect," such as, for example, E345R, E430, or S440S / W, or any other mutations listed in Table 1, can be added to each antibody to provide enhanced CDC and / or ADCC responses specifically against tumor cells expressing at least two antigens.

[0607] Suitable antibodies for bacterial infections include, but are not limited to, those targeting Staphylococcus aureus (e.g., chimeric monoclonal IgG1 paxisumab (BSYX-A110; Biosynexus)), those targeting lipoteichoic acid (LTA) embedded in the Staphylococcus cell wall and described in Baker (Nat Biotechnol. 2006 Dec; 24(12):1491-3) and Weisman et al. (Int Immunopharmacol. 2009 May; 9(5):639-44) (both of which are incorporated herein by reference in their entirety). Example 14 describes a specific implementation using a Staphylococcus aureus antibody variant containing the E345R mutation. However, other mutations in Table 1, including but not limited to E430G and S440W, can be applied in a similar manner to enhance the CDC-mediated activity of antibodies against bacterial antigens.

[0608] Suitable antigens for viral or fungal infections can be any of those described in this invention.

[0609] In one embodiment, the antigen binding to the variant is not human EphA2. In another embodiment, the variant is not derived from human EphA2 mAb 12G3H11 (described in Dall'Acqua et al., above, which is incorporated herein by reference in its entirety). In another embodiment, the antigen binding to the variant is not IL-9. In another embodiment, the variant is not derived from the Fa-hG1 or Fa-hG4 antibody described in WO2007005612 (incorporated herein by reference in its entirety), or any variant thereof. In one embodiment, the antigen binding to the variant is not HIV-1 gp120. In another embodiment, the variant is not derived from the b12 human IgG1κ antibody against gp120.

[0610] In one specific embodiment, the variant is derived from a bispecific parental antibody. The bispecific antibody can belong to any isotype, such as, for example, IgG1, IgG2, IgG3, or IgG4, and can be a full-length antibody or a fragment containing an Fc. Exemplary methods for preparing bispecific antibodies are described in WO2008 / 119353 (Genmab).

[0611] dose

[0612] It should be understood that this invention relates to parental antibodies, and all embodiments described for the first or second parental antibody are also applicable to other parental, first or second parental polypeptides containing the immunoglobulin Fc domain and binding region. The effective dosage and administration regimen for the antibody depend on the disease or symptom to be treated and can be determined by those skilled in the art. Exemplary, non-limiting ranges for therapeutically effective amounts of the antibodies of this invention are about 0.1 to 100 mg / kg, for example about 0.1 to 50 mg / kg, for example about 0.1 to 20 mg / kg, for example about 0.1 to 10 mg / kg, for example about 0.5, about 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0613] The antibody variants of the present invention can also be administered in combination with one or more complement factors or related components to enhance the therapeutic efficacy of the variants and / or compensate for complement depletion. Such complement factors and related components include, but are not limited to, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, and factor B. The combination administration can be simultaneous, separate, or sequential. In one specific embodiment, the present invention provides a kit comprising a pharmaceutical composition containing a variant of the present invention, and at least one complement factor or related component in the same or different pharmaceutical compositions, and instructions for use.

[0614] The antibody variants of the present invention can also be administered in combination therapy, i.e., in combination with other therapeutic agents associated with the disease or symptom to be treated. Therefore, in one embodiment, the antibody-containing drug is used in combination with one or more other therapeutic agents (e.g., cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents). Such combination administration can be simultaneous, separate, or sequential.

[0615] In a further embodiment, the present invention provides a method for treating or preventing a disease (e.g., cancer), the method comprising administering a therapeutically effective amount of a variant of the invention or a pharmaceutical composition thereof to a subject in need of such treatment in conjunction with radiotherapy and / or surgery.

[0616] Preparation method

[0617] It should be understood that the present invention relates to parental antibodies, and all embodiments described with first or second parental antibodies are also applicable to other parental, first or second parental polypeptides containing immunoglobulin Fc domains and binding regions.

[0618] The present invention also provides isolated nucleic acids and vectors encoding variants of any of the foregoing aspects, as well as vectors and expression systems encoding said variants. Suitable nucleic acid constructs, vectors, and expression systems for antibodies and their variants are known in the art and are described in the embodiments. In embodiments where the variant comprises not only a heavy chain (or a fragment thereof containing Fc) but also a light chain, the nucleotide sequences encoding the heavy and light chain portions may be present on the same or different nucleic acids or vectors.

[0619] The present invention also provides a method for generating an antibody variant according to any of the foregoing aspects in a host cell, wherein the variant comprises at least the Fc region of a heavy chain, the method comprising the following steps:

[0620] a) Provide a nucleotide construct encoding the Fc region of the variant.

[0621] b) Express the nucleotide construct in a host cell.

[0622] and

[0623] c) Recover the antibody variant from the cell culture of the host cell.

[0624] In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody will also contain a light chain, and thus the host cell may also express a light chain-encoded construct on the same or a different vector.

[0625] Suitable host cells for antibody recombinant expression are well known in the art and include CHO, HEK-293, PER-C6, NS / 0, and Sp2 / 0 cells. In one embodiment, the host cell is a cell capable of protein Asn-linked glycosylation, such as a eukaryotic cell, a mammalian cell, or a human cell. In a further embodiment, the host cell is a non-human cell that has been genetically engineered to produce glycoproteins with human-like or human-like glycosylation. Examples of such cells are genetically engineered Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325) and genetically engineered Lemnaminor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

[0626] In one embodiment, the host cell is a host cell that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists a large number of such antibody production systems, such as Sp2 / O, NS / O, or transgenic mammary glands (goats), in which only partial removal of the C-terminal lysine is achieved. In one embodiment, the host cell is a host cell with an altered glycosylation mechanism. Such cells have been described in the art and used as host cells for expressing variants of the invention to produce glycosylated antibodies. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and EP1176195; WO03 / 035835; and WO99 / 54342. Other methods for producing modified glycoforms are known in the art and include, but are not limited to, those described below: Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0627] The present invention also relates to antibodies obtained or obtainable by the methods of the present invention as described above.

[0628] In a further aspect, the present invention relates to a host cell capable of generating antibody variants of the present invention. In one embodiment, the host cell has been transformed or transfected with the nucleotide construct of the present invention.

[0629] The present invention is further illustrated by the following examples, which should not be construed as further limitations.

[0630] Example

[0631] Example 1: Design and generation of the 7D8 mutant

[0632] The human monoclonal antibody HuMab-7D8 (described in WO2004 / 035607) was used as the model antibody. It belongs to the human anti-CD20 IgG1 antibody group, which includes oflambumab (HuMax-CD20, 2F2). These antibodies target a unique juxtamembranous epitope on the CD20 molecule and exhibit strong CDC.

[0633] To test the functional relevance of oligomeric Fc-Fc interactions in complement activation and CDC, amino acids in the hydrophobic sheet at the Fc:Fc interface were mutated to potentially disrupt the Fc-Fc lateral interactions and CDC efficacy of 7D8. In the first group of mutants (Table 3), mutations were introduced at sites selected based on the 1HZH crystal structure and described as being exposed in the hydrophobic sheet of the CH2-CH3 domain to alter the charge (Burton Mol Immunol 1985 Mar; 22(3):161-206).

[0634] From the first set of mutants, I253D and H433A were found to have the strongest effect on CDC loss in 7D8 (e.g., Example 5). 1H ZH crystal structures showed that I253 and H433 bind to two distinct pockets at the relative Fc positions of the paired antibodies. Based on these data, a second set of mutations was synthesized around the I253 and H433 positions in the crystal structure to further investigate the importance of residues at the Fc:Fc lateral interface for CDC. The second set of mutations around the I253 and H433 positions that potentially destabilize the Fc:Fc interface and subsequently CDC are listed in Table 4.

[0635] To rule out the possibility that the observed effect on CDC was due to disruption of the C1q direct binding site, a double mutant was generated based on two individual mutants exhibiting CDC loss to test its ability to restore CDC loss caused by a single mutant. The schematic diagram is shown in... Figure 1 The double mutations are listed in Table 5, and their structural diagrams are shown in Figure D. Figure 4 and Figure 5 middle.

[0636] The mutant was prepared using the Quikchange site-directed mutagenesis kit (Stratagene, USA). Briefly, a full-length plasmid DNA template encoding the 7D8 heavy chain with the IgG1m(f) allotype was replicated using forward and reverse primers encoding the desired mutation. The resulting DNA mixture was digested with DpnI to remove the source plasmid DNA and then transformed into *E. coli*. The mutant plasmid DNA isolated from the resulting colonies was verified by DNA sequencing (Agowa, Germany). The mixture of plasmid DNA encoding both the antibody heavy and light chains was transiently transfected into Freestyle HEK293F cells (Invitrogen, USA) using 293fectin (Invitrogen, USA), essentially as described by the manufacturer.

[0637] Table 3: Group 1 mutations involving the introduction of the CH2-CH3 domain of 7D8

[0638]

[0639] (=) No charge

[0640] (-) Negative charge

[0641] (+) positive charge

[0642] (δ+) partial positive charge

[0643] Table 4: Group 2 mutations introduced from the CH2-CH3 domain of 7D8

[0644]

[0645] (=) No charge

[0646] (-) Negative charge

[0647] (+) positive charge

[0648] (δ+) partial positive charge

[0649] Table 5: Double mutations introduced into the CH2-CH3 domain of 7D8 to combine two single mutations that each indicate CDC deletion.

[0650]

[0651] (=) No charge

[0652] (-) Negative charge

[0653] (+) positive charge

[0654] Example 2: Binding of CD20 by the 7D8 mutant on cells

[0655] The binding of purified antibody samples to CD20-positive cells was analyzed by FACS analysis. Group I mutations were tested on Daudi cells (Table 3), and Group II mutations were tested on Raji cells (Table 4). Antibody preparations were serially diluted in RPM 1640 / 0.1% BSA (3-fold dilutions ranging from 0.04 to 10 μg / ml for Group I on Daudi, and 3-fold dilutions ranging from 0.003 to 10 μg / ml for Group II on Raji) in 50 μl of each sample in a polystyrene 96-well round-bottom plate (Greinerbio-one 650101). 5 Cells were incubated at 4°C for 30 minutes. After washing twice in RPM1640 / 0.1% BSA, cells were incubated at 4°C in 100 μl of secondary antibody for 30 minutes. For all experiments on Daudi cells and experiments on Raji cells using the 7D8 antibody, rabbit anti-human IgG (F0056, Dako, Glostrup, Denmark; 1 / 100) conjugated with fluorescein isothiocyanate (FITC) was used as the secondary antibody. For experiments on Raji cells using purified 7D8, goat F(ab')2 anti-human κ light chain (2062-09, Southern Biotech; 1 / 500) conjugated with R-phycoerythrin (R-PE) was used as the secondary antibody. Next, cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 100 μl PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.01 software (GraphPad Software, San Diego, CA, USA) with nonlinear regression (S-shaped dose-response with variable slope).

[0656] The binding of the 7D8 antibody to Daudi cells was not affected by the introduction of point mutations in the CH2-CH3 domain, and this was consistent for all tested mutants and wild-type 7D8. Furthermore, compared to wild-type 7D8, the binding of the 7D8 antibody to Raji cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain, except for E345R. At test concentrations above 0.3 μg / ml, reduced binding of IgG1-7D8-E345R was detected on CD20-positive Raji cells. Reduced binding for H433D and H433R was also detected at the highest antibody concentration tested (10 μg / ml). The reduced binding of IgG1-7D8-E345R, H433D, and H433R can be explained by shielding of secondary antibody epitopes, as direct labeling of E345R and H433R leads to similar or even enhanced binding to Daudi cells. The increased affinity can be explained by the elevated Fc-Fc lateral binding of E345R and H433R compared to wild-type IgG1-7D8.

[0657] The combination of K439E and S440K mutations did not affect the binding of the 7D8 antibody to Raji cells, and the binding was the same as that of single mutant and wild-type 7D8.

[0658] Example 3: C1q binding of the 7D8 mutant to ELISA

[0659] The binding of the 7D8 mutant to C1q was tested in an ELISA, in which purified antibody was coated onto a plastic surface, causing random antibody polymerization. Collected human serum was used as the source of C1q.

[0660] 96-well Microlon ELISA plates (Greiner, Germany) were coated with antibodies serially diluted in PBS (range 0.58–10.0 μg / ml, 1.5-fold dilution) and incubated overnight at 4°C. Plates were washed and blocked with 200 μl / well of 0.5×PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. Plates were incubated sequentially with 3% pooled human serum (Sanquin, catalog number #M0008) at 37°C for 1 hour, with 100 μl / well of rabbit anti-human C1q (DAKO, catalog number #A0136, 1 / 4.000) at room temperature for 1 hour, and with 100 μl / well of porcine anti-rabbit IgG-HRP (DAKO, P0399, 1:10000) as the detection antibody at room temperature for 1 hour, with washing between incubations. Developed with 1 mg / ml 2,2'-azono-bis(3-ethylbenzothiazol-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) for approximately 30 minutes. The reaction was terminated by adding 100 μl of 2% oxalic acid. Absorbance was measured at 405 nm using a microplate reader (Biotek, Winooski, VT). Logarithmic transformation data were analyzed by fitting an sigmoid dose-response curve with a variable slope using GraphPadPrism software. EC5 of each plate relative to the wild-type IgG1-7D8 normalized mutant was calculated. 50 The value was then multiplied by the average of all wild-type IgG1-7D8 data.

[0661] like Figure 6 As shown in Table 6, the point mutations tested, as measured in ELISA, had minimal impact on C1q binding. For the IgG1-7D8-I253D mutant, slightly less efficient C1q binding (higher EC50) was measured in ELISA. 50 (Value). The coating potency of all antibodies was tested and found to be similar for all antibodies.

[0662] Table 6: EC50 of C1q binding in ELISA 50

[0663]

[0664] 1 Calculate the average and SD from at least 3 grains.

[0665] 2Statistical analysis: Dunnett's Multiple Comparison Test (GraphPadPrism 5.01) single-channel ANOVA was used on log-transformed data. Significance was calculated for comparisons with wild-type IgG1-7D8: (na) not applicable, (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001

[0666] Example 4: Binding of the 7D8 mutant to C1q on cells

[0667] An artificial static system for antibody binding and Fc-tail presentation was created by coating antibodies onto a plastic surface. Therefore, complement binding was also tested in cell-based assays, where the binding of C1q to antibody-opsonized CD20-positive B cells was measured by FACS analysis. In experiments using group 1 mutants, Daudi or Raji cells were suspended on ice in 90 μl of RPMI 1640 medium containing 10% FBS (2 × 10⁻⁶ cells / mL). 6 Cells / ml). Add 10 μl of C1q (Complement Technologies, Tyler, TX) at serial concentrations (final concentration range varies between 0-60 μg / ml and 0-140 μg / ml, depending on maximum binding). Then, add 10 μl of purified antibody (final concentration of 10 μg / ml, i.e., saturation concentration) and immediately transfer the reaction mixture to a 37°C water bath and incubate for 1 hour. In experiments using the group 2 mutant, add the test mAb in bulk to Daudi cells, then add various concentrations of C1q to aliquots and incubate the mixture as above. Wash cells three times with PBS / 1% BSA and incubate with FITC-labeled rabbit anti-C1q antibody (DakoCytomation, 10 μg / ml) at room temperature for 30 minutes. Wash cells with PBS / 1% BSA and resuspend in PBS or fix in 2% formaldehyde in PBS. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences), using calibration beads (Spherotech) to convert the average fluorescence intensity into molecules of equivalent soluble fluorescence (MESF). Software (Systat Software Inc., Washington) was used to calculate the dissociation constant (Ki) of C1q binding to CD20-positive cells conditioned with a specified 7D8 antibody. D Value). The mean K was calculated from repeated binding experiments (4 times in Daudi cells and 3 times in Raji cells).D The value, and its K-binding on C1q in cells conditioned with wild-type 7D8. D The values ​​are compared (Tables 7 and 8).

[0668] Group 1 mutants were tested on both Daudi and Raji cells and yielded the same results. Contrary to the C1q ELISA results, most of the tested mutants showed reduced C1q binding affinity (Kb) on both antibody-conditioned Daudi (Table 7A) and Raji (Table 8) cells. D (Increased). Compared to wild-type 7D8, IgG1-7D8-Q311A and H435A showed almost no decrease in C1q binding affinity on conditionated Daudi or Raji cells, while I253A, I253Y, and N434A showed more significant decreases, and I253D and H433A showed very significant decreases. In both cell types, IgG1-7D8-H435R showed a slightly higher C1q binding affinity than wild-type 7D8 (lower K). D However, this is not significant.

[0669] Group 2 mutants were tested on Daudi cells. IgG1-7D8-E345R, E382R, and H433R showed enhanced binding affinity on conditionated Daudi cells compared to wild-type 7D8, through lower K+ binding affinity. D The values ​​are reflected in Table 7B. Compared to wild-type 7D8, all other group 2 mutants showed decreased binding affinity, with G385D, Y436D, Q438D, K439E, and S440K showing a sharp increase in K. D The values ​​(Table 7B) and H433D and Y436C showed a sharp decrease in binding, making it impossible to measure reliable K values. D value.

[0670] The double mutant IgG1-7D8-K439E / S440K showed restored C1q binding on antibody-conditioned Daudi cells, while both single mutants showed decreased C1q binding compared to wild-type 7D8. The binding affinity of the K439E / S440K double mutant was even slightly increased compared to wild-type 7D8 (Table 7C). A mixture of single mutants IgG1-7D8-K439E and IgG1-7D8-K440E completely restored C1q binding, comparable to that of wild-type 7D8 (Table 7C).

[0671] The fact that the IgG1-7D8 mutant showed no change in C1q binding in ELISA (Example 3) contradicts the fact that C1q binding was affected in cell-based assays. This suggests that the CH3 site, which is involved in the Fc:Fc interaction between antibody molecules, does not directly affect C1q binding, but when bound to cells, it affects the kinetic localization of the antibody Fc tail and is therefore an important determinant of C1q binding strength.

[0672] Table 7A: K binding of C1q to antibody-mediated Daudi cells D Value (mutant group 1)

[0673]

[0674] *Comparison with wild-type 7D8 (t-test)

[0675] **(na) is not applicable

[0676] Table 7B: K binding of C1q to antibody-mediated Daudi cells D Value (mutant group 2)

[0677]

[0678]

[0679] *Comparison with wild-type 7D8 (t-test)

[0680] **(na) is not applicable

[0681] ***The average K for 7D8 calculated from experiments 1, 2, 3, 4, 10, and 11 D

[0682] ****Because the binding of these mutants is too weak, reliable fitting curves and K cannot be measured. D value

[0683] Table 7C: K binding of C1q to antibody-mediated Daudi cells D Value (double mutant)

[0684]

[0685] *Comparison with wild-type 7D8 (t-test)

[0686] **(na) is not applicable

[0687] ***The average K for 7D8 calculated from experiments 1, 2, 3, 4, 10, and 11 D

[0688] Table 8: K+ binding of C1q to antibody-mediated Raji cellsD Value (mutant group 1)

[0689]

[0690] *Comparison with wild-type 7D8 (t-test)

[0691] **(na) is not applicable

[0692] Example 5: C1q potency of the 7D8 mutant in CDC assay on CD20-positive Raji cells

[0693] In CDC assays, C1q potency was tested using cells conditioned with the IgG1-7D8 mutant to investigate the effect of observed changes in C1q binding affinity on CDC activity. Therefore, CDC assays were performed using C1q-sublimated normal human serum supplemented with a defined series of C1q concentrations. Cells were placed in 96-well round-bottom plates (Nunc, Rochester, NY) at 0.1 × 10⁻⁶ wells. 6 Raji cells were pre-incubated for 15 minutes at room temperature in 100 μl of RPMI 1640 medium supplemented with 0.1% BSA with 10 μg / ml purified antibody and a series of concentrations of human C1q (0.005, 0.025, 0.1, 0.3, 1.0, 5.0, 30.0 μg / ml). Then, 25 μl of C1q-diluted serum (Quidel, Sandiego, CA) was added, and the cells were incubated at 37°C for 30 minutes or in an incubator for 45 minutes. After incubation, the reaction was terminated by placing the sample on ice. Cell lysis was determined on FACS using the propidium iodide (PI, Sigma Aldrich, Zwijndrecht, Netherlands) live cell rejection assay. The percentage of lysis (%) was determined as follows: % lysis = (number of PI-positive cells / total number of cells) × 100%.

[0694] The lysis of wild-type 7D8 at 30 μg / ml C1q was subtracted from the lysis without C1q and set as 100%. CH4 was calculated using GraphPad Prism software by fitting an sigm-shaped dose-response curve onto the logarithmically transformed data. 50 Value (C1q concentration leading to 50% cleavage). The mutant's CH... 50 The values ​​were standardized relative to wild-type 7D8 (Table 9).

[0695] The data in Table 9 show that, consistent with the measurement of C1q binding affinity, IgG1-7D8-Q311A, E382R, and H435A did not show a decrease in C1q potency; I253A, I253Y, G385D, N434A, and Y436C showed a significant decrease in C1q potency; and I253D, H310K, K322A, H433A, H433D, Y436D, Q438D, K439E, and S440K almost completely lost their ability to induce CDC at all tested C1q concentrations.

[0696] IgG1-7D8-H435R and H433R were slightly more efficient at utilizing C1q than wild-type 7D8, resulting in more efficient CDC. IgG1-7D8-E345R showed a significant improvement in C1q potency, leading to significantly higher CDC cleavage than wild-type 7D8 (Table 9).

[0697] Figure 7 The results showed that the combination of K439E and S440K mutations (both of which, as single mutants, lead to loss of CDC) restored CDC in C1q power assays when both mutations were combined in a single molecule (K439E / S440K double mutant) or when the two single mutants were combined (K439E+S440K mixture).

[0698] Table 9: CH4 efficiency of C1q in CDC assay of Raj cells 50

[0699]

[0700]

[0701] (1) (n) Number of experiments

[0702] (2) Calculate the mean and SD from all the experiments conducted.

[0703] (3) Statistical analysis: One-way ANOVA using Dunnett's Multiple Comparison Test (GraphPadPrism 5.01) was performed on log-transformed data. Significance was calculated for comparisons with wild-type IgG1-7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.

[0704] (4) When the cleavage is less than 50%, CH 50Set to >30μg / mL.

[0705] (5) P-values ​​could not be determined for mutants that did not reach 50% lysis. However, it is assumed that these are significantly different from IgG1-7D8-WT.

[0706] Example 6: CDC assay of 7D8 mutant in CD20-positive cells

[0707] In a round-bottomed 96-hole plate (Nunc, Rochester, NY), 0.1 × 10 6 Cells were pre-incubated with a series of antibody concentrations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 80 μl at room temperature in a shaker for 15 minutes. Next, 20 μl of normal human serum was added as a C1q source (20% final concentration), and the cells were incubated at 37°C for 45 minutes. The reaction was terminated by adding 30 μl of ice-cold RPMI medium supplemented with 0.1% BSA. Cell lysis was confirmed by using propidium iodide on FACS.

[0708] For CDC assays on Daudi cells, EC was calculated by fitting an S-shaped dose-response curve onto logarithmically transformed data using GraphPad Prism software. 50 Value (antibody concentration that results in 50% lysis). The EC value of the mutant... 50 The values ​​were standardized relative to wild-type 7D8 (Tables 10 and 11).

[0709] Table 10 shows that in Daudi cells, IgG1-7D8-I253A, Q311A, E382R, H433R, and H435A showed no difference in CDC compared to wild-type 7D8; IgG1-7D8-I253D, I253Y, H310K, G385D, H433A, H433D, N434A, Y436C, Y436D, Q438D, K439E, S440K, and I253D / H433A were found to have significantly worse CDC (higher EC50) than wild-type 7D8. 50 These antibodies only produce CDC at higher antibody concentrations. The C1q binding defective mutant IgG1-7D8-K322A (included as a control) almost completely loses its ability to induce CDC and fails to achieve EC at the tested concentrations. 50 IgG1-7D8-H435R showed more effective CDC than wild-type 7D8 on Daudi cells. Importantly, consistent with the C1q potency CDC assay, E345R showed significantly better CDC than wild-type 7D8 on Daudi cells, EC50 The value is 10 times lower (Table 10). Figure 8 The results showed that the combination of K439E and S440K mutations (both of which, as single mutants, lead to the loss of CDC) restored CDC when both mutations were combined in a single molecule (K439E / S440K double mutant) or when the two single mutants were combined (K439E+S440K mixture).

[0710] Table 11 shows similar data found for the IgG1-7D8 mutant on Raji cells.

[0711] Table 10: EC2 calculated from CDC assay of Daudi cells 50

[0712]

[0713]

[0714] (1) (n) Number of experiments

[0715] (2) Calculate the mean and SD from all the experiments conducted.

[0716] (3) Statistical analysis: One-way ANOVA using Dunnett's Multiple Comparison Test (GraphPadPrism 5.01) was performed on log-transformed data. Significance was calculated for comparisons with wild-type IgG1-7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.

[0717] (4) When the pyrolysis rate is less than 50%, EC will be used. 50 Set to >30μg / mL.

[0718] (5) For those who have not reached EC 50 The mutants could not be measured for P-values. However, it is assumed that these are significantly different from the wild-type 7D8-WT.

[0719] Table 11: EC calculated from Raji cells using the CDC assay 50

[0720]

[0721]

[0722] (1) (n) Number of experiments

[0723] (2) Calculate the mean and SD from all the experiments conducted.

[0724] (3) Statistical analysis: One-way ANOVA using Dunnett's Multiple Comparison Test (GraphPadPrism 5.01) was performed on log-transformed data. Significance was calculated for comparisons with wild-type 7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.

[0725] (4) When pyrolysis does not reach CH 50 At that time, CH 50 Set to >30μg / mL.

[0726] (5) For those who have not reached EC 50 The mutants could not be measured for P-values. However, it is assumed that these are significantly different from the wild-type 7D8-WT.

[0727] Example 7: Ranking of 7D8 mutants based on their ability to induce CDC

[0728] For the 7D8 mutant tested, correlations were found between C1q binding on Daudi cells (described in Example 4) and C1q potency assays on Raji cells (described in Example 5), and between C1q binding on Daudi cells and CDC assays on both Daudi and Raji cells (described in Example 6) (Correlation Data Table 13). Therefore, K... D The values ​​were ranked according to their ability to induce CDC, as shown in Table 12.

[0729] Table 12: Decreased K based on C1q binding on Daudi cells D The value (which serves as a representative of its ability to induce CDC) ranked all detected 7D8 mutants.

[0730]

[0731]

[0732] *No reliable fitting curve was found. The binding of these mutants was too weak to measure the K-line of the italicized section. D value.

[0733] Table 13: Associations between C1q binding on Daudi cells (Example 4) and C1q potency assay on Raji cells (Example 5), and between C1q binding on Daudi cells and CDC assays on both Daudi and Raji cells (Example 6). Data were log-transformed prior to association analysis.

[0734]

[0735] Example 8: Design and generation of CD38 antibody 005 mutant

[0736] The human monoclonal antibody HuMab 005 is a whole-human IgG1,κ antibody, described in WO2006 / 099875. In this paper, it is used as a model antibody to validate the identified Fc mutation enhancing CDC activity. The mutations tested are listed in Table 14.

[0737] Using the heavy chain of HuMab 005 with IgG1m(f) allotype as a template for mutagenesis, DNA constructs of different mutants were prepared and transiently transfected as described in Example 1.

[0738] Table 14: Mutagen groups introduced into the CH2-CH3 domain of 005 (HuMax-CD38)

[0739]

[0740] (=) No charge

[0741] (-) Negative charge

[0742] (+) positive charge

[0743] (δ+) partial positive charge

[0744] Example 9: Binding of the HuMab-005 mutant to CD38 on cells

[0745] The binding of unpurified antibody samples to CD38-positive Daudi and Raji cells was analyzed using FACS. 10 [units of antibody] were placed in polystyrene 96-well round-bottom plates. 5Cells were incubated in 100 μl of RPM1640 / 0.1% BSA with serially diluted antibody preparations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) at 4°C for 30 min. After washing twice in RPM1640 / 0.1% BSA, the cells were incubated in 50 μl of FITC-conjugated rabbit F(ab')2 anti-human IgG (catalog number F0056; DAKO; 1:150) at 4°C for 30 min. Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 100 μl of PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BDBiosciences). Binding curves were analyzed using GraphPad Prism V5.01 software. Supernatant from simulated transfected cells was used as a negative control.

[0746] The binding of HuMab 005 to Daudi cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound to Daudi cells in a dose-dependent manner. Binding of all tested mutants was similar to that of wild-type HuMab-005, except for 005-E345R, which showed a slightly reduced binding. However, without being bound by any theory, this lower binding could be due to reduced secondary antibody binding, similar to IgG1-7D8-E345 in Example 2. The actual binding affinity of 005-E345 may be similar to or even higher than that of 005-WT; however, we could not confirm this due to the lack of directly labeled antibodies.

[0747] The binding of HuMab-005 to Raji cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound to Raji cells in a dose-dependent manner. The maximum binding of the 005-I253D and H433A mutants was similar to that of wild-type 005, while the binding of the 005-E435R, K439E, S440K mutants, and the combination of 005-K439E+005-S440K was lower than that of wild-type 005. However, without being bound by any theory, this lower binding could be due to reduced secondary antibody binding, similar to IgG1-7D8-E345 in Example 2 (epitope masking).

[0748] Example 10: CDC assay of CD38 antibody 005 mutant on CD38-positive cells

[0749] In a 96-hole round-bottom plate, 0.1 × 10 6Daudi or Raji cells were pre-incubated for 15 minutes at room temperature in a shaker with a series of concentrations of unpurified antibody (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 100 μl. Next, 25 μl of normal human serum was added as a C1q source (20% final concentration), and the cells were incubated at 37°C for 45 minutes. The reaction was terminated by placing the plates on ice. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0750] The ability of the E435R mutation to enhance CDC was further analyzed using different concentrations of normal human serum (NHS) on Wien133 cells. This mutation showed that it enhanced the CDC activity of both 7D8 and 005 antibodies on Daudi and Raji cells. In a round-bottom 96-well plate, 0.1 × 10⁻⁶ H⁺ ions were added to each H⁺ ion. 6 Wien133 cells were pre-incubated for 15 minutes at room temperature in a shaker with a series of concentrations of unpurified antibody (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 50 μl. Next, NHS was added as a source of C1q to achieve a final concentration of 20% or 50% NHS in a 100 μl volume. The reaction mixture was incubated at 37°C for 45 minutes. The plate was placed on ice to terminate the reaction. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0751] It has been found that identifying mutations in the CH2-CH3 domain that lead to the loss of CD20 antibody 7D8 or increase CDC activity have the same effect as 005 antibodies that recognize CD38. Figures 9A-9D The display shows that 005-I253D, H443A, K439E, and S440K are in Daudi ( Figure 9A ) and Raji ( Figure 9B Both cell lines showed complete loss of CDC activity, while the 005-E345R mutant showed a strong enhancement of CDC activity in both cell lines. The combination of 005-K439E + 005-S440K (both single mutants lead to CDC loss) resulted in the recovery of CDC, comparable to the data from 7D8. Surprisingly, 005-E435R even strongly induced CDC in Wien133 cells, while wild-type 005 failed to induce CDC killing (…). Figure 9C ) CDC killing effect of 005-E345R on Wien133 cells was observed at both 20% and 50% serum concentrations. Figure 9CIn 50% serum, 7D8-E345R and 005-E345R also showed enhanced CDC in Raji cells in vitro, similar to their potency in 20% serum. Figure 9D ).

[0752] Because the E345R mutation in the CH2-CH3 region leads to enhanced CDC activity in both the tested CD20 antibody 7D8 and CD38 antibody 005, the E345R mutation is considered a universal antibody modification that can be applied to induce or enhance CDC.

[0753] Example 11: The IgG1 antibody containing the CDC-enhancing mutant E345R is less sensitive to the inhibition of CDC via the Fc-binding peptide DCAWHLGELVWCT than the wild-type antibody.

[0754] Mutating amino acid sites in the hydrophobic sheet of the Fc:Fc interface of IgG revealed that CDC efficacy was either disrupted or enhanced. Further investigation was conducted into the interactions at the Fc-Fc interface, and the potential involvement of oligomeric (e.g., hexacyclic) structures, as observed in the b12 crystal structure, in CDC efficacy. Therefore, a peptide (DCAWHLGELVWCT (SEQ ID NO:7)) targeting a shared binding site in the hydrophobic sheet region on the surface of wild-type IgG Fc was used (Delano et al., Science 2000 Feb 18; 287(5456): 1279-83). Indeed, the shared binding sites on the IgG Fc surface serve as adaptive regions prepared to interact with a wide range of different molecules (Delano et al., Science 2000 Feb 18; 287(5456): 1279-83), consistent with the identity of the core amino acids in the hydrophobic sheets involved in Fc-Fc interactions in the IgG1 b12 crystal structure (Saphire et al., Science 2001 Aug 10; 293(5532): 1155-9). The interactions present at all binding interfaces are mediated by a shared set of six amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435, and Tyr-436) and shared backbone contacts (Delano et al., Science 2000 Feb 18; 287(5456): 1279-83). Therefore, it is expected that Fc-binding peptides can affect Fc-Fc interactions, thereby affecting CDC efficacy.

[0755] In a round-bottomed 96-hole plate, a 0.1×10 6Daudi cells were pre-incubated with 1.0 μg / ml unpurified antibody in 75 μl at room temperature for 10 min using a shaker. 25 μl of a serial concentration (range from 0.06–60 μg / ml final concentration) of the Fc-binding peptide DCAWHLGELVWCT was added to the conditioned cells and incubated at room temperature for 10 min using a shaker. Next, 25 μl of NHS was added as a complement source (20% final concentration), and the cells were incubated at 37°C for 45 min. The reaction was terminated by adding 25 μl of ice-cold RPMI medium (supplemented with 0.1% BSA). 15 μl of propidium iodide was added, and cell lysis was confirmed by FACS analysis.

[0756] Wild type 005 discovered Figure 10A ) or 7D8 ( Figure 10B The Fc-mediated CDC was inhibited in a dose-responsive manner by the Fc-binding peptide DCAWHLGELVWCT. These competitive data further demonstrate that the Fc-Fc interaction located in the hydrophobic IgG plate incorporates CDC potency. Compared to their corresponding wild-type antibodies, both the IgG1-005-E345R and IgG1-7D8-E345R mutants that enhance CDC are less sensitive to competition from the Fc-binding peptide, suggesting that the E345R mutation leads to increased stability of the Fc-Fc interaction and thus enhances CDC.

[0757] Example 12: Antibody-dependent cell-mediated cytotoxicity (ADCC) of CD38 antibody HuMAb 005 variant against CD38-expressing cells.

[0758] Harvest Daudi cells (5 × 10⁻⁶) 6 Cells / ml), washed (twice in PBS, 1200 rpm, 5 min) and collected in 1 ml of RPMI 1640 medium supplemented with 10% enhanced fetal bovine serum (Cosmic Calf Serum, CCS) (HyClone, Logan, UT, USA), the RPMI 1640 medium containing 200 μCi 51 Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, Netherlands). The mixture was incubated in a shaking water bath at 37°C for 1 hour. After washing the cells (twice in PBS, 1200 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted using the trypan blue exclusion method, and diluted to 1×10⁻⁶. 6 Cells / ml concentration.

[0759] Meanwhile, peripheral blood mononuclear cells (PBMCs) were isolated from the fresh erythrocyte sedimentation rate buffy coat (Sanquin, Amsterdam, Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte isolation medium; Lonza, Verviers, France). After resuspending the cells in RPMI 1640 medium supplemented with 10% CCS, cell counts were performed using the trypan blue exclusion method, and the concentration was increased to 1 × 10⁻⁶. 7 Cells / ml.

[0760] For ADCC experiments, 50 μl of the solution was added to a 96-well microtiter plate. 51 Cr-labeled Daudi cells (5,000 cells) were incubated with 15 μg / ml CD38 antibody IgG1-005 or mutant IgG1-005-E345R in 100 μl of RPMI medium supplemented with 10% CCS. After 10 minutes at room temperature, 50 μl of PBMCs (500,000 cells) were added to achieve an effector-to-target ratio of 100:1. 51 Cr-labeled Daudi cells (5,000 cells) were incubated with 100 μl of 5% Triton X100 to determine the maximum amount of cell lysis. 5,000 cells were then incubated with Triton X100. 51 Cr-labeled Daudi cells were incubated in 150 μl of culture medium without any antibodies or effector cells to determine the amount of spontaneous lysis. The level of antibody-independent cell lysis was determined by incubating 5,000 Daudi cells with 500,000 PBMCs without antibodies. Cells were then incubated at 37°C for 4 hours in 5% CO2. To determine the amount of cell lysis, cells were centrifuged (1200 rpm, 3 min), and 75 μl of the supernatant was transferred to a microtube, where a gamma counter was used for counting. 51 Cr release. The percentage of antibody-mediated lysis was calculated using the measured count per minute (cpm) as follows:

[0761] (cpm sample - cpm fragmentation independent of Ab) / (cpm maximum fragmentation - cpm spontaneous fragmentation) × 100%

[0762] Table 15 shows the EC50 values ​​of IgG1-005-wt and IgG1-005-E345R calculated in the ADCC assays performed. Four samples were tested. In all four tested samples, IgG1-005-E345R showed a significantly lower EC50 value than IgG1-005-wt. 50 value.

[0763] Table 15: EC calculated from four experiments 50 value

[0764]

[0765]

[0766] Figure 11 The results showed that, compared to the wild-type antibody HuMab-005, the mutant IgG1-005-E345R demonstrated enhanced ADCC efficacy, inducing ADCC at lower concentrations.

[0767] Example 13: FcRn binding and pharmacokinetic analysis of the 7D8 mutant compared to wild-type 7D8

[0768] Neonatal Fc receptors (FcRn) contribute to the long plasma half-life of IgG by protecting it from degradation. After antibody internalization, FcRn binds to the antibody's Fc region in the endosome, where the interaction is stable in a slightly acidic environment (pH 6.0). Upon recovery to the plasma membrane (where the environment is neutral (pH 7.4)), the interaction is lost, and the antibody is released back into circulation. This affects the plasma half-life of IgG.

[0769] The ability of the 7D8 mutant IgG1-7D8-E354R to interact with FcRn derived from mice, macaques, and humans was tested in ELISA. All incubations were performed at room temperature. 5 μg / ml (100 μl / well) of recombinantly produced biotinylated FcRn extracellular domains (mouse, human, or macaque) (FcRnECDHis-B2M-BIO) was coated onto 96-well plates and diluted in PBST with 0.2% BSA; incubation for 1 hour. Plates were washed three times with PBST, and wild-type IgG1-7D8 or IgG1-7D8-E354R was added in a 3-fold serial dilution (in PBST / 0.2% BSA, pH 6.0), and the plates were incubated for 1 hour. Plates were washed with PBST / 0.2% BSA at pH 6.0. Add goat-anti-human IgG (Fab'2)-HRP (Jackson Immuno Research, catalog number: 109-035-097) diluted in PBST / 0.2% BSA at pH 6.0 and incubate the plates for 1 hour. After washing, add ABTS as substrate and incubate the plates in the dark for 30 minutes. Read the absorbance at 405 nm using an EL808 ELISA reader.

[0770] The mice in this study were housed in barrier units at the Central Laboratory Animal Facility (Utrecht, Netherlands) and fed with freely available water and food in filter-covered cages. All experiments were approved by the Utrecht University Animal Ethics Committee.

[0771] To analyze the pharmacokinetics of the 7D8 mutant in vivo, SCID mice (CB-17 / IcrCrl-scid-BR, Charles-River) were intravenously injected with 100 μg (5 mg / kg) of wild-type 7D8, IgG1-7D8-E354R, -S440K, or K322A; 3 mice in each group.

[0772] Blood samples of 50 μl were collected from the saphenous vein at 10 min, 4 h, 24 h, 2 days, 7 days, 14 days, and 21 days after antibody administration. The blood was collected in heparin-containing vials and centrifuged at 10,000 g for 5 minutes. The plasma was stored at -20°C until the mAb concentration was determined.

[0773] Human IgG concentrations were determined using a sandwich ELISA. Mouse mAb anti-human IgG-κ clone MH16 (#M1268, CLB Sanquin, Netherlands) coated at a concentration of 2 μg / ml onto 96-well Microlon ELISA plates (Greiner, Germany) was used as the capture antibody. After blocking the plates with PBS supplemented with 2% chicken serum, the samples were added and serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 2% chicken serum) and incubated at room temperature (RT) in a plate shaker for 1 hour. The plates were then incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA) and developed with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Roche, Mannheim, Germany). The absorbance was measured at 405 nm using a microplate reader (Biotek, Winooski, VT).

[0774] SCID mice were chosen because they have low plasma IgG concentrations and therefore relatively low IgG clearance. This provides a PK model that is highly sensitive to detecting changes in clearance (due to reduced binding of the Fcγ-part to the neonatal Fc receptor (FcRn)).

[0775] Use GraphPad PRISM version 4 (Graphpad Software) to perform statistical tests.

[0776] Figures 12A-12CBoth wild-type HuMab-7D8 and IgG1-7D8-E345R showed good binding to FcRn in mice, humans, and macaques. IgG1-7D8-E345R showed slightly better binding than wild-type 7D8.

[0777] Figure 13 Plasma concentrations were displayed over time. Wild-type HuMab-7D8 showed no difference in plasma concentration (clearance) over time compared to any of IgG1-7D8-E345R, -S440K, or K322A.

[0778] Example 14: Enhancing the bactericidal activity of IgG antibodies against bacteria expressing Fc-binding surface proteins using the stable Fc-Fc mutant E345R.

[0779] The complement cascade system is an important host defense mechanism against pathogens, which can be divided into three different activation pathways to recognize pathogens: i) the classical antibody-mediated pathway, which is activated after C1q binding to an antibody that binds to the pathogen; ii) the lectin pathway; and iii) the alternative pathway, in which the complement system directly recognizes and is activated by the pathogen in the absence of an antibody. These three pathways converge on the steps of C3 cleavage and C3b deposition. Microorganisms have evolved a variety of complement evasion mechanisms, one of which is mediated by protein A (Joiner Ann. Rev. Microbiol. (1988) 42:201-30; Foster Nat Rev Microbiol (2005) Dec; 3(12):948-58). Protein A was first identified in the cell wall of Staphylococcus aureus and is well known for its binding to the Fc region of IgG (Deisenhofer et al., Biochem (1981) 20, 2361-70; Uhlen et al., J. Biol. Chem (1984) 259, 1695-1702). To date, the antiphage effect of protein A and its role in the pathogenesis of Staphylococcus aureus can be explained by the interaction between protein A and IgG, which leads to incorrect antibody orientation to be recognized by neutrophil Fc receptors (Foster Nat Rev Microbiol (2005) Dec; 3(12): 948-58).

[0780] Example 11 shows that CDC mediated by B cell-specific IgG1 antibodies is inhibited by the competitive Fc-binding peptide DCAWHLGELVWCT. This peptide targets a common binding site on IgG Fc that coincides with the binding sites of protein A, protein G, and rheumatoid factor (Delano et al., Science 2000 Feb 18; 287(5456):1279-83). Based on these data, it is believed that the protein A-mediated bacterial complement escape mechanism functions through competitive Fc binding, leading to instability of Fc-Fc interactions between microbial-specific antibodies and thus inhibiting antibody-mediated complement activation. Furthermore, Example 11 also shows that B cell-specific IgG1 antibodies containing the CDC-enhanced E345R mutation are less sensitive to inhibition of CDC by the competitive Fc-binding peptide DCAWHLGELVWCT than parental wild-type antibodies. By extrapolating these results to Fc-binding proteins expressed by microorganisms, the enhanced Fc-Fc interaction stability of IgG1 due to the E345R mutation makes it less likely for microbial-specific antibodies to inhibit complement through pathogen escape strategies by competing with microbial surface proteins (such as protein A) for Fc binding. Therefore, introducing the E345R mutation into IgG antibodies against bacteria leads to increased C3b deposition on bacteria and enhanced bactericidal activity compared to parental wild-type antibodies.

[0781] As an in vitro measure of complement-mediated bacterial killing, both neutrophil phagocytosis and plasma C3a production (which is consistent with C3b deposition on bacteria) can be measured, as described below. In fact, C3b deposition on Staphylococcus aureus has been described as leading to enhanced phagocytosis and being associated with cytotoxicity (Rooijakkers et al., Nature Immunology 2005:6, 920-927).

[0782] FITC-labeled Staphylococcus aureus was achieved by incubating exponentially growing bacterial cultures with 100 μg / ml FITC in 0.1 M carbonate buffer (pH 9.6) at 37°C for 1 hour. Human polymorphonuclear cells (PMNs) were isolated using a Ficoll gradient. FITC-labeled bacteria were conditioning with a series of specific antibodies, with or without E345R. The concentration of FITC-labeled bacteria was increased by 1 × 10⁻⁶ FITC. 8Phagocytosis was induced in vitro by conditioning FITC-labeled bacteria and human PMN in a total volume of 200 μl in the presence of 25% IgG-reduced serum as a complement source, incubated at 37°C with vigorous shaking for 25 min. Cells were fixed and erythrocytes were lysed by incubation at room temperature with BD FACS lysis solution for 15 min. After washing, phagocytosis was measured by FACS. Neutrophil populations were selected using forward and side scatter gating, and phagocytosis was represented by the mean fluorescence in the neutrophil population. Alternatively, C3a generation in the sample was measured by ELISA as a measure of complement activation and C3b deposition.

[0783] It is expected that Staphylococcus aureus-specific antibodies containing the E345R mutation will induce more complement activation and neutrophil phagocytosis than the parental wild-type antibody. An example of an antibody that can be used for such experiments is the chimeric monoclonal IgG1 pargeximab (BSYX-A110; Biosynexus), which targets lipoteichoic acid (LTA) embedded in the cell wall of Staphylococcus aureus (Baker, Nat Biotechnol. 2006 Dec; 24(12):1491-3; Weisman et al., Int Immunopharmacol. 2009 May; 9(5):639-44).

[0784] Example 15: Application of limiting CDC activation to CDC-repressive mutations in target cells simultaneously bound by a mixture of two different therapeutic monoclonal antibodies.

[0785] As described in Example 6, the CD20 antibody 7D8 mutants K439E and S440K, as monoclonal antibodies, reduced CDC efficacy. Mixing 7D8 antibodies containing these mutations restored CDC. Therefore, effective CDC is limited to cells that simultaneously bind two mutant antibodies. As described in Example 10, the CD38 antibody 005 mutants K439E and S440K, as monoclonal antibodies, reduced CDC efficacy. Mixing 005 antibodies containing these mutations restored CDC. Therefore, effective CDC is limited to cells that simultaneously bind two mutant antibodies.

[0786] It is advantageous to restrict effective CDC induction to target cells that simultaneously express two specific antigens, utilizing their combined expression to improve the selectivity of CDC induction. To restrict CDC induction to cells simultaneously binding CD20 and CD38 antibodies, 7D8-K439E and 005-S440K pairs, or 7D8-S440K and 005-K439E pairs, are added individually or in a 1:1 mixture in the CDC assay. In a round-bottom 96-well plate, 0.1 × 10⁻⁶... 6Daudi or Raji cells were pre-incubated for 15 minutes at room temperature in a shaker with a series of concentrations of unpurified antibody or antibody mixtures (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 100 μl. Then, 25 μl of normal human serum was added as a complement source (20% final concentration), and the cells were incubated at 37°C for 45 minutes. The plates were placed on ice to terminate the reaction. 10 μl of propidium iodide was added, and cell lysis was confirmed on FACS. 7D8-K439E, 005-S440K, and 005-K439E were expected to exhibit limited CDC potency. Simultaneous addition of 7D8-K439E and 005-S440K was expected to restore effective CDC specific to both CD20 and CD38 expression cells. Similarly, it is expected that the mixture of 7D8-S440K and 005-K439E will restore effective CDC specific to both CD20 and CD38 cells.

[0787] Example 16: Enhancing the specificity of enhanced CDC by combining E345R with complementary repressive mutations K439E and S440K in a mixture of two different monoclonal antibodies.

[0788] As described in Example 6, the CD20 antibodies 7D8 mutants K439E and S440K, as monoclonal antibodies, reduced CDC efficacy. Mixing 7D8 antibodies containing these mutations restored CDC. Therefore, effective CDC is limited to cells that simultaneously bind to two mutant antibodies. As described in Example 10, the CD38 antibodies 005 mutants K439E and S440K, as monoclonal antibodies, reduced CDC efficacy. Mixing 005 antibodies containing these mutations restored CDC. Therefore, effective CDC is limited to cells that simultaneously bind to two mutant antibodies.

[0789] It is advantageous to limit CDC-induced enhancement to target cells that simultaneously express two specific antigens, which utilizes their combined expression to improve the selectivity of enhanced CDC-induced enhancement. It is also advantageous to limit CDC-induced enhancement to target cells that simultaneously bind a mixture of at least two different antibodies, said antibodies binding the same cell surface antigen simultaneously on two different epitopes, or on two cross-competing, similar, or identical epitopes.

[0790] Therefore, to limit enhanced CDC induction to cells simultaneously binding to both CD20 and CD38 antibodies, the CDC enhancing mutant E345R was combined with the CDC repressive mutant in antibodies 7D8-E345R / K439E, 7D8-E345R / S440K, 005-E345R / S440K, and 005-E345R / K439E. These antibodies were added individually or mixed 1:1 in CDC assays as follows. In a round-bottom 96-well plate, 0.1 × 10⁻⁶... 6 Wien133 cells (other cell types, such as Daudi or Raji cells, can also be used) are incubated with serial concentrations of unpurified antibody (final concentrations of 0.056–10,000 ng / ml of 3-fold diluted 7D8-E345R / K439E, 7D8-E345R / S440K, 005-E345R / S440K, or 005-E345R / K439E) or antibody mixtures (final concentration of 0.01 μg / ml CD20 antibody mixed with 0–333 ng / ml of 3-fold diluted CD38 antibody; or 3.3 μg / ml CD38 antibody mixed with 0.0056–1,000 ng / ml of 3-fold diluted CD20 antibody) in a total volume of 100 μl at room temperature in a shaker for 15 minutes. Next, 25 μl of normal human serum was added as a complement source (20% final concentration), and the mixture was incubated at 37°C for 45 minutes. The plate was placed on ice to terminate the reaction. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0791] A series of concentrations of 005-E345R / K439E or 005-E345R / S440K antibody were mixed with a fixed concentration of 0.01 μg / ml of 7D8 double mutant antibody (e.g., from...). Figure 14A The maximum concentration of minimum CDC for Wien133 cells was determined as a single agent to prepare complementary combinations 005-E345R / K439E+7D8-E345R / S440K or 005-E345R / S440K+7D8-E345R / K439E. Figure 14C The results showed that, in the presence of fixed concentrations of complementary 7D8-E345R / K439E or 7D8-E345R / S440K CD20 antibodies, the 005 double-mutant CD38 antibody dose-dependently induced CDC. The CDC potency of these complementary combinations (…) Figure 14C It is comparable to the 005-E345R single mutant (enhanced) antibody as a single agent. Figure 14BConversely, in the presence of irrelevant antibody b12, both 005-E345R / K439E and 005-E345R / S440K showed almost no CDC in the series of concentrations tested (comparable to 005-E345R / K439E or 005-E345R / S440K as single agents). Figure 14B (as shown in the image).

[0792] A series of concentrations of 7D8-E345R / K439E or 7D8-E345R / S440K antibody were mixed with a fixed concentration of 3.3 μg / ml of 005 double mutant antibody (e.g., from...). Figure 14B The drug identified in the study showed little but limited efficacy against Wien133 cells as a single agent (CDC) to prepare complementary combinations 7D8-E345R / K439E+005-E345R / S440K or 7D8-E345R / S440K+005-E345R / K439E. Figure 14D The results showed that the 7D8 double mutant CD20 antibody induced CDC very effectively in the presence of complementary 005-E345R / K439E or 005-E345R / S440KCD38 antibodies, even at the lowest concentrations tested, as if there were only a few 7D8 double mutant antibody molecules per cell. To eliminate the contribution of the increased Fc tail density on the cell membrane to the observed enhanced CDC from the mixture of 7D8 and 005 antibodies with complementary K439E and S440K mutations, antibody combinations with non-complementary mutations were also tested. Figure 14D The results show that non-complementary combinations exhibit far lower CDC efficiency than complementary combinations, due to the less efficient Fc-Fc interactions compared to complementary combinations.

[0793] These data suggest that (enhanced) CDC induction by therapeutic antibodies can be limited to cells that simultaneously bind a mixture of two complementary antibodies, in which case they have different antigen specificities, thereby enhancing target cell specificity by requiring co-expression of both antigens.

[0794] like Figure 14A and 14BAs can be seen, compared to 7D8-E345R alone, 7D8-E345R / K439E, 005-E345R / S440K, 7D8-E345R / S440K, and 005-E345R / K439E exhibit limited CDC efficiency. Furthermore, the mixture of 7D8-E345R / K439E and 7D8-E345R / S440K achieves enhanced CDC potency compared to wild-type 7D8 antibody as a single agent. Similarly, the mixture of 005-E345R / K439E and 005-E345R / S440K also achieved enhanced CDC potency compared to wild-type 005 antibody as a single agent (data not shown).

[0795] Example 17: Use of limiting effective CDC activation to CDC repressive mutations in antibody complexes specifically composed of therapeutically administered antibodies.

[0796] As described in Example 6, the CD20 antibody 7D8 double mutant K439E / S440K restored the CDC efficacy reduced by the K439E or S440K single-point mutant. As described in Example 10, the CD38 antibody 005 double mutant K439E / S440K restored the CDC efficacy suppressed by the K439E or S440K single-point mutant. As observed, the single-point mutation disrupts the Fc:Fc interaction when there is an unmutated amino acid on the facing side of the Fc:Fc interface. Introducing a compensatory mutation on the facing side of the Fc:Fc interface restores the CDC efficacy. Therefore, effective CDC is clearly limited to antibody complexes specifically composed of antibodies containing these two mutations.

[0797] In another embodiment, CDC induction is limited to antibody complexes specifically composed of therapeutically administered antibodies. To limit CDC induction to cells that bind only to therapeutic CD20 or CD38 antibodies, the CDC repressive mutations K439E and S440K are combined into antibodies 7D8-K439E / S440K or 005-K439E / S440K. These antibodies are added individually to the CDC assay with or without non-target-specific IgG as follows: In a round-bottom 96-well plate, 0.1 × 10⁻⁶ 6 One Daudi or Raji cell was incubated with unpurified antibody or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 100 μl at room temperature in a shaker for 15 minutes. Next, 25 μl of normal human serum was added as a complement source (20% final concentration), and the mixture was incubated at 37°C for 45 minutes. The reaction was terminated by placing the plates on ice. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0798] It is expected that 7D8-K439E / S440K will induce CDC with similar potency to the wild-type 7D8 antibody. The addition of non-specific IgG to 7D8-K439E / S440K is not expected to affect its CDC-inducing potency. Similarly, it is expected that 005-K439E / S440K will achieve CDC with similar potency to the wild-type HuMAb 005. The addition of non-specific IgG to 005-K439E / S440K is not expected to affect its CDC-inducing potency.

[0799] Example 18: Use of enhanced CDC activation limited to CDC repressive mutations in antibody complexes specifically composed of therapeutically administered antibodies.

[0800] As described in Example 6, the CD20 antibody 7D8 double mutant K439E / S440K restored the CDC efficacy reduced by the K439E or S440K single-point mutant. As described in Example 10, the CD38 antibody HuMAb 005 double mutant K439E / S440K restored the CDC efficacy suppressed by the K439E or S440K single-point mutant. As observed, the single-point mutation disrupts the Fc:Fc interaction when there is an unmutated amino acid on the facing side of the Fc:Fc interface. Introducing a compensatory mutation on the facing side of the Fc:Fc interface restores the CDC efficacy. Therefore, effective CDC is clearly limited to antibody complexes specifically composed of antibodies containing these two mutations.

[0801] In another embodiment, CDC-induced enhancement is limited to antibody complexes specifically composed of therapeutically administered antibodies. Mutations capable of forming CDC-induced antibody complexes can be identified using serum antibodies that are nonspecific to the antigen of interest by screening and selecting mutations that stimulate Fc:Fc interactions for CDC stimulation. To limit enhanced CDC induction to cells specifically bound by CD20 complexes or CD38 antibodies, the CDC-enhancing mutation E345R is combined with a CDC-repressive mutation into antibodies 7D8-E345R / K439E / S440K or 005-E345R / K439 / S440K. These antibodies are added individually to the CDC assay with or without non-target-specific IgG as follows: In a round-bottom 96-well plate, 0.1 × 10⁻⁶ 6One Daudi or Raji cell was incubated with unpurified antibody or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ml) in a total volume of 100 μl at room temperature in a shaker for 15 minutes. Next, 25 μl of normal human serum was added as a complement source (20% final concentration), and the mixture was incubated at 37°C for 45 minutes. The reaction was terminated by placing the plates on ice. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0802] Compared to wild-type HuMAb 7D8, 7D8-E345R / K439E / S440K is expected to induce CDC with enhanced efficiency. Adding non-specific IgG to 7D8-E345R / K439E / S440K is not expected to affect the efficiency of CDC induction compared to wild-type 7D8 antibody. Similarly, 005-E345R / K439E / S440K is expected to achieve CDC with enhanced efficiency compared to wild-type 005 antibody. Adding non-specific IgG to 005-E345R / K439E / S440K is not expected to affect the efficiency of CDC compared to wild-type 005 antibody.

[0803] Example 19: Identification of mutants that stimulate Fc:Fc interaction-mediated antibody oligomerization as detected by CDC assay using mutant screening methods

[0804] As described in Examples 6 and 10, amino acid mutations stimulating CDC were identified in multiple cell lines expressing various levels of the said antigens for antibodies recognizing two different target antigens (CD20 and CD38). Surprisingly, the single-point mutation E345R was confirmed to be sufficient to confer anti-CD38 antibody 005 to CDC-dependent lysis of Wien133 cells, which, in wild-type IgG1 form, could not lyse these cells via CDC.

[0805] Other mutations at or around the Fc:Fc interface can stimulate oligomerization and CDC in a similar manner. Alternatively, mutations can indirectly stimulate oligomerization, for example, by inducing Fc:Fc interactions through allosteric changes.

[0806] To determine whether other amino acid mutations could stimulate Fc-mediated antibody oligomerization, a library of anti-CD38 IgG1-005 mutants was screened using the CDC assay, both individually and in pairs, to select amino acid pairs that interact, for example, at the Fc:Fc interface. However, the same strategy can be applied to other antibodies, such as another IgG1 or IgG3 antibody.

[0807] A concentrated library of mutations at the locations shown in Table 16 was generated. The mutations were introduced into the IgG1-005 Fc region using the Quikchange site-directed mutagenesis kit (Stratagene, USA). In brief, for each desired mutation location, a full-length plasmid DNA template with the 005 heavy chain of the IgG1m(f) allotype was replicated using forward and reverse primers encoding degenerate codons at the desired location. The resulting DNA mixture was digested with DpnI to remove the source plasmid DNA and used for transformation of *E. coli*. The resulting colonies were collected and cultured, from which plasmid DNA was isolated and re-transformed into *E. coli* to obtain cloning colonies. The mutant plasmid DNA isolated from the resulting colonies was verified by DNA sequencing (LGC Genomics, Berlin, Germany). The expression cassette was amplified from plasmid DNA by PCR, and a DNA mixture containing both the IgG1-005 mutant heavy chain and the wild-type light chain was transiently transfected into Freestyle HEK293F cells (Invitrogen, USA) using 293fectin (Invitrogen, USA), essentially as described by the manufacturer. Supernatants containing the antibody mutants were collected. The mutant antibody supernatants were screened individually and in paired mixtures using the CDC assay as follows.

[0808] In a 96-hole round-bottom plate, 0.1 × 10 6 One Daudi or Wien-133 cell (other cell types, such as Raji cells, can also be used) was pre-incubated with 1.0 μg / ml unpurified antibody in a total volume of 100 μl on a shaker at room temperature for 15 minutes. Next, 30 μl of normal human serum was added as a complement source (30% final concentration), and the mixture was incubated at 37°C for 45 minutes. The reaction was terminated by placing the plate on ice. 10 μl of propidium iodide was added, and cell lysis was confirmed by FACS.

[0809] Mutations described in Tables 16, 17, and 18 were selected based on their ability to enhance oligomerization (as detected by CDC efficacy), whether as a single mutant or mixed with other mutants (e.g., in the case of mutations crossing the Fc:Fc interface). Further screening of mutations was conducted based on their ability to not impair FcRn, protein-A or protein-G binding, ADCC, ADCP, or other Fc domain-mediated effector functions. Combining such stimulating point mutations into an Fc domain could further stimulate oligomerization and CDC efficacy.

[0810] To assess its ability to inhibit oligomerization (as determined by CDC in Daudi cells), mutations in the CH2-CH3 region of the CD38 antibody 005 were tested. The lysis of the mutant antibody was compared to that of wild-type 005 (whose lysis was set to 100%). An inhibition cutoff value was set at ≤66% lysis. In this manner, most of the tested mutations inhibited CDC (see Table 16).

[0811] To assess its ability to inhibit oligomerization (as determined by CDC in Wien133 cells), mutations in the CH2-CH3 region of the introduced CD38 antibody 005 were tested (Table 17). Wild-type CD38 antibody 005 failed to induce CDC in Wien133 cells. Mutants exhibiting ≥39% cell lysis were evaluated as enhanced. Completely unexpectedly, almost all obtained amino acid substitutions of E345 and E430 stimulated cell lysis by CDC. To confirm this result, amino acids E345, E430, and S440 were substituted by site-directed mutagenesis with each possible mutation, and their ability to enhance oligomerization (as determined by CDC in Wien133 cells using fresh human serum batches) was tested, producing slightly more effective lysis (Table 18). Again, all E345 and E430 substitutions induced effective CDC in Wien133 cells.

[0812] The following preferred mutations caused ≥39% cell lysis in Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E3 45Y, D / E356G, D / E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I , E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.

[0813]

[0814]

[0815]

[0816] Example 20: In vivo potency of IgG1-7D8-E345R in a subcutaneous B-cell lymphoma xenograft model

[0817] The in vivo antitumor potency of the IgG1-7D8-E345R antibody was evaluated in a subcutaneous model using Raji-luc#2D1 cells. These cells showed ~300,000 CD20 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. Cells were cultured in RPMI containing 10% enhanced fetal bovine serum (HyClone, Logan, UT), penicillin and streptomycin, 1% (v / v) sodium pyruvate and 1 μg / ml puromycin (P-8833, Sigma, Zwijndrecht). Cells were collected at the logarithmic phase (approximately 70% confluence). Female SCID mice (CB-17 / IcrPrkdc-scid / CRL) (Charles-River) aged 6 to 11 weeks were used. On day 0, 5 × 10⁶ cells were added to 200 μl of PBS. 6 Raji-luc#2D1 cells were subcutaneously injected into the right abdomen of each mouse. Tumor formation was monitored using a caliper. The tumor was considered closed when the average tumor volume reached 100 mm. 3 Around day 7, mice were divided into groups (n=9) and treated by intraperitoneal (ip) injection of a single dose of 50 μg antibody (2.5 mg / kg) into each mouse. All antibody samples were supplemented with irrelevant antibody b12 to obtain a total antibody concentration of 0.5 mg / ml. The treatment groups are shown in Table 18. Seven days after treatment, blood samples were obtained to determine serum human IgG levels to verify correct antibody administration. Tumors were measured using a PLEXX caliper at least twice a week until the endpoint tumor volume reached 1500 mm. 3 The tumor may show signs of ulceration or even severe clinical signs.

[0818] Table 18: Treatment Groups and Dosages

[0819]

[0820] Figure 15A This shows the mean tumor growth on day 22 when all groups were still intact. Wild-type antibody IgG1-7D8 slightly inhibited tumor growth compared to the negative control antibody IgG1-b12, although this was not statistically significant. Only IgG1-7D8-E345R significantly inhibited tumor growth compared to the negative control antibody IgG1-b12 (one-way ANOVA analysis, p < 0.01).

[0821] Figure 15B The tumor size was shown to be less than 700 mm. 3Kaplan-Meier plot of the percentage of mice. Tumor formation was significantly delayed in mice treated with IgG1-7D8-E345R antibody compared with mice treated with the negative control antibody IgG1-b12 (Mantel-Cox analysis, p < 0.01), but not in mice treated with wild-type IgG1-7D8.

[0822] These data show that the E345R mutation enhances the in vivo antitumor efficacy of the CD20 antibody 7D8.

[0823] Example 21: In vivo potency of IgG1-005-E345R in a subcutaneous B-cell lymphoma xenograft model

[0824] The in vivo antitumor potency of IgG1-7D8-E345R was evaluated in a subcutaneous model using Raji-luc#2D1 cells. These cells showed ~150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The tumor seeding and measurement protocol was essentially the same as described in Example 20. On day 0, 5 × 10⁶ cells were inoculated into 200 μl of PBS. 6 One Raji-luc#2D1 cell was subcutaneously (sc) injected into the right abdomen of SCID mice. When the average tumor volume was 100 mm², the cells were used. 3 Around day 7, mice were divided into groups (n=7) and treated by intraperitoneal injection of a single dose of 500 μg antibody (25 mg / kg) into each mouse. The treatment groups are shown in Table 19. Tumors were measured until the endpoint of 1500 mmHg. 3 Or until the tumor shows signs of ulceration or until severe clinical signs are observed to avoid significant discomfort.

[0825] Figure 16A The figure shows the mean tumor growth on day 21 when all groups were still intact. Wild-type antibody IgG1-005 slightly...

Claims

1. A method for enhancing complement-dependent cytotoxicity (CDC) of a parent antibody, said parent antibody being a bispecific antibody comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of human IgG1, the second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of human IgG1, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and said method comprising: (1) Introducing the E345R or E345K mutation into the first and second CH2-CH3 regions of the Fc region of the human IgG1 heavy chain, or (2) Introducing an E345R or E345K mutation into the first and second CH2-CH3 regions of the human IgG1 heavy chain Fc region, and wherein further amino acid mutations are performed in the first CH2-CH3 region and in the second CH2-CH3 region, wherein the further amino acid mutation in the first CH2-CH3 region corresponds to a K409R mutation in the human IgG1 heavy chain Fc region, and wherein the further amino acid mutation in the second CH2-CH3 region corresponds to an F405L mutation in the human IgG1 heavy chain Fc region. The further amino acid mutations in the first CH2-CH3 region are different from the further amino acid mutations in the second CH2-CH3 region, and the amino acid numbers are based on EU-index numbers.

2. A method for enhancing complement-dependent cytotoxicity (CDC) of a combination of first and second bispecific antibodies, wherein each of the first and second bispecific antibodies comprises a first polypeptide and a second polypeptide, the first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of human IgG1, the second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of human IgG1, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and each bispecific antibody comprises an Fc domain of an immunoglobulin, wherein the method comprises: (1) Introducing the E345R or E345K mutation into the first and second CH2-CH3 regions of the Fc region of the human IgG1 heavy chain, or (2) Introducing an E345R or E345K mutation into the first and second CH2-CH3 regions of the human IgG1 heavy chain Fc region, and wherein further amino acid mutations are performed in the first CH2-CH3 region and in the second CH2-CH3 region, wherein the further amino acid mutation in the first CH2-CH3 region corresponds to a K409R mutation in the human IgG1 heavy chain Fc region, and wherein the further amino acid mutation in the second CH2-CH3 region corresponds to an F405L mutation in the human IgG1 heavy chain Fc region. The further amino acid mutations in the first CH2-CH3 region are different from the further amino acid mutations in the second CH2-CH3 region, and the amino acid numbers are based on EU-index numbers.

3. The method of claim 2, wherein the further amino acid mutation in the second CH2-CH3 region of the Fc region of the human IgG1 heavy chain is K409R.

4. The method of claim 2, wherein the further amino acid mutation in the first CH2-CH3 region of the Fc region of the human IgG1 heavy chain is F405L.

5. The method according to any one of claims 1-4, wherein the method does not alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the first polypeptide and the second polypeptide or the parent antibody according to claim 1.

6. The method according to any one of claims 1-4, wherein, as determined by ELISA, the method does not alter the binding of the first polypeptide and the second polypeptide or the parent antibody according to claim 1 to the neonatal Fc receptor (FcRn).

7. The method of claim 6, wherein the method does not increase or decrease the binding of the first polypeptide and the second polypeptide or the parent antibody of claim 1 to the neonatal Fc receptor (FcRn) by more than 20%.

8. The method of claim 6, wherein the method does not increase or decrease the binding of the first polypeptide and the second polypeptide or the parent antibody of claim 1 to the neonatal Fc receptor (FcRn) by more than 10%.

9. The method of claim 6, wherein the method does not increase or decrease the binding of the first polypeptide and the second polypeptide or the parent antibody of claim 1 to the neonatal Fc receptor (FcRn) by more than 5%.

10. The method of claim 6, wherein the method does not increase or decrease the plasma clearance rate of the first polypeptide and the second polypeptide or the parent antibody of claim 1 by more than 2.5 times.

11. The method of claim 6, wherein the method does not increase or decrease the plasma clearance rate of the first polypeptide and the second polypeptide or the parent antibody of claim 1 by more than 2.0 times.

12. The method of claim 6, wherein the method does not increase or decrease the plasma clearance rate of the first polypeptide and the second polypeptide or the parent antibody of claim 1 by more than 1.5 times.

13. The method of claim 6, wherein the method does not increase or decrease the plasma clearance rate of the first polypeptide and the second polypeptide or the parent antibody of claim 1 by more than 1.2 times.

14. A variant of a parental antibody, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of human IgG1, the second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of human IgG1, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or different antigens, wherein... (1) The E345 mutation in the first and second CH2-CH3 regions is an E345R or E345K mutation; or (2) The E345 mutation in the first and second CH2-CH3 regions is an E345R or E345K mutation, and wherein further amino acid mutations are performed in the first CH2-CH3 region and in the second CH2-CH3 region, wherein the further amino acid mutation in the first CH2-CH3 region corresponds to the K409R mutation in the Fc region of the human IgG1 heavy chain, and wherein the further amino acid mutation in the second CH2-CH3 region corresponds to the F405L mutation in the Fc region of the human IgG1 heavy chain. The further amino acid mutations in the first CH2-CH3 region are different from those in the second CH2-CH3 region, and the amino acid numbers are based on EU-index numbers.

15. A variant of claim 14, wherein the mutation of the first polypeptide at F405 is F405L in the Fc region of the human IgG1 heavy chain.

16. A variant of claim 14, wherein the mutation of the second polypeptide in K409 is K409R in the Fc region of the human IgG1 heavy chain.

17. A variant of any one of claims 14 to 16, wherein said variant is a full-length human antibody.

18. A variant of claim 14, wherein the full-length human antibody is a full-length human IgG1 antibody.

19. A composition comprising first and second bispecific antibodies, said first and second bispecific antibodies comprising a first polypeptide and a second polypeptide, said first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of human IgG1, said second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region of human IgG1, wherein said first and second antigen-binding regions bind to different epitopes on the same antigen or different antigens, wherein, (1) The mutations in the first and second CH2-CH3 regions corresponding to E345 are mutations corresponding to E345R or E345K mutations; or (2) The mutations in the first and second CH2-CH3 regions corresponding to E345 are mutations corresponding to E345R or E345K mutations, and further amino acid mutations are performed in the first CH2-CH3 region and in the second CH2-CH3 region, wherein the further amino acid mutation in the first CH2-CH3 region corresponds to the K409R mutation in the Fc region of the human IgG1 heavy chain; wherein the further amino acid mutation in the second CH2-CH3 region corresponds to the F405L mutation in the Fc region of the human IgG1 heavy chain, and The further amino acid mutations in the first CH2-CH3 region are different from those in the second CH2-CH3 region, and the amino acid numbers are based on EU-index numbers.

20. A composition comprising a variant of any one of claims 14 to 18 or a composition of claim 19 and a pharmaceutically acceptable carrier.

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