BR122022020571B1 - METHOD FOR INCREASING COMPLEMENT-DEPENDENT CYTOTOXICITY - Google Patents

Abstract

Described herein are related polypeptides and antibodies that comprise a variant Fc domain. The variant Fc domain provides stabilized Fc:Fc interactions when the polypeptides, antibody or antibodies are bound to their target, antigen or antigens on the surface of a cell, thereby providing enhanced complement-dependent cytotoxicity (CDC).

Description

FIELD OF INVENTION

[001] The present invention relates to Fc domain-containing polypeptides, such as antibodies, having increased complement-dependent cytotoxicity (CDC) and may also have other modified actuator functions that result from one or more amino acid modifications in the Fc domain.

BASICS OF THE INVENTION

[002] The actuator functions mediated by the Fc region of an antibody allow the destruction of foreign entities, such as the death of pathogens and the release and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) is initiated by binding of the Fc region to cells bearing the Fc receptor receptor, whereas complement-dependent cytotoxicity ( CDC) is initiated by binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

[003] Each IgG antibody contains two binding sites for C1q, one in each heavy chain (Fc) constant region. A single IgG molecule in solution, however, does not activate complement when the affinity of monomeric IgG for C1q is very weak (Kd ~10-4 M) (Sledge et al., 1973 J. Biol. Chem. 248,2818- 13; Hughes-Jones et al., 1979 Mol. Immunol. 16.697701). Antigen-driven association of IgG can lead to very strong binding of the multivalent C1q molecule (Kd ~10-8 M) and complementary activation (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM exists naturally in covalently linked penta- or hexamers and binding parameters of pentamers or hexamers of cellularly expressed or immobilized antigen can effectively evoke CDC. Antigen binding is a requirement for inducing a conformational change in IgM to expose the C1q binding binding sites (Feinstein et al., 1986, Immunology Today, 169-174).

[004] It has also been suggested that IgG complement activation can achieve complement activation by the formation of hexameric ring structures, through the interaction of the CH2/CH3 domains of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69). Evidence supporting the existence of such hexameric IgG structures has been observed in two-dimensional crystals (Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, D. M. ed.), pp17.1-17.5. Blackwell, Edinburgh; Pinteric et al., 1971 Immunochem. 8, 1041-5) and three-dimensional, as well as for IgG1, IgG2a and IgG4 and human F in solution (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115). A hexameric ring formation was also observed in the crystal structure of the human IgG1K b12 antibody directed against HIV-1 gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug 10;293(5532),1155-9). In the hexamer ring the b12 hexamer ring, six accessible C1q binding sites were presented on the hexamer surface, one from each of the six antibodies, while the other six binding sites faced downwards.

[005] C1q looks like a bundle of tulips with six globular heads, containing the antibody combining regions, attached to six collagen rods (Perkins et al., 1985 Biochem J. 228, 13-26; Poon et al. , 1983 J Mol Biol. 168, 563-77; Reid et al., 1983 Biochem Soc Trans 11, 1-12; Weiss et al., 1986 J. Mol. Biol. 189, 573-81). C1q has been observed to adapt into the b12 hexameric assembly of the 1HZH crystal structure, so that each of those six globular heads is in contact with each 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”, Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, California. November 2000). Mutations in selected amino acids at the Fc interfaces observed between b13 antibodies related by symmetry in the crystal structure were observed to decrease the avidity of C1q binding, indicating the contribution of these amino acids to the Fc:Fc interaction.

[006] WO 2006/104989 describes altered antibody Fc regions and their uses.

[007] WO 2005/047327 describes neonatal Fc receptor binding polypeptide variants, dimeric Fc binding proteins and methods related thereto.

[008] WO 2010/106180 describes Fc variants having increased binding to the neonatal Fc receptor (FcRn).

[009] WO 2005/070963 describes polypeptide Fc region variants and uses thereof.

[0010] WO 2006/053301 describes Fc variants with altered binding to FcRn.

[0011] US 2011/0123440 describes altered antibody Fc regions and their uses. Altered Fc regions have one or more amino acid substitutions.

[0012] US 2008/0089892 describes Fc region variants of the polypeptide and compositions comprising these Fc region variants.

[0013] US 2010/0184959 describes methods of providing an Fc polypeptide variant with altered recognition of an Fc ligand and/or an actuator function.

[0014] US 2010/015133 describes methods of producing polypeptide by regulating polypeptide association.

[0015] US 2010/105873 describes an integrated method for generating multi-domain protein therapeutics.

[0016] US 6,737,056 describes polypeptide variants with altered actuator function.

[0017] Previous efforts have been made to identify antibody Fc variants with an enhanced actuator function or other modified properties. Such studies have focused, for example, on exchange segments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72) or amino acid substitutions in the junction region (Dall 'Acqua et al., 2006 J Immunol 177, 1129-1138) at or near the C1q binding site in the CH2 domain, centered around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642). For example, Moore et al. (2010 mAbs 2(2), 181-189)) describes testing various combinations of S267E, H268F, S324T, S239D, I332E, G236A and I332E for enhanced actuator function through CDC or ADCC. Other Fc mutations that affect binding to Fc receptors (WO 2006/105062, WO 00/42072, U.S. Patent 6,737,056 and U.S. Patent 7,083,784) or physical properties of antibodies (WO 2007/005612 A1) have also been suggested.

[0018] Despite these and other advances in the technique, however, there remains a need for new and improved improved antibody-based therapeutics.

SUMMARY OF THE INVENTION

[0019] The present invention provides polypeptide and antibody variants having enhanced complement-dependent cytotoxicity (CDC) and may also have other enhanced actuator functions as compared to sense polypeptide precursors/antibody. Without being limited by theory, it is believed that the variants are capable of a more stable binding interaction between the Fc regions of two polypeptide/antibody molecules, thereby providing a more avid surface leading to enhanced actuator function, such as an increased or more specific CDC response. Particular variants are also characterized by an enhanced ADCC response, ADCP response and/or other enhanced actuator functions. This subtle mechanism of polypeptide/antibody design can be applied, for example, to increase the efficacy or specificity of antibody-based therapeutics, as described here.

[0020] Thus, in one aspect the present invention relates to a method of increasing the complement-dependent cytotoxicity (CDC) of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, which method comprises introducing a mutation to the precursor polypeptide in one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of a human IgG1 heavy chain.

[0021] In another aspect, the present invention relates to a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises one or more mutations selected from the group corresponding to, E430S , E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor (FcRn ).

[0022] The invention also provides the use of one or more such mutations to increase complement-dependent cytotoxicity (CDC) mediated by the polypeptide or antibody when bound to its antigen, for example, on the surface of an antigen-expressing cell, a cell membrane or a virion.

[0023] In one aspect, referred to here as a “simple mutant”, a variant has increased CDC and may also have increased other actuator functions compared to the precursor polypeptide or antibody.

[0024] In one aspect, referred to herein as a “double mutant”, the variant comprises at least two mutations in said segment and has improved CDC and may have other improved actuator functions as compared to a variant comprising only one of said at least two mutations.

[0025] In one aspect, referred to herein as a “mixed mutant”, the variant provides an increased CDC and may also have other increased actuator functions when used in combination with a second variant of the same or different polypeptide or antibody comprising a mutation in a different amino acid residue in said segment, as compared to one or more of the variant, second variant and the precursor polypeptide or precursor antibody only.

[0026] Typically, the mutation is an amino acid substitution, such as a mutation that exchanges a precursor amino acid residue for one having a different size and/or physicochemical property that promotes the formation of a new intermolecular Fc:Fc bond or increases the strength of the interaction of an existing pair. Exemplary amino acid residues for the mutation according to the invention are shown in Tables 1 and 2A and B, together with exemplary amino acid substitutions. Non-limiting illustrations of different aspects of the invention are provided in Figure 1.

[0027] These and other aspects of the invention, particularly various uses and therapeutic applications for the polypeptide and antibody variants, are described in other additional details.

Brief Description of the Drawings

[0028] Figure 1: (A) Schematic representation of IgG molecules in hexamer formation. The dotted circle illustrates two adjacent Fc:Fc interaction pairs of two neighboring IgG molecules. The arrow in the box illustrates the direction in which illustrations in B, C and D are viewed: neighboring Fc molecules are rotated 90° (in the plane of the drawing) and viewed from the Fab members in the direction of the CH3 domains. (B) Observed effect of oligomerization-enhancing mutations in CDC. A schematic representation illustrating Fc:Fc interaction pairs with increased efficiency according to the single mutant and double mutant aspects of the invention. (C) Observed effect of mutations that inhibit oligomerization in CDC. A schematic representation illustrating how at least two oligomerization-inhibiting mutations that compensate for each other can be, combined into one molecule (double mutant aspect) or separated into two molecules (mixed mutant aspect), to restore or enhance the interaction of Fc:Fc according to the double mutant and mixed mutant aspects of the invention. Mixed mutants achieve activation of the specific actuator function dependent on the binding of both antibodies, which can recognize different targets. (D) Theoretical binding effect of C1q inhibition mutations in CDC. Schematic representation of Fc:C1q interactions, highlighting that if mutations inhibit C1q binding, these cannot be combined or mixed to restore CDC activity, because C1q cannot compensate for defects introduced into the antibody.

[0029] Figure 2: Sequence alignment of human IgG1, IgG1f, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM Fc segments corresponding to residues P247 to K447 in the IgG1 heavy chain, using the software Clustal 2.1, as numbered by the EU index as presented in Kabat. The sequences shown represent residues 130 to 330 of the human IgG1 heavy chain constant region (SEQ ID NO: 1; UniProt Accession No. P01857) and the allotypic variant IgG1m(f); residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID No.: 2; UniProt accession No. P01859) and residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID No.: 2; UniProt accession No. P01860) and residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO: 4; UniProt accession No. P01861) and residues 225-428 of the IgE constant region (Uniprot Accession No. P01854) and residues 133- 353 of the IgA1 constant region (Uniprot Accession No. P01876) and residues 120-340 of the IgA2 constant region (Uniprot Accession No. P01877) and residues 230-452 of the IgM constant region (Uniprot Accession No. P01871) and residues 176 -384 of the IgD constant region (Uniprot Accession No. P01880).

[0030] Figure 3A and B: Sequence alignment of anti-EGFr 2F8 antibody in a main chain of IgG1 (SEQ ID NO: 3), IgG4 (SEQ ID NO: 5) and (partial) IgG3 (SEQ ID NO °: 6). Amino acid numbering according to Kabat and according to the EU index are described (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0031] Figure 4: Detailed view of the K439/S440 interactions between the Fc of adjacent molecules (Fc and Fc', respectively) in a multimeric (e.g., hexameric) arrangement, which illustrates the interaction between Fc and Fc' molecules unmodified wild type.

[0032] Figure 5: Detailed view of K439/S440 interactions between the Fc of adjacent molecules (Fc and Fc', respectively) in a multimeric (e.g., hexameric) arrangement illustrating the interaction between Fc and Fc' variant molecules which comprise K439E and S440K mutations.

[0033] Figure 6: C1q ELISA binding with Fc:Fc 7D8 mutants. The indicated antibody concentration series were coated on the microtiter plate reservoirs and incubated with a fixed concentration C1q. The efficiency for binding C1q was comparable to wild-type 7D8 for all coated mutants except I253D. A representative of at least 3 experiments is shown.

[0034] Figure 7: CDC mediated by 7D8 variants in CD20 positive Raji cells. Raji cells were incubated with the 7D8 mutants (K439E, S440K, K439E/S440K Double mutant, K439E + S440K mix) and a series of C1q concentrations to test the efficacy of CDC by measuring cell lysis. A representative graph of repeated experiments is shown.

[0035] Figure 8: CDC mediated by 7D8 mutants (7D8-WT, K439E, S440K, double mutant K439E/S440K, mix of K439E + S440K) in CD20 positive Daudi cells. A concentration series of 7D8 mutants were tested for their efficacy to induce CDC.

[0036] Figure 9: CDC mediated by CD38 HuMAb 005 antibody mutants in CD38 positive cells. (A) Efficacy of CDC in Daudi cells by a concentration series of 005 mutants. (B) CDC efficacy in Raji cells by a concentration series of HuMAb 005 mutants. (C) CDC efficacy of the E345R mutant of HuMAb 005 with 20% or 50% NHS in Wien133 cells. (D) CDC efficacy of E345R mutants of HuMAb 005 and 7D8 with 20% or 50% NHS in Raji cells. Unpurified antibody samples isolated from transient transfection were tested. As a negative control, supernatant from mock-transfected cells was used.

[0037] Figure 10: CDC for wild-type and E345R mutants of CD38 antibody HuMAb 005, (A) and CD20 antibody HuMAb 7D8 (B) in a competition experiment with an Fc-binding peptide. Cell lysis was measured after CDC in antibody-opsonized Daudi cells incubated with a concentration range of the Fc-binding peptide DCAWHLGELVWCT (SEQ ID NO: 7). Unpurified antibody samples isolated from transient transfections were used. as a negative control, supernatant from mock-transfected cells was used.

[0038] Figure 11: ADCC of Daudi cells expressing CD38 by wild-type antibody CD38 HuMAb 005 and mutant IgG1-005-E345R. ADCC of PBMC from a donor is shown, described as % lysis.

[0039] Figure 12: Binding of wild-type IgG1-7D8 and mutant IgG1-7D8-E345R to human, cynomolgus and mouse FcRn, as determined by ELISA at pH 6.

[0040] Figure 13: Plasma concentrations of wild-type IgG1-7D8 and -E354R, -S440K and K322A variants following intravenous injection into SCID mice.

[0041] Figure 14A, B, C and D: CDC in CD20 and CD38 positive Wien133 cells.

[0042] Figure 15A and B: Assessment of the in vivo efficacy of IgG1-7D8-E345R in a subcutaneous xenograft model with Raji-luc #2D1 cells.

[0043] Figure 16A and B: Assessment of the in vivo efficacy of IgG1-005-E345R in a subcutaneous xenograft model with Raji-luc #2D1 cells.

[0044] Figure 17: CDC in Wien133 cells in CD38 positive, EGFR negative by CD38/EGFR bispecific antibody with the E345R mutation.

[0045] Figure 18A and B: CDC in Wien133 cells or CD20 positive, CD38 negative Raji cells by bispecific antibody with CD20/CD38 with and without the E345R mutation.

[0046] Figure 19: CDC in EGFR positive A431 cells by the EGFR 2F8 antibody with the E345R mutation.

[0047] Figure 20A and B: CDC mediated by E345R mutant antibodies.

[0048] Figure 21: Analysis of colonization of TF antibodies (FITC) with the lysosomal marker LAMP1 (APC).

[0049] Figure 22A-D: Introduction of E345R resulted in enhanced CDC-mediated killing compared to wild-type rituximab tested in different B cell lines.

[0050] Figure 22E: Introduction of E345R resulted in increased maximum CDC-mediated killing compared to wild-type rituximab, independent of expression levels of the complement regulatory proteins CD46 (A), CD55 (B) or CD59 (C) in lines of different B cells with comparable CD20 expression levels.

[0051] Figure 23: CDC kinetics. E345R antibodies result in faster and more substantial target cell lysis by CDC than compared to wild-type antibodies.

[0052] Figure 24: CDC kinetics. Introduction of the E345R mutation into the bispecific CD38xCD20 antibody results in faster and more substantial CDC-mediated target cell lysis.

[0053] Figure 25 A-B: CDC kinetics. Introduction of the E345R mutation into bispecific antibodies CD38xEGFR (A) and CD20xEGFR (B) that bind monovalently to EGFR-negative Raji cells, results in faster and more substantial CDC-mediated target cell lysis.

[0054] Figure 26A-F: CDC in Wien133 cells by an antibody combination of a wild-type antibody with a mutant antibody containing (A-C) E345R and Q386K or (D-F) E345R, E430G and Q386K. IgG1-b12 mutants did not bind Wien133 cells and were used as negative control antibodies.

[0055] Figure 27: CDC efficacy of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing the E345R mutation.

[0056] Figure 28: Introduction of the Fc-Fc stabilizing E345R mutation into the wild-type antibody CD38 005 results in enhanced killing of primary CLL cells in an ex vivo CDC assay (mean ± standard error of the mean).

[0057] Figure 29: Binding of FcRn from wild-type IgG1-005 and IgG1-005 mutants to human, mouse and cynomolgus FcRn at pH 6.0, as determined by ELISA.

[0058] Figure 30: Efficacy of CDC in 20% normal human serum of various rituximab mutants, wild-type rituximab and irrelevant negative control antibody IgG1-b12 in Ramos and SU-DHL-4 cell lines.

[0059] Figure 31: Generation of C4d in normal human serum from IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S and IgG1- Wild-type 005-S440Y and heat-aggregated IgG (HAG) (positive control) as determined by the Micro Vue C4d-fragment ELISA.

[0060] Figure 32A/B: Plasma release rate 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 as determined by total human IgG ELISA (Figure 32A) and by human CD38-specific ELISA (Figure 32B).

DETAILED DESCRIPTION OF THE INVENTION

[0061] As described here, surprisingly, mutations in amino acids that are not directly involved in Fc:C1q binding can, however, increase the CDC of an antibody and can also improve other Fc-mediated actuator functions of the antibody. This supports the hypothesis that antibody molecules such as IgG1 antibodies can form oligomeric structures that are ultimately bound by C1q. Furthermore, while some mutations have been observed to decrease CDC induction, some combinations of such mutations in the same or different antibody molecules have resulted in restored CDC induction and shown additional specificity for antibody oligomerization and thereby promoting induction. more specific CDC guidelines. Particular mutations that enhance the CDC response have also been characterized by an improved ADCC response, improved avidity, improved internalization and in vivo efficacy in a mouse tumor model system as shown in the Examples. These discoveries enable novel antibody-based therapeutics with enhanced CDC induction capabilities, more selective CDC induction, and/or other improved actuator functions.

[0062] The polypeptide variants, including the antibody variants, of the invention all comprise a binding region and a full-length or partial Fc domain of an immunoglobulin that comprises one or more mutations in the segment corresponding to amino acid residues E345 to S440 in IgG1. Without being limited by theory, the identified mutations are believed to result in a more effective and/or more specific CDC induction based on three different principles, schematically represented in Figure 1 and referred to here as “single mutant”, “double mutant”. ” and “mixed mutants”.

[0063] The enhanced C1q and/or CDC effects of the variants of the invention are primarily only detectable in assays that allow antibody oligomers to form, such as in cell-based assays when the antigen is not fixed but is present on a membrane. fluid. Furthermore, it can be verified according to the principles shown in Figure 1C that these effects result from a more stable antibody oligomer and not from a modification of a direct C1q binding site.

Definitions

[0064] The term “simple mutant” should be understood as a variant of the present invention having increased CDC and may also have other enhanced actuator functions compared to the precursor polypeptide or antibody.

[0065] The term “double mutant” should be understood as a variant that comprises at least two mutations in said segment and has improved CDC and may also have other enhanced actuator functions as compared to a variant that comprises only one of said at least two mutations.

[0066] The term “mixed mutant” should be understood as a variant providing an increased CDC and optionally also other enhanced actuator functions when used in combination with a second variant of the same or different polypeptide comprising a mutation in an amino acid residue. different in said segment, as compared to one or more of the variant, second variant and the precursor polypeptide or antibodies alone.

[0067] The term “polypeptide comprising an Fc domain of an immunoglobulin and a binding region” refers, in the context of the present invention, to a polypeptide comprising an Fc domain of an immunoglobulin and a binding region that is capable of to bind to any molecule, such as a polypeptide, for example, present in a cell, bacteria or virion. The Fc domain of an immunoglobulin is defined as the fragment of an antibody that would typically be generated following digestion of an antibody with papain (which is known to one skilled in the art) that includes the two CH2-CH3 regions of an immunoglobulin and a connecting region, for example, a joint region. The constant domain of an antibody heavy chain defines the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc domain mediates the actuating functions of antibodies with cell surface receptors called Fc receptors and complement system proteins. The binding region may be a polypeptide sequence, such as a protein or protein ligand, receptor, an antigen binding region or a ligand binding region to a cell, bacteria or virion. If the binding region is, for example, a receptor, the "polypeptide comprising an Fc domain of an immunoglobulin and a binding region" can be prepared as a fusion protein of the Fc domain of an immunoglobulin and said binding region . If the binding region is an antigen binding region the "polypeptide comprising an Fc domain of an immunoglobulin and a binding region" may be an antibody, such as a chimeric, humanized or human antibody or a heavy chain only antibody or a ScFv-Fc fusion. The polypeptide comprising an Fc domain of an immunoglobulin and a linker region may typically comprise a connecting region, for example, a joining region and two CH2-CH3 regions of the heavy chain of an immunoglobulin, thus the “polypeptide that comprises an Fc domain of an immunoglobulin and a binding region” may be a “polypeptide comprising at least one Fc domain of an immunoglobulin and a binding region”. The term “Fc domain of an immunoglobulin” means, in the context of the present invention, that a connecting region, e.g., antibody subtype-dependent joint, and the CH2 and CH3 region of an immunoglobulin are present, e.g., an IgG1 , human IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM or IgE. The polypeptide is not limited to human origin but may be of any origin, such as, for example, mouse or cynomolgus origin.

[0068] The term “CH2 region” or “CH2 domain” as used herein is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region can be from any of the other subtypes as described here.

[0069] The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341 to 447 according to the EU numbering system. However, the CH3 region can also be from any of the other subtypes as described here.

[0070] The term “immunoglobulin” refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of light molecular weight (L) chains and a pair of heavy (H) chains, all the four potentially interconnected by bisulfite bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1, CH2 and CH3. The heavy chains are interconnected through bisulfite bonds in the so-called “joint region”. Each light chain is typically comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of a domain, CL. The VH and VL regions can further be subdivided into regions of hypervariability (or hypervariable regions that can be hypervariable in sequence and/or form of structurally defined arcs), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved , called structure regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy 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 context, the amino acids of constant region sequences are numbered here according to the EU index (described in Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91 to 3242, pp 662,680,689 (1991)).

[0071] The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule or a derivative thereof, having the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about of eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about three, four, five, six, seven or more days, etc., or any other defined functionally relevant period (such as sufficient time to induce, promote, intensify and/or modulate a physiological response associated with antibody binding to antigen and/or sufficient time for the antibody to recruit an actuator activity). The antibody of the present invention comprises an Fc domain of an immunoglobulin and an antigen-binding region. An antibody generally contains two CH2-CH3 regions and a connecting region, e.g., a joint region, e.g., at least one Fc domain. In this way, the antibody of the present 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 a binding domain that interacts with an antigen. The constant or “Fc” regions of antibodies can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as trigger cells) and components of the complement system, such as C1q, the first component in the pathway. classic complement activation. An antibody can also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody having specificities 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 targets, they may be on the same or different cells or cell types. As indicated above, unless otherwise stated or clearly contradicted by the context, the term antibody herein includes a fragment of an antibody comprising at least a portion of an Fce region that retains antigen-specific binding capacity. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples and binding fragments encompassed within the term "Ab" or "antibody" include, without limitation, monovalent antibodies (described in WO2007059782 by Genmab); heavy chain antibodies, consisting of only two heavy chains and naturally occurring in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), engineered strand exchange domain (SEED or seed body) which are similar asymmetric and bispecific antibody molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); Fc?Adp (Regeneron, WO2010151792), Azimetric Structure (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), immunoglobulin double variable domain antibodies (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); double domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone/Eli Lilly), Knobs-into antibody formats -holes (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); electrostatic conduction antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); IgG1 and IgG2 bispecifics (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual-targeting domain antibodies (GSK/Domantis), Two-in-one antibodies that recognize two targets (Genentech, NovImmune), cross-linked MAbs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), Bispecific as IgG (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74) and DIG-body and PIG-body (Pharmabcine) and Dual affinity whitening molecules (Fc-DART or Ig-DART, from Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia) , methods with common light chain (Crucell/Merus, US7262028) or common heavy chains (KÀBodies from NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc domain such as scFv fusions, such as BsAb from ZymoGenetics/BMS), HERCULES from Biogen Idec (US007951918), SCORPIONS from Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672 -92), scFv fusion from Novartis, scFv fusion from Changzhou Adam Biotech Inc (CN 102250246), TvAb from Roche (WO 2012025525, WO 2012025530), mAb2 from f-Star (WO2008/003116) and dual 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 polyclonals) for example generated by technologies exploited by Symphogen and Merus (Oligoclonics) and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially have any isotype.

[0072] The term “full-length antibody” when used herein refers to an antibody (e.g., a precursor antibody or variant antibody) containing all heavy and light chain constant and variable domains and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype.

[0073] The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0074] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or others, as used herein refer to a preparation of Ab molecules of simple molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to Abs that exhibit a simple binding specificity that have constant and variable regions derived from human germline immunoglobulin sequences. Human mAbs can be generated by a hybridoma that includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a transgene repertoire. light chain, rearranged to produce a functional human antibody and fused to an immortalized cell.

[0075] As used herein, "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM or any allotypes thereof such as IgG1m(za) and IgG1m(f)) which is encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be combined with a kappa (K) or lambda (X) light chain.

[0076] The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding with only one of the antibody binding domains to an antigen, for example, it has a simple antibody-antigen penetration and thus is not capable of antigen cross-linking.

[0077] As used herein, the term “target” is in the context of the present invention to be understood as a molecule in which the binding region of the polypeptide comprising an Fc domain and a binding region, when used in the context of binding of an antibody includes any antigen toward which the raised antibody is directed. The term "antigen" and "target" can be used interchangeably with respect to an antibody and has the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0078] As used herein, the term "binding" in the context of binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10-6 M or less, e.g. 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10-10 M or less, or about 10-11 M or less when determined for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte and binds to the predetermined antigen with an affinity corresponding to a KD that is at least at least ten times smaller, such as at least 100 times smaller, e.g., at least 1,000 times smaller, such as at least 10,000 times smaller, e.g., at least 100,000 times smaller than its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount by which the affinity is lower is dependent on the KD of the antibody, so when the KD of the antibody is much lower (i.e., the antibody is highly specific), then the amount by which the affinity for the antigen is lower than that the affinity for a non-specific antigen can be at least 10,000-fold. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0079] A “variant” or “antibody variant” or “precursor antibody variant” of the present invention is an antibody molecule that comprises one or more mutations as compared to a “precursor antibody”. The different terms may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary precursor antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, or any combination thereof. Similarly, a “variant” or “a variant of a polypeptide comprising an Fc domain of an immunoglobulin and a binding region” or “a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region” of The present invention is a “polypeptide comprising an Fc domain of an immunoglobulin and a binding region”, which comprises one or more mutations as compared to a “precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region”. The different terms may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary mutations include amino acid deletions, insertions and amino acid substitutions in the precursor amino acid sequence. Amino acid substitutions can exchange a naturally occurring amino acid for another naturally occurring amino acid, or for a non-naturally occurring amino acid derivative. Amino acid substitution can be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables: Amino Acid Residue Classes for Conservative Substitutions Alternative conservative amino acid residue substitution classes Alternative physical and functional classifications of amino acid residues

[0080] In the context of the present invention, a substitution in a variant is indicated as: Original amino acid - position - substituted amino acid; The three-letter code, or a one-letter code, are used, including the Xaa and X codes to indicate the amino acid residue. Accordingly, the annotation “E345R” or “Glu345Arg” means that a variant comprises a substitution of glutamic acid with Arginine at a variant amino acid position corresponding to the amino acid at position 345 in the precursor antibody.

[0081] Where a position such as is not present in an antibody, but a variant comprises an insertion of an amino acid, for example: Position - substituted amino acid; the annotation, for example, “448E” is used.

[0082] Such annotation is particularly relevant in connection with modifications in the series of homologous polypeptides or antibodies.

[0083] Similarly when the identity of the substitution of amino acid residues is secondary: Original amino acid - position; or “E345”.

[0084] For a modification where the original amino acids and/or substituted amino acids may comprise more than one, but not all amino acids, the substitution of glutamic acid for Arginine, lysine or Tryptophan at position 345: “Glu345Arg,Lys,Trp” or “E345R,K,W” or “E345R/K/W” or “E345 a R, K or W” May be used interchangeably in the context of the invention.

[0085] Furthermore, the term “a substitution” encompasses a substitution in any of the other nineteen natural amino acids, or in other amino acids, such as unnatural amino acids. For example, an E amino acid substitution 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. This is, by the way, equivalent to the indication 345X, where the X indicates any amino acid. These replacements may also be indicated E345A, E345C, etc, or E345A,C,ect, or E345A/C/ect. The same analogy applies to each and every position mentioned above, to specifically include in this any one of such substitutions.

[0086] An amino acid or segment in a sequence that “matches” an amino acid or segment in another sequence is one that (i) aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar , typically at default settings and (ii) has a sequence identity to SEQ ID NO: 1 of at least 50%, at least 80%, at least 90%, or at least 95%. For example, the sequence alignments shown in Figures 2 and 3 can be used to identify any amino acid in the IgG2, IgG3 or IgG4 Fc sequence that corresponds to a particular amino acid in the IgG1 Fc sequence.

[0087] The present invention relates to variants, viz. precursor polypeptides and precursor antibodies, and/or variant polypeptides and variant antibodies, having a certain degree of identity of amino acids P247 to K447 of SEQ ID NO: 1, 2, 3, 4 and 5, such precursor antibodies and/or variants being then “homologous antibodies” are indicated.

[0088] For the purposes of the present invention the degree of identity between the two amino acid sequences, as well as the degree of identity between two nucleotide sequences, is determined by the “align” program which is a Needleman-Wunsch alignment (i.e. a global alignment). The program is used for aligning polypeptide as well as nucleotide sequences. The default BLOSUM50 count matrix is used by polypeptide alignments and the default identity matrix is used by nucleotide alignments, the first residue penalty of a gap is -12 for polypeptides and -16 for nucleotides. The penalties for additional residues from a cleft are -2 for polypeptides and -4 for nucleotides.

[0089] “Align” is part of the FASTA package version v20u6 (see W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448 and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology 183:63-98). FASTA protein alignments use the Smith-Waterman algorithm with no limitation on gap size (see “Smith-Waterman algorithm”, T. F. SmitH and M. S. Waterman (1981) J. Mol. Biolo. 147:195-197).

[0090] As used herein, the term "actuator cell" refers to an immune cell that is involved in the actuator phase of an immune response, as exposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for example, lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), dead cells, natural dead cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells and basophils. Some actuator cells express Fc receptors (FcRs) or complement receptors and carry out specific immune functions. In some embodiments, an actuator cell such as, for example, a naturally dead cell, is capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcRs are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to antigen-presenting cells. In some embodiments, ADCC can still be intensified by the antibody driven by classical complement activity resulting in the deposition of activated C3 fragments in the target cell. C3 cleavage products are ligands for complementary receptors (CRs), such as CR3, expressed in myeloid cells. Recognition of complement fragments by CRs on actuator cells may promote enhanced Fc receptor-mediated ADCC. In some embodiments the antibody leads to classical complement activation which leads to C3 fragments in the target cell. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, an actuator cell may phagocytose the target antigen, target particle, or target cell. The expression of a particular FcR or complement receptor in an effective cell can be regulated by humoral factors such as cytokines. For example, FCYRI expression has been observed to be upregulated by interferon γ (IFN Y) and/or G-CSF. This enhanced expression increased the cytotoxic activity of cells carrying FCYRI against antigens. An actuating cell can subject a target antigen to phagocytosis or the target cell to phagocytosis or target cell lysis. In some embodiments the antibody that leads to classical complement activation leads to a C3 fragment in the target cell. These C3 cleavage products can promote phagocytosis directly by the actuating cells or indirectly by enhancing antibody-mediated phagocytosis.

[0091] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of introducing transcription of a segment of the nucleic acid linked to the vector. One type of vector is a "plasmid", which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which the nucleic acid segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and are therefore replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes in which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of the vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0092] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to the cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to a particular individual cell, but also to the progeny of such a cell. Because certain modifications may occur in the course of generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the precursor cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomes such as CHO cells, HEK-293 cells, PER.C6, NS0 cells and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic cells such as plant cells and fungi.

[0093] The term "transfectome", as used herein, includes recombinant eukaryotic host cells that express the Ab or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, plant cells, or fungi , including yeast cells.

[0094] The term “preparation” refers to preparations of antibody variants and mixtures of different antibody variants that may have an increased ability to form oligomers when interacting with antigen associated with a cell (e.g., an antigen expressed in cell surface), a cell membrane, a virion, or other structure, thereby enabling increased C1q binding, complement activation, CDC, ADCC, ADCP, other Fc-mediated actuator function, internalization, submodulation, apoptosis, conjugate uptake of antibody drug (ADC), avidity or a combination of any of these. Exemplary assays are provided in the Examples for, for example, C1q binding avidity (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25 and 35); ADCC (Example 12), in vivo efficacy (Example 20, 21), plasma release rates (Example 37), FcRn binding (Example 34), and target-independent liquid phase complement activation (Example 36). Variants according to the aspects herein referred to as “single mutant”, “double mutant” and “mixed mutants”, are described in further detail below, together with exemplary processes for their preparation and methods of use.

[0095] As used herein, the term “affinity” is the strength of the binding of one molecule, e.g., an antibody, to another, e.g., a target or antigen, at a single site, such as the monovalent bond of a individual antigen binding site of an antibody to an antigen.

[0096] As used herein, the term “avidity” refers to the combined strength of multiple binding sites between the two structures, such as between the antigen binding sites of antibodies that simultaneously interact with a target or for example, between the antibody and C1q. When more than one binding interaction is present, the two structures will only dissociate when all binding sites are present and in this way, the rate of dissociation will be lower than for individual binding sites and therefore provide a greater total actuating binding force. (avidity) compared to the binding strength of the individual binding sites (affinity).

[0097] As used herein, the term “oligomer” refers to a molecule that consists of more than one but a limited number of monomer units (e.g., antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to indicate the number of monomer units in the oligomer, for example, a tetramer being composed of four units and a hexamer of six units.

[0098] The term “oligomerization”, as used herein, is intended to refer to the process that converts monomers to a limited degree of polymerization. In this, it is observed that oligomerization of the Fcs domain occurs upon target binding by Fc domain-containing polypeptides, such as antibodies, preferably, but not limited to, on a cell surface. Antibody oligomerization can be assessed, for example, using a cell surface C1q binding assay (as described in Examples 4 and 9), C1q assay efficacy (as described in Example 5), and complement-dependent cytotoxicity in Examples 6, 10 and 19).

[0099] The term “C1q binding”, as used herein, is intended to refer to C1q binding in the context of C1q binding to an antibody bound to its antigen. Antibody binding to its antigen is understood to occur both in vivo and in vitro in the context described herein. C1q binding can be assessed, for example, by using the antibody immobilized on an artificial surface (e.g., plastic on ELISA plates, as described in example 3) or by using it bound to a predetermined antigen on a cell or virion surface. (as described in examples 4 and 9). The binding of C1q to an antibody oligomer is understood here as a multivalent interaction resulting in high avidity binding.

[00100] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is triggered by the binding of the complement component C1q to an antibody bound to its antigen. C1q is the first protein in the early events of the classical complement cascade, which involves a series of cleavage reactions that culminate in the formation of an enzymatic activity called C3 convertase, which cleaves the complement component C3 into C3b and C3a. C3b covalently binds to C5 in the membrane to form C5b, which in turn triggers the last events of complement activation in which the terminal complement components C5b, C6, C7, C8 and C9 accumulate in the membrane attack complex (MAC). The complement cascade results in the creation of pores due to which it causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be assessed by using C1q efficacy (as described in Example 5), CDC kinetics (as described in Examples 28, 29, and 30), CDC assays (as described in Examples 6, 10, 19, 25, 27, 33 and 35) or by cellular deposition of the C3b and C4b method described in Beurskens et al April 1, 2012 vol. 188 no. 7 3532-3541.

[00101] The term “complement-dependent cytotoxicity” (“CDC”), as used herein, is intended to refer to the process of antibody-mediated complement activation leading to lysis of the antibody bound to its target in a cell or virion as a result of the pores in the membrane that are created by MAC assembly. CDC can be assessed by in vitro assay such as a CDC assay in which normal human serum is used as a source of complement, as described in example 6, 10, 19, 25, 27, 33 and 35 or the efficacy of the assay. C1q, as described in example 5, in which normal human serum was limited in C1q.

[00102] The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein is intended to refer to the mechanism of killing of target cells or virions coated with the antibody by cells expressing Fc receptors that recognize the constant region to the binding antibody. ADCC can be determined using methods such as, for example, the ADCC assay described in Example 12.

[00103] The term “antibody-dependent cellular phagocytosis” (“ADCP”) as used herein is intended to refer to the mechanism of elimination of target cells or virions coated by the antibody by internalization by phagocytes. Target cells or virions coated by the internalized antibody are contained in a vesicle called a phagosome, in which they then fuse with one or more lysosomes to form a phagolysosome. ADCP can be assessed by using an in vitro cytotoxicity assay with macrophages as actuator cells and video microscopy as described by van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685. Or as described in example 14 for, for example, S. aureus phagocytes by PMN.

[00104] The term “complement-dependent cellular cytotoxicity” (“CDCC”) as used herein is intended to refer to the mechanism of killing of target cells or virions by cells expressing complement receptors that recognize complement cleavage products. 3 (C3) that are covalently bound to target cells or virions as a result of antibody-mediated complement activation. The CDCC can be evaluated in a similar manner as described by the ADCC.

[00105] The term “plasma half-life” as used herein indicates the time it takes to reduce the concentration of the polypeptide in blood plasma by one half of its initial concentration during elimination (after the distribution phase). For antibodies the distribution phase will typically be 1 - 3 days during which phase there is about a 50% decrease in blood plasma concentration due to redistribution between plasma and tissues. The plasma half-life can be measured by methods known in the art.

[00106] The term “plasma release rate” as used herein is a quantitative measurement of the rate at which a polypeptide is removed from the blood upon administration to a living organism. The release rate and plasma can be calculated as the dosage/AUC (ml/day/kg), where the AUC value (area under the curve) is determined from the concentration time curves according to Example 37.

[00107] The term “submodulation”, as used herein, is intended to refer to the process that decreases the number of molecules, such as antigens or receptors, on a cell surface, for example, by binding an antibody to a receptor.

[00108] The term “internalization”, as used herein, is intended to refer to any mechanism by which an antibody or Fc-containing polypeptide is internalized into a target-expressing cell from the cell surface and/or the surrounding medium, by example, through endocytosis. Internalization of an antibody can be assessed using a direct assay measuring an amount of the internalized antibody (such as, for example, the lysosomal colocalization assay described in example 26).

[00109] The term “antibody drug conjugate” as used herein refers to an antibody or Fc-containing polypeptide having specificity for at least one malignant cell type, a drug and a linker linking the drug to e.g. the antibody. The linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody drug conjugate kills the malignant cell.

[00110] The term “antibody drug conjugate absorption” as used herein refers to the process in which antibody drug conjugates are bound to a target on a cell following absorption/engulfment of the cell membrane and is therefore removal of the cell. Absorption of the antibody drug conjugate can be assessed as “antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay” as described in WO 2011/157741.

[00111] The term “apoptosis” as used herein refers to the process of programmed cell death (PCD) that can occur in a cell. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include bleb formation, cell contraction, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Binding of an antibody to a certain receptor can induce apoptosis.

[00112] Fc receptor binding can be indirectly measured as described in example 12.

[00113] The term “FcRn” as used herein is intended to refer to the neonatal Fc receptor which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk across the intestinal epithelium of the newborn rodent into the bloodstream of newborns. Studies still suggest a similar receptor in humans. In humans, however, it is observed in the placenta to help facilitate the transport of IgG from mothers to the developing fetus and has been shown to play a role in monitoring IgG turnover. FcRn binds IgG at acidic pH 6.0-6.5 but not at neutral pH or higher. Therefore, FcRn can bind IgG from the intestinal lumen (within the intestine) at a slightly acidic pH and ensure efficient unidirectional transport to the basolateral site (within the body) where the pH is neutral to basic (pH 7.0-7. 5). This receptor also plays a role in the recovery of adult IgG through its occurrence in the endocytosis pathway in endothelial cells. FcRn receptors in acidic endosomes bind to pinocytosis through internalized IgG, recycling the cell surface, releasing it at the basic pH of the blood, therefore preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the longer half-life of IgG in blood compared to other isotypes. Examples 13 and 34 describe an assay that shows IgG binding to FcRn at pH 6.0 in ELISA.

[00114] The term “Protein A” as used herein is intended to refer to the 56 kDa MSCRAMM surface protein originally observed in the cell wall of Staphylococcus aureus bacteria. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity and a two-component system called ArlS-ArlR. It was observed that in the biochemical search for its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains folded into a three-helix bundle. Each domain is capable of binding proteins from many mammalian species, but notably IgGs. It binds to the heavy chain Fc region of most immunoglobulins (overlapping the conserved binding site of FcRn receptors) and also interacts with the Fab region of the human VH3 family. Despite these interactions in serum, IgG molecules bind to bacteria through their Fc region solely through their Fab regions, whereby the bacteria interrupt opsonization, complement activation and phagocytosis.

[00115] The term “G Protein”, as used herein is intended to refer to an immunoglobulin binding protein expressed in group C and G Streptococcal bacteria very similar to protein A but with different specificities. It is a 65-kDa (G protein G148 G) and 58 kDa (G protein C40) cell surface protein that has been observed to be used in antibody purification through its binding to the Fc region.

Methods of affecting CDC of a polypeptide

[00116] It is understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a link region.

[00117] In one aspect the present invention relates to a method of increasing the complement-dependent cytotoxicity (CDC) of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, which method comprises introducing a mutation to the precursor polypeptide at one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of a human IgG1 heavy chain.

[00118] In one embodiment the precursor polypeptide may be a precursor antibody comprising an immunoglobulin Fc domain and an antigen binding region.

[00119] Introducing a mutation to a precursor polypeptide in accordance with the method or use of the present invention results in a variant polypeptide (which may also be referred to as a “variant” herein). Thus, the methods of the present invention can be carried out to obtain any variant or variant polypeptide as described herein.

[00120] A variant polypeptide obtained from a method or use of the present invention has an increased CDC compared to the precursor polypeptide. Typically, the effect of a polypeptide on an effective function can be determined by an EC50 value, which is the concentration of the polypeptide required to achieve half-maximal lysis.

[00121] Maximum lysis is the lysis obtained when a saturation amount of the polypeptide is used in which saturation is intended to refer to the amount of the polypeptide in which all targets for the polypeptide are bound by the polypeptide.

[00122] The term “increase in CDC”, “improvement in CDC”, “increase in an effective function”, or “improvement in an effective function”, refers in the context of the present invention that there is a decrease in the EC50 value of the variant polypeptide compared to the precursor polypeptide. The decrease in EC50 value may be, for example, at least or about 2 times, such as at least or about 3 times, or at least or about 5 times, or at least or about 10 times. Alternatively, “increased CDC”, “enhanced CDC”, “increased effective function”, or “enhanced effective function” means that there is an increase in the maximum amount of lysed cells (where the total amount of cells is displayed at 100%) for example, from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100% under conditions where the precursor polypeptide lyses less than 100% of all cells.

[00123] A variant should be tested for increased or improved effective function by cloning the IgG1-005 or IgG1-7D8 heavy chain variable domain into a variant and testing its efficacy in CDC assays, such as described by Daudi (example 6) and Wien (example 10). Using an IgG1-7D8 variable domain HC and Daudi cells, an increase would be defined by more than 2-fold EC50 being less than the EC50 of IgG1-7D8 under the condition studied, such as about 2-fold, about 3-fold, about 5 times, about 10 times or more than 10 times the lowest EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, an increase would be defined by more than 2-fold EC50 being less than the EC50 of IgG1-005 under the condition studied, such as about 2-fold, about 3-fold, about 5 times, about 10 times or more than 10 times the lowest EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, an increase would be defined by more than 2-fold EC50 being less than the EC50 of IgG1-7D8 under the condition studied, such as about 2-fold, about 3-fold, about 5 times, about 10 times or more than 10 times lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, an increase would be defined by an increase in maximum lysis ranging from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100%. An increase in CDC efficacy should also be defined by more than 2 times the EC50 less than the EC50 of IgG1-005 under the condition studied, such as about 2 times, about 3 times, about 5 times, about of 10-fold or greater than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed under conditions where Wien133 cell lysis is detectable.

[00124] The inventors of the present invention surprisingly observed that mutations at these specific positions have an improved effect on the variant CDC antibody, which is obtained by introducing one or more mutations into a precursor antibody in accordance with the method of the present invention (e.g. as shown in example 19). Without being bound by theory, it is believed that by replacing one or more amino acids from the group mentioned above the oligomerization of the positions is stimulated. The antibodies bind with higher avidity (exemplified by example 2; direct labeling of IgG-7D8-E345R results in increased binding of Daudi cells compared to IgG-7D8-WT) which causes the antibodies to bind for a longer period of time cells and therefore different effective functions are enabled, e.g., increased C1q binding, C1q CDC efficacy, ADCC, internalization, ADCP, and/or in vivo efficacy. These effects were exemplified by example 4 (C1q binding in cells), example 5 (C1q efficacy in a CDC assay), example 6, 7, 27, 28, 29 and 35 (CDC assay), example 12 (ADCC), example 26 (internalization), example 21 and 22 (in vivo efficacy), plasma release rate (example 37), FcRn binding (example 34), and target-independent liquid phase complement activation (example 36).

[00125] In this way, mutation of an amino acid residue 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 may also be referred to as the “single mutant” aspect or “CDC-enhancing mutations” in the context of the present invention.

[00126] Thus, in one embodiment, in the CDC increase method the mutation in one or more amino acid residues are selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain.

[00127] In a preferred embodiment, in the CDC increase method the mutation in one or more amino acid residues are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00128] In one embodiment, the precursor polypeptide is a precursor antibody that comprises an Fc domain of an immunoglobulin and an antigen-binding region.

[00129] In another aspect, the present invention also relates to a method of increasing CDC and antibody-dependent cell-mediated cytotoxicity (ADCC) of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, which method comprises introducing a mutation to the precursor polypeptide at one or more amino acid residues corresponding to E430X, E345X and S440W in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a naturally occurring amino acid.

[00130] 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 an IgG1 heavy chain human.

[00131] In a preferred embodiment, the mutation in one or more amino acid residues are selected from the group corresponding to positions E345R, E430T and E430F in the Fc region of a human IgG1 heavy chain.

[00132] In one embodiment, at least one other effective function of the antibody, such as complement activation of C1q binding, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc gamma receptor binding, protein A binding , G protein binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, antibody-mediated complement receptor binding of an opsonized antibody, antibody-mediated phagocytosis (ADCP), internalization, apoptosis, and/or Binding to the complement receptor of an opsonized antibody is also increased, as is ADCC.

[00133] In one embodiment, the precursor polypeptide is a precursor antibody that comprises an Fc domain of an immunoglobulin and an antigen-binding region.

[00134] In one embodiment, the CDC of the precursor antibody is increased when the precursor antibody is bound to its antigen on a cell that expresses the antigen, on a cell membrane, or on a virion.

[00135] In one embodiment, the precursor antibody is a monospecific, bispecific or multispecific antibody.

[00136] Still in one aspect, the present invention relates to a method of increasing the complement-dependent cytotoxicity (CDC) of a precursor antibody that is a bispecific antibody that comprises a first polypeptide that comprises a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same or different antigens and wherein the method comprises introducing a mutation to the first and/or second region of CH2-CH3 in one or more amino acid residues selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain and wherein the first CH2-CH3 region comprises an additional amino acid mutation at a position selected from that corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of an IgG1 heavy chain human and wherein the second CH2-CH3 region comprises an additional amino acid mutation at a position selected from that corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain and wherein the additional amino acid mutation in the first region of CH2-CH3 is different from the additional amino acid mutation in the second region of CH2-CH3.

[00137] In one embodiment, the mutation in one or more amino acid residues are selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a heavy chain of human IgG1.

[00138] In a preferred embodiment, the mutation in one or more amino acid residues are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00139] In one embodiment, the method comprises introducing a mutation in only one of the first or second polypeptide of the specific antibody.

[00140] In one embodiment, the method comprises introducing a mutation in both the first and second polypeptides of the specific antibody.

[00141] In a preferred embodiment, the additional amino acid mutation of the first CH2-CH3 region is in the position corresponding to K409, such as K409R, in the Fc region of a human IgG1 heavy chain and in which the amino acid mutation The additional second CH2-CH3 region is at the position corresponding to F405, such as F405L, in the Fc region of a human IgG1 heavy chain.

[00142] The inventors of the present invention have also shown that introducing a mutation to a precursor antibody at a corresponding amino acid residue at K439 or S440 in the Fc region of a human IgG1 heavy chain decreases the effective function of the precursor antibody (examples 5, 6 and 10).

[00143] As shown in example 6, the amino acid substitution of position K439E or S440K as “single mutants” decreased CDC as compared to any of the first mutations according to the method of the present invention.

[00144] A variant antibody obtained from said method of decreasing an effective function has a decreased effective function compared to the precursor antibody. Typically, the effect of an antibody on effective function can be measured by the EC50 value, which is the concentration of antibody required to obtain half the maximum lysis value.

[00145] Maximum lysis is the lysis obtained when a saturating amount of antibody is used in which saturation is intended to refer to the amount of antibody in which all antigens for the antibody are bound by the antibody.

[00146] The term “decrease an effective function” refers in the context of the present invention that there is an increase in the EC50 value of the variant antibody compared to the precursor antibody. The increase in EC50 value may be, for example, at least or about 2 times, such as at least or about 3 times, or at least or about 5 times, or at least or about 10 times. Alternatively, “decrease in effective function” means that there is a decrease in the maximum amount of cells lysed, e.g., 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% under conditions where the precursor antibody lyses less than 100% of all cells.

[00147] A variant should be tested for diminished effective function by cloning the IgG1-005 or IgG1-7D8 heavy chain variable domain into the variant and testing its efficacy in CDC assays, as described for Daudi cells (example 6) and Wien133 cells (example 10). Using an IgG1-7D8 HC variable domain and Daudi cells, a decrease should be defined by more than 2 times the EC50 lower than the EC50 of IgG1-7D8 under the condition studied, such as about 2-fold, about 3-fold , about 5 times, about 10 times or more than 10 times lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, a decrease would be defined by more than 2-fold EC50 being less than the EC50 of IgG1-005 under the condition studied, such as about 2-fold, about 3-fold, about 5 times, about 10 times or more than 10 times lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, a decrease would be defined by more than 2-fold EC50 being less than the EC50 of IgG1-7D8 under the condition studied, such as about 2-fold, about 3-fold, about 5 times, about 10 times or more than 10 times lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, a decrease would be defined by a decrease in maximum lysis ranging 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%. A decrease in CDC efficacy should also be defined by more than 2 times the EC50 less than the EC50 of IgG1-005 under the condition studied, such as about 2 times, about 3 times, about 5 times, about of 10-fold or greater than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed under conditions where Wien133 cell lysis is detectable.

[00148] Still in one aspect, the invention relates to a method according to the invention and the embodiments described therein, which method comprises introducing the mutation in one of more positions other than S440Y and S440W and further introducing a mutation (i) at each of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W, (ii) at each of the amino acid residues corresponding to K447 and 448 in the Fc region of a human IgG1 heavy chain, such as K447K/R/H and 448E/D in the Fc region of a human IgG1 heavy chain, preferably K447K and 448E in the Fc region of a human IgG1 heavy chain. human IgG1, or (iii) at each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of a human IgG1 heavy chain, such as K447D/E, 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain, preferably K447E, 448K and 449P in the Fc region of a human IgG1 heavy chain.

[00149] With regard to the embodiment in which an additional mutation is introduced as described in step (ii) or (iii) above it should be noted that under normal circumstances the lysine at position K447 is cleaved during antibody production in cells . This can be avoided by protecting position K447 by adding one or more additional amino acid residues (such as 448 or 448/449). This is further described in WO 2013/004841 (Genmab A/S).

[00150] In one embodiment, the method comprises introducing the mutation in one of more positions other than S440Y and S440W and further introducing a mutation in each of the amino acid residues corresponding to K439 and/or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in in S440 is not S440Y or S440W.

[00151] In a preferred embodiment, the mutation in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc region of a human IgG1 heavy chain. Human IgG1 is S440K/R.

[00152] In one embodiment, the precursor polypeptide is a precursor antibody that comprises an Fc domain of an immunoglobulin and an antigen-binding region.

[00153] In one embodiment, the precursor antibody is a monospecific, bispecific, or multispecific antibody. The bispecific antibody can be any of the embodiments described herein.

[00154] Furthermore, any of the mutations listed in table 1 can be introduced by the bispecific antibody. Example 24 shows that introducing the E345R mutation into a bispecific CD20xEGFR antibody enhances CDC efficacy. Examples 23, 29 and 30 also describe some of the bispecific antibodies that comprise a mutation according to the present invention.

[00155] The introduction of mutations in both the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain in a precursor antibody, with the proviso that the mutation in S440 is not S440Y or S440W is also referred to herein as the “double mutant” aspect. The S440Y and S440W mutations have, as described elsewhere, been observed to increase CDC when introduced into a precursor polypeptide.

[00156] Also as described elsewhere the inventors of the present invention have observed introducing an identified mutation at an amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain results in a decrease in effective function (examples 5, 6, 10). However, when inhibition of mutations in either the amino acid residues corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain are introduced the decrease in effective function is restored, thereby rendering the effective function of the precursor antibody without a mutation similar. in the K439 and S440 mutations. However, the presence of the K439 and S440 mutations is, without being limited by any theory, believed to restrict the induction of effective functions to the oligomeric complexes exclusively corresponding to the unique antibodies comprising both the K439 and S440 mutations. Thus, if both K439 and S440 mutations are included in a therapeutic antibody, it is believed, without being bound by any theory, that when such therapeutic antibodies are administered to a patient the induction of effective functions is limited to oligomeric antibody complexes containing therapeutic antibodies comprising the K439/S440 mutations but not containing the patients' own antibodies, which do not comprise the K439 and S440 mutations, therefore limiting any potential side effects caused by the interaction of a therapeutic antibody with the patients' own antibodies.

[00157] When combining mutations at position K439 and/or S440 with the first mutation, CDC intensification is obtained and CDC specificity is increased. In a similar manner, increased CDC enhancement specificity can be obtained by introducing the mutations described in embodiments (ii) and (iii) above.

[00158] In another aspect, the present invention relates to a method of increasing the complement-dependent cytotoxicity (CDC) of a combination of at least one first and a second precursor polypeptide, wherein the at least first and second polypeptide precursor each comprises an Fc domain of an immunoglobulin and a binding region, wherein the method comprises introducing to the at least first and/or second precursor polypeptides a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain.

[00159] In one embodiment, the method comprises introducing to the at least first and/or second precursor polypeptides a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R , E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain.

[00160] In a preferred embodiment, the method comprises introducing to the at least first and/or second precursor polypeptides a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00161] In one embodiment, the method comprises introducing a mutation that may be the same or different to both the first and second precursor polypeptides.

[00162] In yet another embodiment, the method comprises (i) introducing a mutation to the first precursor polypeptide at one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y , S440Y and S440W in the Fc region of a human IgG1 heavy chain, (ii) provide the second precursor polypeptide that does not comprise a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain.

[00163] In one embodiment, the method comprises introducing to the first precursor polypeptide or a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E345K or E345Q in the Fc region of a human IgG1 heavy chain.

[00164] In yet another embodiment, the mutation in one or more positions is other than S440Y and S440W and in which the method further comprises the steps of (i) introducing the first precursor polypeptide or a second mutation in the residue of amino acid corresponding to position K439 in the Fc region of a human IgG1 heavy chain and (ii) introducing to the second precursor polypeptide or a second mutation in the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is not S440Y or S440W; wherein steps (i) and (ii) may alternatively be; (iii) introducing to the first precursor polypeptide or a second mutation in the amino acid residue corresponding to position S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is not S440Y or S440W; (iv) introducing to the second precursor polypeptide or a second mutation in the amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain.

[00165] The second precursor polypeptide can be any precursor polypeptide that by itself does not provide sufficient CDC response upon binding to the target cell.

[00166] Therefore, without being bound by theory, it is believed that said method provides a first variant polypeptide comprising a mutation in one or more amino acid residues in accordance with the above list and thereby in which the variant polypeptide has CDC response. increased and provides a second variant polypeptide that does not comprise such mutations, a CDC response from the second precursor polypeptide is obtained.

[00167] The method of combining a first antibody comprising one of said mutations capable of increasing CDC with a second antibody that is not modified according to the invention, as shown in example 31 results in an increased CDC of the combination. Thus, this method can in one embodiment be used to combine a therapeutic antibody, such as the second antibody, which has been proven to be safe but not sufficiently efficient (or whereby increased efficiency is desirable) with a first antibody that comprises a mutation and therefore resulting in a combination that is effective.

[00168] Examples of suitable second antibodies that do not comprise a mutation in an amino acid residue selected from that corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the IgG1 heavy chain human, includes but is not limited to any of the following; (90Y) clivatuzumab tetraxetan; (90Y) tacatuzumab tetraxetan; (99mTc) fanolesomab; (99mTc) nofetumomab Merpentan; (99mTc) pintumomab; 3F8; 8H9; abagovomab; abatacept; abciximab; Actoxumab; adalimumab; adecatumumab; afelimomab; aflibercept; Afutuzumab; alacizumab pegal; albiglutide; ALD518; alefacecept; alemtuzumab; Alirocumab; altumomab; Altumomab pentetate; alvircept sudotox; amatuximab; AMG714/HuMax-IL15; anatumomab mafenatox; Anrukinzumab (= IMA-638); apolizumab; arcitumomab; aselizumab; atacicept; atinumab; Atlizumab (= tocilizumab); atorolimumab; baminercept; Bapineuzumab; basiliximab; bavituximab; bectumomab; Belatacept; belimumab; benralizumab; bertilimumab; besilesomab; bevacizumab; Bezlotoxumab; biciromab; bifarcept; bivatuzumab; Bivatuzumab mertansine; blinatumomab; blosozumab; brentuximab vedotin; briakinumab; briobacept; brodalumab; canakinumab; cantuzumab mertansine; cantuzumab ravtansine; caplacizumab; capromab; Capromab pendetide; carlumab; catumaxomab; CC49; cedelizumab; Certolizumab pegal; cetuximab; Ch.14.18; citatuzumab bogatox; cixutumumab; Clazakizumab; clenoliximab; Clivatuzumab tetraxetan; conatumumab; conbercept; CR6261; crenezumab; dacetuzumab; daclizumab; dalantercept; dalotuzumab; daratumumab; Demcizumab; denosumab; Detumomab; Dorlimomab aritox; drozitumab; dulaglutide; ecromeximab; eculizumab; edobacomab; edrecolomab; efalizumab; efungumab; elotuzumab; elsilimomab; enavatuzumab; enlimomab; enlimomab pegal; enokizumab; ensituximab; epitumomab; epitumomab cituxetan; epratuzumab; erlizumab; ertumaxomab; etanercept; 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; gevokizumab; girentuximab; glembatumumab; Glembatumumab vedotin; golimumab; Gomiliximab; GS6624; anti-CD74 antibodies; anti-cMet antibodies as described in WO 2011/110642; anti-Her2 antibodies as described in WO 2011/147986 or WO 2011/147982; anti-IL8 antibodies as described in WO 2004/058797; anti-TAC antibodies as described in WO 2004/045512; anti-tissue factor (TF) antibodies as described in WO 2010/066803 or WO 2011/157741; ibalizumab; ibritumomab tiuxetan; icrucumab; igovomab; Imciromab; inclacumab; indatuximab ravtansine; infliximab; inolimomab; inotuzumab ozogamicin; intetumumab; iodine (124I) girentuximab; ipilimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; lemalesomab; lenercept; lerdelimumab; lexatumumab; libivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; lumiliximab; maptumumab; maslimomab; matuzumab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minretumomab; mirococept; mitumomab; mogamulizumab; morolimumab; motavizumab; moxetumomab; pasudotox; Muromonab-CD3; nacolomab tafenatox; namilumab; naptumomab stafenatox; narnatumab; natalizumab; nebacumab; necitumumab; nerelimomab; nimotuzumab; Nivolumab; Nofetumomab; merpentan; obinutuzumab; Ocaratuzumab; ocrelizumab; odulimomab; ofatumumab; olaratumab; olokizumab; omalizumab; onartuzumab; onercept; oportuzumab monatox; oregovomab; otelixizumab; oxelumab; ozoralizumab; pagibaximab; 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-IGF1R; RG1512/HuMax-pSelectin; rilonacept; rilotumumab; rituximab; robatumumab; roledumab; romosozumab; Rontalizumab; rovelizumab; ruplizumab; samalizumab; sarilumab; satumomab; Satumomab pendetide; secukinumab; sevirumab; sibrotuzumab; sifalimumab; siltuximab; siplizumab; sirukumab; solanezumab; solitomab; Sonepcizumab; sontuzumab; sotatercept; stamulumab; sulesomab; suvizumab; tabalumab; Tacatuzumab tetraxetan; tadocizumab; talizumab; tanezumab; taplitumomab paptox; tefibazumab; telimomab aritox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; Ticilimumab (= tremelimumab); tigatuzumab; TNX-650; Tocilizumab (= atlizumab); toralizumab; torapsel; tositumomab; tralokinumab; trastuzumab; trastuzumab emtansine; TRBS07; trebananib; tregalizumab; tremelimumab; tucotuzumab celmoleukin; tuvirumab; ublituximab; urelumab; urtoxazumab; ustekinumab; vapaliximab; vatelizumab; vedolizumab; veltuzumab; vepalimomab; vesencumab; visilizumab; volociximab; Vorsetuzumab mafodotin; votumumab; zalutumumab; zanolimumab; ziralimumab; and zolimomab aritox.

[00169] The first and second variant antibodies will have preference for oligomerization with one another compared to any naturally occurring or wild-type antibody as shown in example 10.

[00170] In one embodiment, the mutation in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain human is S440K/R.

[00171] Therefore, the increase in specificity is in relation to “CDC induction”. Thus, said method is in one embodiment a method of increasing the specificity of inducing an effective function by a combination of at least one first and one second precursor polypeptide.

[00172] To carry out the method of increasing specificity, or specificity of inducing an effective function, by a combination of at least one first and a second precursor polypeptide, a combination of a first variant and a second variant polypeptide is obtained.

[00173] To introduce a mutation in K439 or S440 of a precursor polypeptide, a variant polypeptide thus obtained has a decreased effective function compared to the precursor polypeptide. However, as also described elsewhere herein, the mutation at K439 and S440 are capable of complementing each other or restoring the effective function of a polypeptide comprising both mutations. This ability of the mutations at K439 and S440 to complement each other can be similarly utilized in two polypeptides. Thus, when a mutation at K439 is introduced into a first precursor polypeptide and a mutation at S440 is introduced into a second precursor polypeptide, or vice versa, the decrease in effective function is no longer seen as the first and second polypeptide variants are used in combination. The term "specificity enhancement" or "specificity enhancement" refers in the context of the fact that an effective response is induced by a combination of a first variant polypeptide comprising a mutation at K439 and a second variant polypeptide comprising a mutation at S440. is greater than the effective response induced by the first variant polypeptide comprising a mutation in K439 or the second variant polypeptide comprising a mutation in S440.

[00174] By introducing an amino acid substitution at both K439 and S440 the specificity of oligomerization is increased.

[00175] When combining mutations at position K439 and/or S440 with the first mutation, CDC intensification is obtained and CDC specificity is increased.

[00176] In one embodiment the at least first and second precursor polypeptides bind to the same binding site or, with respect to antibodies, the same epitope.

[00177] In one embodiment the at least first and second precursor polypeptides bind to different binding sites on the same target or, with respect to antibodies, to different epitopes on the same antigen.

[00178] In one embodiment the at least first and second precursor polypeptides bind to different epitopes on different targets.

[00179] In one embodiment the first and second precursor polypeptides are first and second precursor antibodies, which have the same or different VL and VH sequences.

[00180] In one embodiment, the combination of at least one first and one second precursor polypeptide comprises a first precursor polypeptide and a second polypeptide.

[00181] In one embodiment, specificity is increased when a combination of the first and second precursor polypeptides is bound to its binding site or antigen on a cell expressing the antigen, on a cell membrane, or on a virion .

[00182] Whereas, in another aspect the present invention also relates to the use of a mutation in two or more amino acid residues of a polypeptide to increase the specificity of, for example, CDC induced by, the polypeptide when bound to its antigen in a cell expressing the antigen, in a cell membrane, or in a virion, in which a first mutation is in an amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain; a second mutation is in an amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain.

[00183] In one embodiment, the first and second precursor polypeptides are a first and a second precursor antibody each comprising an Fc domain of an immunoglobulin and an antigen-binding region.

[00184] In one embodiment, the first and second precursor antibody is a monospecific, bispecific or multispecific antibody.

[00185] In one embodiment, the first and/or second precursor antibody is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide comprising a second CH2-CH3 region and a second antigen-binding region, wherein said first and second antigen-binding regions bind different epitopes on the same or different antigens and wherein said first CH2-CH3 region comprises an additional amino acid mutation at a position selected from that corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of a human IgG1 heavy chain and wherein the second CH2-CH3 region comprises a mutation of additional amino acid at a position selected from that corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain and wherein the additional amino acid mutation in the first CH2-CH3 region is different from that additional amino acid mutation in the second region of CH2-CH3.

[00186] In a preferred embodiment, the first CH2-CH3 region comprises an additional amino acid mutation at the position corresponding to K409, such as K409R, in the Fc region of a human IgG1 heavy chain and wherein the second region of CH2-CH3 comprises an additional amino acid mutation at the position corresponding to F405, such as F405L, in the Fc region of a human IgG1 heavy chain.

[00187] By carrying out this method, a combination of at least one first and one second variant antibody is obtained. The at least first and second variant antibodies obtained or this method combined when the increased CDC compared to a combination of the first and second precursor antibodies.

[00188] The term “increased CDC” should be understood as described herein.

[00189] The first and/or second precursor antibody can be any precursor antibody as described herein.

[00190] Methods of increasing CDC of a combination of a first and a second antibody may be in particular carried out in order to obtain a first and/or second variant antibody that has any of the characteristics of a variant antibody as described in this .

[00191] In one embodiment the at least first and second precursor antibodies bind to the same epitope.

[00192] In one embodiment the at least first and second precursor antibodies bind to different epitopes on the same antigen.

[00193] In one embodiment the at least first and second precursor antibodies bind to different epitopes on different targets.

[00194] In one embodiment the first and second precursor antibodies have the same or different VL and VH sequences.

[00195] In one embodiment the combination of at least one first and one second precursor antibody comprises a first precursor antibody and a second antibody.

[00196] In one embodiment the combination of at least one first and one second precursor antibody comprises other precursor antibodies, such as a third, fourth or fifth precursor antibody.

[00197] In one embodiment the first and second bispecific or multispecific precursor antibodies are the same or different antibodies. In one embodiment the first and second bispecific or multispecific precursor antibodies bind to different epitopes on the same or different antigen. Thus, in one embodiment said at least first and second precursor antibodies are bispecific or multispecific antibodies that bind different epitopes on the same antigen or different antigens.

[00198] In one embodiment of the methods and/or uses of the present invention the precursor antibody, this is a precursor antibody, a first precursor antibody or a second precursor antibody, may contain mutations other than those of the present invention that have been observed affect an actuator function. Such other mutations may be introduced at the same time as the mutations of the present invention that affect an effective function or they may be introduced sequentially, the methods or uses of the present invention are not limited to the simultaneous or sequential introduction of mutations. Bispecific antibodies can be any bispecific antibody and the methods and uses of the present invention are not limited to any particular bispecific format since it is anticipated that different formats can be used.

[00199] In one embodiment, the method does not alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the precursor polypeptide or precursor antibody.

[00200] In one embodiment, the method does not alter the binding of the precursor polypeptide or precursor antibody to the neonatal Fc receptor (FcRn) as determined by the method described in Example 34.

[00201] In one embodiment, the method does not increase or decrease the binding of the precursor polypeptide or precursor antibody to the neonatal Fc receptor (FcRn) by more than 30%, such as greater than 20%, 10% or 5 % as measured by a change in absorbance at OD405 nm as determined by the method described in Example 34.

[00202] In one embodiment, the method does not increase the evident affinity of the precursor polypeptide or precursor antibody to the mouse neonatal Fc receptor (FcRn) by more than a factor of 0.5 or does not decrease the evident affinity of the polypeptide precursor or precursor antibody to mouse FcRn by more than a factor of 2 as determined by the method described in Example 34.

[00203] In one embodiment, the method does not alter the plasma release rate of the precursor polypeptide or precursor antibody as determined by the method described in Example 37.

[00204] In one embodiment, the method does not increase or decrease the plasma release rate of the precursor polypeptide or precursor antibody by more than a factor 3.0, such as more than a factor 2.5, factor 2 ,0, factor 1.5, or factor 1.2, as determined by the method described in Example 37.

[00205] In one embodiment, the method does not alter target-independent liquid phase complement activation of the variant as determined by the method as determined by the method described in Example 36.

[00206] In one embodiment, the method does not alter the plasma half-life of the precursor polypeptide or precursor antibody.

[00207] Any of the mutations or combinations described herein can be introduced in accordance with the method of the present invention.

[00208] Selected exemplary or preferred amino acid substitution mutations can be tested in appropriate ways allowing oligomer formation of antigen-bound antibodies and detecting complement activation of enhanced C1q binding, CDC, ADCC and/or internalization, such as those described in the examples. For example, the binding avidity of C1q can be determined in accordance with an assay similar to that described in Example 4, using cells that express the antigen for the antibody variant. Exemplary CDC assays are provided in Examples 5, 6, 10, 16, 19, 22, 23, 24, 25, and 35. An exemplary ADCC assay is provided in Example 12. An exemplary internalization assay is provided in Example 26. Finally , for discrimination between mutations in amino acid residues directly involved in C1q binding from mutations affecting 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 release rates can be compared according to an assay described in example 37, comparison of FcRn binding according to example 34 and target-independent liquid phase complement activation can be evaluated according to the assay in example 36.

[00209] In one embodiment the mutation in one or more amino acid residues may be an amino acid substitution, an amino acid deletion or an amino acid insertion.

[00210] In one embodiment the mutation in one or more amino acid residues is an amino acid deletion.

[00211] In one embodiment the mutation in one or more amino acid residues is an amino acid insertion.

[00212] In a particular embodiment, the mutation in one or more amino acid residues is an amino acid substitution.

[00213] In one embodiment the mutation in one or more amino acid residues can be selected from any of the amino acid substitutions, amino acid deletions listed in Table 1.

[00214] Thus, in an 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; in particular E345A, D, G, H, K, N, Q, R, S, T, Y or W or more particularly E345D, K, N, Q, R or W or even more particularly E345R, Q, N, K or Y. In another preferred embodiment, E345X is E345K or E345Q.

[00215] In another additional embodiment E430X can be E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W or Y ; in particular E430T, S, G, F or H. In another preferred embodiment, E430X is E430G or E430S. In another embodiment, the mutation is not in an amino acid residue directly involved in C1q binding, optionally as determined by comparing C1q binding in an ELISA assay according to example 3 with C1q binding in an cell-based assay according to example 4.

[00216] In one embodiment, the one or more mutations is a mutation, that is, no more than one mutation is introduced to the precursor antibody.

[00217] In another embodiment, the method or use according to the present invention comprises introducing a mutation in at least two, such as two, three four, five or more of the amino acid residues in Table 1.

[00218] Any of the combinations of mutations described here can be introduced in accordance with the method of the present invention.

[00219] In one embodiment, the method comprises introducing to the precursor polypeptide more than one mutation, such as two, three, four or five, in particular two or three mutations in 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 more than one of the amino acid residues corresponding to E345X, E430X, S440Y and S440W in the Fc region of a human IgG1 heavy chain can be mutated, such as two or all of E345X, E430X, S440Y and S440W. , optionally in combination with a mutation in one or more other amino acids listed in Table 1. The at least two mutations may be any amino acid residue substitution of position E345 in combination with any amino acid residue substitution of position E430 or S440Y or S440W or may be any amino acid substitution of position E430 in combination with any amino acid residue of position S440Y or S440W. In a further embodiment, two or three mutations are introduced into the precursor antibody at amino acid residues selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[00220] Such 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 in the Fc region of a human IgG1 heavy chain.

[00221] In the methods or uses according to the present invention, CDC is increased when the antibody is bound to its antigen.

[00222] Without being linked to any theory, it is believed that CDC is increased when the antibody is linked to its antigen, where the antigen is on a cell that expresses the antigen, cell membrane or virion. In one embodiment, the Fc region of an IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO: 1.

[00223] The precursor polypeptide or precursor antibody can be any precursor polypeptide or any precursor antibody as described herein. The precursor polypeptide and the precursor antibody in this context are also intended to be the first precursor and the second precursor polypeptide and the first precursor and the second precursor antibody.

[00224] In one embodiment, the precursor antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

[00225] In one embodiment the precursor antibody is a human full-length antibody, such as a human full-length IgG1 antibody.

[00226] In one embodiment, the precursor antibody, first precursor antibody and second precursor antibody is a human IgG1 antibody, for example, the IgG1m(za) or IgG1m(f) allotype, optionally comprising an Fc region that comprises SEQ ID NO: 1 or 5.

[00227] In one embodiment, the precursor antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO: 2.

[00228] In one embodiment, the precursor antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO: 3.

[00229] In one embodiment, the precursor antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO: 4.

[00230] In one embodiment, the precursor antibody is a bispecific antibody.

[00231] In one embodiment, the precursor antibody is any antibody as described herein, for example, a fragment antibody comprising at least part of an Fc Region, monovalent antibodies (described in Genmab's WO2007059782); heavy chain antibodies, consisting of only two heavy chains and naturally occurring in e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), engineered strand exchange domain (SEED or seed body) which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); Fc?Adp (Regeneron, WO2010151792), Azimetric Structure (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), immunoglobulin double variable domain antibodies (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); -holes (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); electrostatic guide antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); IgG1 and IgG2 bispecifics (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual-targeting domain antibodies (GSK/Domantis), Two-in-one antibodies that recognize two targets (Genentech, NovImmune), cross-linked MAbs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like bispecifics (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74) and DIG-body and PIG-body (Pharmabcine) and dual affinity bleaching molecules (Fc-DART or Ig-DART, from Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia) , methods with common light chain (Crucell/Merus, US7262028) or common heavy chains (KÀBodies by NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc domain such as scFv fusions, such as BsAb from ZymoGenetics/BMS), HERCULES from Biogen Idec (US007951918), SCORPIONS from Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672- 92), scFv fusion from Novartis, scFv fusion from Changzhou Adam Biotech Inc (CN 102250246), TvAb from Roche (WO 2012025525, WO 2012025530), mAb2 from f-Star (WO2008/003116) and dual scFv fusions. Also It should be understood that the term antibodies, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) e.g. generated by technologies exploited by Symphogen and Merus (Oligoclonics) and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially have any isotype.

[00232] In another embodiment, the antigen is expressed on the surface of a cell.

[00233] In another embodiment, the cell is a human tumor cell.

[00234] In yet another embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c -Met, HERV envelope protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.

[00235] In another embodiment, the antigen is associated with the cell membrane.

[00236] In another embodiment, the antigen is associated with the virion, optionally in which the antigen is comprised in the protein coat or a lipid envelope of the virion.

[00237] In another embodiment, the antibody is a human antibody, optionally binding at least one antigen selected from CD20 and CD38.

[00238] In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally comprising a variable heavy and/or light chain region of at least one of 7D8 and 005.

[00239] In any use according to the described invention, the antibody without any mutations of the present invention can be any precursor antibody. Thus, use herein provides any variants of such precursor antibodies.

[00240] In one embodiment the effective function is Fc receptor binding, for example, including Fc gamma receptor binding.

[00241] In one embodiment the effective function is internalization of Fc-containing polypeptide.

[00242] In one embodiment the effective function is a combination of complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[00243] As used herein, the term “C1q binding”, when used in the context of a variant or antibody of a precursor antibody, includes any mechanism of the first component in the classical pathway of complement activation mediated by the binding of the variant or antibody to tissues. or host factors, including various cells of the immune system (such as trigger cells). C1q binding of an antibody can be assessed using an ELISA (such as, e.g., C1q binding ELISA used in Examples 3 and 4) or C1q efficacy can be assessed by a CDC assay (such as, e.g. , the CDC assay used in example 5). In a further embodiment, the C1q binding avidity of the antibody is determined according to an assay described in example 4.

[00244] In all methods according to the described invention, the antibody without any mutations of the present invention can be any precursor antibody. Therefore, the method herein provides any variants of such precursor antibodies.

[00245] The precursor antibody, the first precursor antibody, the second precursor antibody or and variants thereof obtained by the methods and/or uses of the present invention may bind any target as described herein.

[00246] Examples of antigens or targets that the invention can be directed against are; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; carcinoma-associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Crypto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin chain II beta; FLT1; LEAF1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein homolog 90 [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); human respiratory syncytial virus, F glycoprotein; ICAM1; IFNA1; IFNA1; bispecific IFNB1; IgE Fc; IGF1R; IGHE connection region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin 2 receptor bta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, LPS lipopolysaccharide domain; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serum IATS O11; RSV (human respiratory syncytial virus, F glycoprotein); ROR1; RTN4; SELL; SELP; STEAP1; subunit II B of Shiga-like toxin [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipotechoic acid; alpha-beta T cell receptor; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1 and Vimentin.

[00247] In the main aspect, the present invention relates to a method of inducing CDC against a cell, cell membrane or virion that expresses a target to which a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region binds. , which comprises (i) providing the precursor polypeptide or a combination of at least one first precursor polypeptide and a second precursor polypeptide that has been mutated in accordance with any of the embodiments described herein and (ii) contacting a preparation of the mutated precursor polypeptide of step (i) or the mutated combination of at least one first precursor polypeptide and a second precursor polypeptide of step (i) with the cell, cell membrane or virion expressing an antigen in the presence of human complement or an actuator cell.

[00248] In one embodiment any or all polypeptide precursor, first precursor polypeptide and the second precursor polypeptide may be an antibody.

[00249] In another embodiment, the method increases an additional effective response selected from ADCC, Fc gamma receptor binding, protein A binding, G protein binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), cytotoxicity complement-enhanced, antibody-mediated binding to the complement receptor of an opsonized antibody, and any combination thereof.

[00250] In yet another embodiment, the method also induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[00251] In yet another embodiment, the method also induces Internalization of Fc-containing polypeptide.

[00252] In one embodiment, the cell is a human tumor cell or a bacterial cell.

[00253] In another embodiment, the IgG1 antibody precursor is a human IgG1 antibody.

[00254] In another embodiment, the first and second antigens are separately selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), EpCAM and MRP3.

[00255] In another embodiment, the first and second precursor antibodies are fully human, optionally in which the first and second precursor antibodies bind the separately selected antigens of CD20 and CD38.

[00256] In yet another embodiment, the first and second precursor antibodies are separately selected from 7D8 and 005.

[00257] In yet another embodiment, the cell is a bacterial cell.

[00258] In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. Epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis.

[00259] In another embodiment, the first and/or second antigen is lipoteichoic acid (LTA), optionally wherein at least one of the first and second precursor antibodies is pagibaximab.

[00260] In another embodiment, the antigen is expressed in a virion.

[00261] In another embodiment, the first and second antibodies bind the same antigen.

[00262] In another embodiment, the first and second antibodies comprise the same VH sequence, VL sequence or both VH sequence and VL sequence.

[00263] For the purposes of the present invention, the target cell that expresses or is otherwise associated with an antigen can be any prokaryotic or eukaryotic cell. The exemplary antigen-expressing cell includes, but is not limited to, mammalian cells, particularly human cells, such as human cancer cells, and unicellular organisms, such as bacteria, protozoa, and unicellular fungi, such as yeast cells. Cellular membranes comprising or otherwise associated with an antigen include partial and/or disrupted cell membranes derived from a cell expressing the antigen. An antigen associated with a virion or viral particle may be comprised of or otherwise associated with the protein coat and/or a lipid envelope of the virion.

[00264] The target cell may, for example, be a human tumor cell. Suitable tumor antigens include any described target or antigen, but are not limited to, erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74 , CD138, CXCR5, c-Met, HERV envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFR, L1-CAM, AXL, Tissue Factor (TF), EpCAM and MRP3. Preferred antigens include CD20, CD38, HER2, EGFR, IGFR, CD25, CD74 and CD32. Exemplary antibodies include anti-CD20 antibody 7D8 as described in WO 2004/035607, anti-CD38 antibody 005 as described in WO 06/099875, anti-CD20 antibody 11B8 as described in WO 2004/035607, anti-CD38 antibody 003 as described in WO 06/099875, anti-EGFr 2F8 antibody as described in WO 02/100348. Examples of other particular antibodies are provided herein.

[00265] Alternatively, the target cell may be a bacterial cell, such as, for example, S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis. Exemplary antigens include Lipotechoic Acid (LTA) and exemplary antibodies include pagibaximab.

[00266] Alternatively, the target may be present on the surface of a virus, fungal cell or other particle, such as, for example, West Nile virus, Dengue virus, hepatitis C virus (HCV), human immunodeficiency virus ( HIV), human papillomavirus, Epstein-Barr virus, Herpesvirus, poxvirus, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus and Histoplasma.

[00267] In one embodiment, contact step (ii) takes place in vitro.

[00268] In one embodiment, contact step (ii) takes place in vivo.

[00269] In another embodiment, step (ii) comprises the variants to a patient.

[00270] In a further embodiment, the patient suffers from cancer, a bacterial infection or a viral infection. The contact step (ii) of the above-mentioned embodiments can take place in vitro or in vivo. In the latter case, step (ii) may further comprise administering the preparation or preparations to a patient, optionally a patient suffering from cancer or a bacterial infection. Additional details on therapeutic applications are provided below.

[00271] The first and second antibodies comprise antigen-binding regions that can bind to the same or different epitope. Such epitopes can be on the same or different targets.

[00272] In one embodiment, the first and second antibodies bind different epitopes on different targets. Such targets may be expressed in the same cell or cell type or may be expressed in different cells or cell types. In such an embodiment, the enhancement of an effective function is directed only with respect to cells or cell types that express both targets and thereby reduces the risks of any collateral damage of cells or cell types that are not the cause. of a disease to be treated.

[00273] Without being bound by any theory, it is believed that CDC enhancement can be restricted to target cells that express two specific targets/antigens simultaneously, as long as the first and second antibodies bind the epitopes found on the same cell , thereby exploiting the combined expression of targets to improve the selectivity of enhanced CDC induction.

[00274] In cases where targets are expressed in different cells or cell types, it is believed, without being linked by theory, that administration in any order of the first and second antibodies will improve CDC enhancement and possibly also other effective functions by “recruiting” a second cell or cell type that expresses the second target.

[00275] In an embodiment in which a combination of a first and a second antibody are used, step (ii) can be carried out simultaneously, separately or sequentially by contacting the cell with the first and second mutated precursor antibodies in the presence of human complement and/or an actuator cell.

[00276] The invention also provides a method of inducing a CDC or other effective response, such as ADCC, against a target cell, cell membrane, virion or the particle associated with the antigen to which an IgG1 or IgG3 antibody binds, which comprises the steps of (i) providing an antibody variant that comprises a mutation at K439 that is K439E and a mutation at S440 that is S440K or S440R in the Fc region of the antibody and (ii) contacting a preparation of the variant with the cell in the presence of a human complement and/or an actuator cell

[00277] The invention also provides a method of inducing a CDC or other effective response, such as ADCC, against a target cell, cell membrane or virion that expresses a first antigen to which a first IgG1 antibody binds and a second antigen to which a second antibody binds, which comprises the steps of (i) providing a first variant which is the first antibody comprising a mutation of a K439E and a second variant which is the second antibody comprising a S440K or S440R mutation and (ii ) in a simultaneous, separate or sequential manner by contacting the cell with preparations of the first and second variants in the presence of human complement or an actuator cell.

[00278] In separate and specific embodiments, the first and second antibodies bind (i) different antigens; (ii) different epitopes on the same antigen, (iii) the same epitope or an antigen and (iv) the same epitope or an antigen and comprises the same VH and/or VL sequences. Other methods

[00279] In another main aspect, the invention relates to a method of identifying a mutation in an antibody that enhances the effective function of the antibody to bind C1q, which comprises the steps of (i) preparing at least one antibody comprising a mutation in one or more amino acids selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain; (ii) evaluating the C1q activity of the antibody when bound to the cell surface expressing the antigen as compared to the precursor antibody and (iii) selecting a mutation of any variant having an increased C1q avidity.

[00280] In one embodiment, the at least one antibody comprises one or more amino acid substitution(s) selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, such such as E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00281] In yet another main aspect, the invention relates to a method of identifying a mutation in a precursor antibody that increases the ability of the antibody to induce a CDC response, which comprises the steps of (i) preparing at least a precursor antibody variant comprising a mutation in one or more amino acids selected from the group corresponding to E430X, E345X, S440Y or S440W in the Fc region of a human IgG1 heavy chain; (ii) evaluate the response of CDC induced by the variant when bound to the surface of a cell expressing the antigen, in the presence of actuating cells or complement, as compared to the precursor antibody and (iii) select a mutation of any variant having an increased response from CDC.

[00282] In one embodiment, the at least one antibody comprises one or more amino acid substitution(s) selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the region Fc of a human IgG1 heavy chain, such as E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

Polypeptides of the present invention Polypeptide precursors

[00283] As described herein, the present invention inter alia relates to variants of precursor polypeptides that comprise one or more mutations in the CH3 region of an immunoglobulin, for example, in the heavy chain of the antibody. The “precursor polypeptides” can be “precursor antibodies”. "Precursor" antibodies, which may be wild-type antibodies, to be used as the starting material of the present invention prior to modification may be, for example, produced by the first hybridoma method described by Kohler et al., Nature 256, 495 (1975) or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581,597 (1991). Monoclonal antibodies can be obtained from a suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for example, in the form of cells expressing the antigen on the surface or a nucleic acid that encodes an antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals, such as rabbits, rats, dogs, primates, etc.

[00284] Precursor 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, for example, HuMAb mice, which carry parts of the human immune system rather than the mouse system. The mouse HuMAb contains a human immunoglobulin gene minisite that encodes the non-rearranged Khuman heavy (µ and γ) and light chain immunoglobulin sequences, along with targeted mutations to inactivate the endogenous µ and K chain sites (Lonberg, N. et al., Nature 368, 856 859 (1994)). Consequently, mice exhibit reduced expression of mouse IgM or k in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG, k monoclonal antibodies (Lonberg , N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536 546 (1995)). The HuMAb mouse preparation is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al ., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187. Splenocytes from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[00285] Furthermore, human antibodies of the present invention or antibodies of the present invention to other species can be identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, mammalian display, yeast and other techniques known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. A particular strategy, described in example 17, can be applied to any antibody to prepare and obtain a variant of the invention using phage display.

[00286] The precursor antibody is not limited to antibodies having, for example, a natural human Fc domain but this may also be an antibody having mutations other than those of the present invention, such as, for example, mutations affecting glycosylation or allows the antibody to be a bispecific antibody. By the term “natural antibody” is meant any antibody that does not comprise any genetically introduced mutations. An antibody comprising naturally occurring modifications, e.g., different allotypes, therefore, should be understood as a "natural antibody" within the meaning of the present invention and may therefore be understood as a precursor antibody. Such antibodies can serve as a template for the one or more mutations according to the present invention and thereby provide the variant antibodies of the invention. An example of a precursor antibody that comprises mutations other than those of the present invention is the bispecific antibody as described in WO2011/131746 (Genmab), using reducing conditions to promote the exchange of half a molecule of two antibodies that comprise regions of CH3 similar to IgG4, thus forming bispecific antibodies without the concomitant formation of aggregates. Other examples of precursor antibodies include but are not limited to bispecific antibodies, such as heterodimeric bispecifics: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); Fc?Adp (Regeneron); Knobs-into-holes (Genentech); Electrostatic guide (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azimetric structure (Zymeworks); mAb-Fv (Xencor) and LUZ-Y (Genentech). Other exemplary antibody precursor formats include, without limitation, a wild-type antibody fragment, a full-length or Fc-containing antibody, a human antibody, or any combination thereof.

[00287] The precursor antibody can bind any target, examples of such targets or antigens of the invention can be, and are not limited to, directed against are; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; carcinoma-associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Crypto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin chain II beta; FLT1; LEAF1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein homolog 90 [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 arch IIIB gp120 V3; HLA-DRB (HLA-DR beta); human respiratory syncytial virus, F glycoprotein; ICAM1; IFNA1; IFNA1; bispecific IFNB1; IgE Fc; IGF1R; IGHE connection region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin 2 receptor bta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, LPS lipopolysaccharide domain; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serum IATS O11; RSV (human respiratory syncytial virus, F glycoprotein); ROR1; RTN4; SELL; SELP; STEAP1; subunit II B of Shiga-like toxin [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipotechoic acid; alpha-beta T cell receptor; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1 and Vimentin.

[00288] The precursor antibody can be any human antibody of any isotype, for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM and IgD, optionally a human full-length antibody, such as an IgG1 antibody full human length. The precursor antibody may comprise a sequence according to any one of SEQ ID NO: 1, 2, 3, 4 and 5.

[00289] Monoclonal antibodies, such as precursors and/or variants, for use in the present invention, can be produced, for example, by the first hybridoma method described by Kohler et al., Nature 256, 495 (1975) or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for example, in the form of cells expressing the antigen on the surface or a nucleic acid. that encodes an antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of human or non-human mammals, such as mice, dogs, primates, etc.

[00290] In one embodiment, the antibody is a human antibody. Human monoclonal antibodies directed against any antigen can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. Such transgenic and transchromosomal mice may include mice referred to herein as HuMAb® mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.”

[00291] HuMAb® mice contain human immunoglobulin gene minilocuses encoding non-rearranged human heavy (µ and Y) chain and K light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous µ and K chain sites (Lonberg, N. et al., Nature 368, 856-859 (1994)). Consequently, mice exhibit reduced expression of mouse IgM or k mice and in response to immunization, the introduced human light and heavy chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG,k monoclonal antibodies. (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The HuMAb® mouse preparation is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

[00292] HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in their endogenous light chain (cap) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a disruption of CMD in its endogenous heavy chain genes endogenous heavy chain genes (as described in example 1 of WO 01/14424) and a human KCo5 kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845 -851 (1996)). Additionally, Hco7 mice have a human HCo7 heavy chain transgene (as described in US 5,770,429), HCo12 mice have a human HCo12 heavy chain transgene (as described in example 2 of WO 01/14424), HCo17 mice have a human HCo17 heavy chain transgene (as described in example 2 of WO 01/09187) and HCo20 mice have a human HCo20 heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and kappa light chain transgenes in a background homozygous for the disruption of the endogenous mouse heavy chain and kappa light chain sites.

[00293] In the KM mouse strain, the endogenous mouse light chain gene was homozygous disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene was homozygous interrupted as described in example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of hCF chromosome fragment 14 (SC20) as described in WO 02/43478. HCo12-Balb/C mice can be generated by crossing HCo12 with KCo5 [J/K](Balb) as described in WO/2009/097006.

[00294] Splenocytes from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[00295] Furthermore, any antigen binding regions can be obtained from human antibodies or antibodies from other species through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display and other techniques, using techniques are well known in the art and the resulting molecules may be subjected to further maturation, such as affinity maturation, as such 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) (ribosomal display), Parmley and Smith , Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), 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 display technologies are used to produce antibodies that are not human, such antibodies may be humanized.

[00296] A mutation according to the present invention can be, but is not limited to, the deletion, insertion or substitution of one or more amino acids. Such an amino acid substitution may occur with any naturally occurring or non-naturally occurring amino acid.

“Simple mutants”

[00297] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to other precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00298] Antibody or polypeptide variants in accordance with a “single mutant” aspect of the present invention comprise a mutation, typically an amino acid substitution, in one or more amino acid residues shown in Table 1, which lists each amino acid residue, numbered according to an EU index in a human IgG1 antibody, together with the amino acid at the corresponding position in an IgG2, IgG3 and IgG4 antigen precursor and “Exemplary” and “Preferred” amino acid substitution. The IgG2 segment corresponding to residues 126 to 326, the IgG3 segment corresponding to residues 177 to 377, and the IgG4 segment corresponding to residues 127 to 327 in IgG1 are shown in Figure 2. Table 1: Exemplary mutation sites and amino acid substitutions for the “simple mutant” appearance

[00299] As seen in Table 1, the amino acid substitution that resulted in increased cell lysis of Wien133 cells in example 19 are included as “Preferred Substitutions”.

[00300] In one aspect the present invention relates to a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises one or more mutations selected from the group corresponding to E430G, E430S , E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor. (FcRn) can be determined by the method described in example 34.

[00301] In another aspect, the present invention relates to a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises one or more mutations selected from the group corresponding to E430G , E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that increase or decrease binding of the variant to the receptor. Neonatal Fc (FcRn) by more than 30%, such as greater than 20%, 10% or 5% as measured by a change in absorbance OD405 nm as determined by the method described in Example 34.

[00302] In another aspect the present invention relates to a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that increase the precursor affinity of the precursor antibody to the Fc receptor mouse neonatal (FcRn) by more than a factor of 0.5 or does not decrease the evident affinity of the precursor polypeptide or precursor antibody to mouse FcRn by more than a factor of 2, as determined by the method described in Example 34.

[00303] In one embodiment, the one or more mutations are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00304] In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the variant.

[00305] In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the release and plasma rate of the variant as determined in the methods described in example 37.

[00306] In another embodiment, the variant does not contain any additional mutations in the Fc domain that increase or decrease the plasma release rate of the variant by more than a factor of 3.0, such as by more than a factor 2.5, factor 2.0, factor 1.5, or factor 1.2 as determined by the methods described in example 37.

[00307] In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the serum half-life of the variant.

[00308] In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter liquid phase complement activation independent of the variant's target as determined by the method described in Example 36.

[00309] In one embodiment, the variant does not contain any additional mutations in the Fc domain.

[00310] In one embodiment, the variant comprises only one mutation.

[00311] In one embodiment the variant polypeptide may be a variant antibody comprising an Fc domain of an immunoglobulin and an antigen binding region.

[00312] In a specific embodiment, the amino acid substitution is E345R.

[00313] As shown in the examples, the antibody variants CD38 antibody HuMab-005 and -003 (as described in WO WO 2006/099875) and/or CD20 antibody HuMab-7D8 and -11B8 (as described in WO 2004/035607) and rituximab and/or EGFR antibody HuMab-2F8 (as described in WO 2002/100348) comprising one of these amino acid substitutions having higher binding complement activation of C1q and/or CDC than wild-type HuMab 005 and 7D8, respectively.

[00314] It should be understood that the variant may also comprise one of the “Exemplary Substitutions” mutations listed in Table 1. The variant may also comprise more than one mutation, such as two, three, four, five or six of any of the mutations listed in Table 1.

[00315] In addition to the indicated mutations, the variant may have any of the characteristics as described for the precursor antibody. In particular, this may be a human antibody. The variant can also be mutations of any IgG subtype.

[00316] When bound to its antigen on the surface of a cell that expresses the antigen, on a cell membrane, on a virion or on another particle or the antigen is associated with a virion, optionally wherein the antigen is comprised in the coat of protein or a lipid envelope of the virion, such an antibody variant may have compared to the precursor antibody at least one of (i) antibody-mediated CDC, (ii) antibody-mediated complement activation, (iii) C1q binding, (iv) oligomer formation, (v) oligomer stability or a combination of any of (i) to (v) increased. In an embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment the variant also increased ADCC compared to the precursor polypeptide or precursor antibody. In a further embodiment the variant retains the same or similar release rate compared to the precursor polypeptide or precursor antibody. In a further embodiment the variant does not have a plasma release rate that is increased or decreased by more than a factor of 3.0, such as by more than a factor 2.5, factor 2.0, factor 1 .5, or factor 1.2 as determined in the method as described in example 37 when compared to the precursor polypeptide or precursor antibody.

[00317] Without being linked by any specific theory, the effect caused by substitution amino acids in the indicated positions, with the amino acid residues of the present invention may, for example, cause the effect by itself, be involved in contact with the Fc domain of another molecule directly or can be mutated to interact with another Fc domain directly or indirectly affects the intermolecular Fc:Fc interaction. Thus, substitutions are believed, without being bound by theory, to directly or indirectly enhance the binding between antibody molecules in oligomeric form, to enhance the stability of the oligomeric structure, such as a hexameric, pentameric, tetrameric, trimeric or dimeric structure. For example, the amino acid substitution may be one that promotes or strengthens the formation of new intermolecular Fc:Fc bonds, such as, but not limited to, Van der Waals interactions, hydrogen bonds, charge-charge interactions, or stacking interactions. aromatic or one that promotes increased entropy in the Fc:Fc interaction by the release of water molecules. Furthermore, with reference to Table 1, “Exemplary substitutions” can be selected based on size and physicochemical properties that engage in or promote intermolecular Fc:Fc interactions or intramolecular interactions. “Preferred Substitutions” can be selected based on size and optimal physicochemical properties to engage in or stimulate intermolecular Fc:Fc interactions or intramolecular interactions.

[00318] In one embodiment, the variant may comprise additional mutations selected from Table 1.

[00319] 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.

[00320] In any embodiments where such a mutation in at least two amino acids is comprised in the variant, this may be present in each of the heavy chains of the variant or one of the two may be comprised in one of the heavy chains and the other may be be included in the other heavy chain, respectively, or vice versa.

[00321] In one embodiment, the mutation in two amino acid residues is a deletion, insertion or substitution. Such amino acid substitution can occur with any naturally occurring or artificial amino acids.

[00322] Mutations according to the present invention may each be, but not limited to, a deletion, insertion or substitution of one or more amino acids. Such an amino acid substitution can occur with any naturally occurring or non-naturally occurring amino acid.

[00323] Thus, in one embodiment, the mutation in at least one amino acid residue is a deletion.

[00324] In another embodiment, the mutation in at least one amino acid residue is an insertion.

[00325] In another embodiment, the mutation in at least one amino acid residue is a substitution.

[00326] Exemplary specific combinations of a mutation in two amino acid residues are E345R/E430T, E345R/S440Y, E345R/S440W, E345R/E430G, E345Q/E430T, E345Q/S440Y, E345Q/S440W, E430T/S440Y and E43 0T/ S440W.

[00327] Apart from mutations in one or more amino acids according to embodiments of the invention, the IgG heavy chain may comprise additional mutations known in the art, for example, mutations that further improve effective functions. Such additional mutations include CDC enhancing mutations, Fc-gamma receptor binding or FcRn binding, and/or enhancing effective functions mediated by the Fc-gamma receptor.

[00328] In one embodiment, a variant according to the invention further comprises a known CDC enhancer modification, for example, an exchange of segments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72); one or more amino acid substitutions in the joint region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138), and/or one or more amino acid substitutions at or near the C1q binding site in the CH2 domain , centered around residues D270, K322, P329 and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642). For example, in one embodiment, a variant according to the invention further comprises a combination of any of the amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E, providing enhanced effective function via CDC or ADCC ( Moore et al., 2010 mAbs 2(2), 181-189)). Other Fc mutations that affect binding to Fc receptors (described in WO 2006/105062, WO 00/42072, U.S. Patent 6,737,056 and U.S. Patent 7,083,784) or physical properties of antibodies (described in WO 2007/005612 A1) may also be used in variants of the invention.

[00329] In one embodiment, a variant according to the invention further comprises modifications that enhance the effective binding function of the Fc-gamma receptor and/or mediated by the Fc-gamma receptor. Such modifications include (i) reducing the amount of fucose in CH2-linked glycosylation (glyco-engineering) (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 joint or CH2 regions of antibodies (protein engineering) (Lazar GA, et al., Proc Natl Acad Sci U S A 2006; 103: 4005 -10).

[00330] In one embodiment, a variant according to the invention is further designed into the FcRn binding site, for example, to extend the half-life (t1/2) of IgG antibodies. Such modifications include (i) mutations N434A and T307A/E380A/N434A (Petcova et al. Int Immunol. 2006 Dec;18(12):1759); (ii) a substitution of one or more of Pro238, Thr256, Thr307,Gln311, Asp312, Glu380, Glu382 and Asn434 on an alanine residue that improves FcRn binding (Shields RL, et al. J. Biol. Chem. 2001 ;276:6591) and (iii) an amino acid substitution or amino acid substitution combination selected from M252Y/S254T/T256E, M252W, M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S, G385D/Q386P/N389S , G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, N434F/Y436H, H433R/N434Y/Y436H, M252Y/S254T/T256E-H433K/N434F/Y436H or M252Y/S254T/ T256E- G385R/Q386T/P387R/N389P in IgG1, which increases affinity for FcRn (Dall'Acqua et al., supra).

“Double Mutant”

[00331] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptide comprising an Fc domain of an immunoglobulin and a linker region.

[00332] As described above and further below, the present invention also relates to a “double mutant” aspect, in which two mutations, each individually, decrease an effective function but together restore the effective function at the level of precursor antibody. When used together with the variant specificity is increased. Antibody variants according to a “double mutant” appearance comprise two mutations, typically amino acid substitution, in the specific amino acid residue interaction pair K439 and S440, K447 and 448 or K447, 448 and 449.

[00333] Thus, in one aspect the present invention relates to a variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises a first mutation selected from the group corresponding to E430G , E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, such as E430G, E430S, E345K or E345Q, in the Fc region of a human IgG1 heavy chain and a second mutation selected from the group corresponding to (i ) an amino acid residue corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W and if the first mutation is S440Y or S440W the second mutation will be at the residue of amino acid corresponding to K439 in the Fc region of a human IgG1 heavy chain, (ii) an amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain, or (iii ) an amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain. Table 2A and B show “Exemplary Substitutions” and “Preferred Substitutions” for the “double mutant” (Table A) and “mixed mutant” (Table 2B) aspects. Table 2A: Exemplary mutation sites and amino acid substitution for the “double mutant” appearance Table 2B: Exemplary mutation sites and amino acid substitution appearance for “mixed mutants” (Ab1 + Ab2)

[00334] In one embodiment, the variant comprises a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y and a second mutation in an amino acid residue corresponding to K439 and S440 in the region Fc of a human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y and S440W.

[00335] It is considered by the present invention that the variant may also comprise one of the amino acid residue substitutions, such as K439E or S440K, such as the variant comprising a mutation in K439, optionally without any mutation in S440.

[00336] In one embodiment, the invention relates to the variant, wherein the mutation in K439 is an amino acid substitution in an amino acid selected from E and D, such as K439E.

[00337] In another embodiment, the variant comprises a mutation in S440, optionally without any mutation in K439.

[00338] In one embodiment, the invention relates to the variant, wherein the mutation in S440 is an amino acid substitution in an amino acid selected from K and R, such as S440K.

[00339] In one embodiment, the variant comprises mutations in both K439 and S440.

[00340] In another embodiment, the mutation in K439 is selected from K439 to D, E or R, such as K439D/E and the mutation in S440 is selected from S440 to D, E, K and R, such as S440K/R.

[00341] In another embodiment, the mutation in K439 is selected from K439D and K439E and the mutation in S440 is selected from S440K and S440R.

[00342] In another embodiment, the variant comprises K439E and S440K mutations.

[00343] In one embodiment, the precursor polypeptide is a precursor antibody comprising an Fc domain of an immunoglobulin and an antigen-binding region.

[00344] As described in examples 4 to 6, antibody variants comprising only one of the K439E and S440K mutations had a dramatically increased KD for C1q, reflecting decreased complement activation and/or CDC capacity. Surprisingly, it was observed that HuMAb 7D8 or 005 antibody variants comprising both mutations had restored or increased binding of C1q or CDC. Without being bound by any specific theory, the underlying mechanism can perhaps be explained by respective mutations that sterically compensate for each other, as illustrated in Figures 4 and 5.

[00345] In one embodiment the precursor polypeptide, and thus a variant thereof, may be an antibody comprising an Fc domain of an immunoglobulin and an antigen-binding region.

[00346] In another embodiment, the variant comprising a mutation at both positions K439 and S440 as described herein has an increase in an effective Fc-mediated function selected from complement-dependent cytotoxicity (CDC), complement activation of C1q binding, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding including Fc gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), cell-dependent cytotoxicity complement (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target submodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof, as compared to the precursor antibody or a variant antibody comprising a mutation in only one of K439 and S440.

[00347] The invention also provides the use of K439E and S440K mutations in an antibody to restore one or more of (i) antibody-mediated CDC, (ii) antibody-mediated complement activation, (iii) C1q binding avidity , (iv) oligomer formation, (v) oligomer stability, or a combination of any of (i) to (v), compared to the precursor antibody, which may, for example, be a wild-type antibody or a variant antibody comprising only one of the K439E or S440K mutations. In an embodiment of (iv) or (v), the oligomer is a hexamer.

[00348] In one embodiment, the variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.

Mixed mutants

[00349] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a link region.

[00350] As described above, the inventors of the present invention also observed that there are mutations that, by themselves, decrease an effective function but when used together, the effective function is restored, for example, mutations at positions K439 and S440 in the region Human IgG1 heavy chain Fc. This concept can also be used to ensure the formation of pairs of two different antibodies, thereby introducing K439 into one antibody and S440 into the other. Thus, antibody variants according to a “mixed mutant” aspect comprise one mutation, but one that typically leads to a reduced or greatly reduced Fc:Fc interaction between identical Fc molecules. However, when the “mixed mutant” antibody variants of the invention are capable of forming pairs with each other; provide restored or even increased CDC, complement activation of C1q binding, oligomer formation and/or oligomer stability for the specific antibody variant pair, compared to, for example, each variant alone or a mixture of the precursor antibody or precursor antibodies. In one embodiment of the invention, the oligomer is a hexamer. In one embodiment, the antibody variant pair also or alternatively has another retained or enhanced effective function, such as complement activation of C1q binding, antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn binding, of Fc receptor including Fc gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, polypeptide internalization Fc-containing, target submodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination of these. This aspect of the invention provides diverse applications not only where the strength but also the selectivity in complement activation of C1q binding, CDC or other effective function can be regulated.

[00351] Exemplary mutation sites for each antibody variant in a “mixed mutant” pair are not shown in Table 2B. Specifically, the invention provides a variant of an antibody comprising an antigen-binding region and an Fc domain of an immunoglobulin, which variant comprises a mutation in a residue in the Fc region of the human IgG1 heavy chain corresponding to one of K439 and S440. .

[00352] In one embodiment, the mutation is at K439 and is an amino acid substitution in an amino acid selected from E or D, such as K439E. In one embodiment, the mutation is at S440 and is an amino acid substitution in an amino acid selected from K or R, such as S440K.

[00353] In one embodiment, the variant comprises an amino acid mutation only in the position corresponding to K439 and not the S440 position in the fc region of an IgG1 heavy chain.

[00354] In one embodiment, the variant comprises an amino acid mutation only in the position corresponding to S440 with the proviso that the mutation in S440 is not S440Y or S440W and does not comprise an amino acid mutation in the position corresponding to K439 in the Fc region of an IgG1 heavy chain.

[00355] Thus, in one embodiment the present invention also refers to a variant comprising a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain and a second mutation in an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain.

[00356] In another embodiment, the present invention also refers to a variant comprising a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y in the Fc region of a chain human IgG1 heavy chain and a second mutation in an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the second mutation is not S440Y or S440W.

[00357] In one embodiment, the two embodiments described above can be combined in the aspect of the “mixed mutant” pair according to the present invention.

[00358] Each variant in a “mixed mutant” pair may still comprise a mutation in an amino acid listed in Table 1.

[00359] In one embodiment of the present invention, the “mixed mutant” pair comprises a first variant of a precursor antibody and a second second variant of a precursor antibody, wherein the first variant comprises a first Fc domain of an immunoglobulin and an antigen-binding region, wherein said first variant comprises (i) a first mutation in one or more amino acid residues other than a mutation in K439 selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G , E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, in the Fc region of a human IgG1 heavy chain and a second mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain and in which the second variant comprises a second Fc domain of an immunoglobulin and an antigen binding region, wherein said second variant comprises (i) a first mutation in one or more amino acid residues other than a mutation in S440 selected from the corresponding group to E430X and E345X, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y, in the Fc region of a human IgG1 heavy chain and (ii) a second mutation at the position corresponding to S440 in the Fc region of a IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W.

[00360] Other exemplary “mixed-mutant” pairs may further comprise, and are not limited to, any of the following pairs; a first variant comprising the K447E mutation and a second variant comprising the K447/P448 mutation; a first variant comprising the K447E mutation and a second variant comprising the K447/K448/P449 mutation.

[00361] In one embodiment, the mutation is a deletion, insertion or substitution. Such an amino acid substitution can occur with any naturally or non-naturally occurring amino acids.

[00362] In one embodiment, the mutation is a deletion.

[00363] In another embodiment, the mutation is an insertion.

[00364] In another embodiment, the mutation is a substitution of an amino acid.

[00365] In a particular embodiment, the first variant and/or the second variant comprises a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

[00366] For example, in one embodiment, one variant in a “mixed mutant” pair comprises one of E430G, E430S, E345K or E345Q along with K439E mutations, while the other variant comprises one of E430G, E430S, E345K or E345Q together with the S440K mutations, thus provides both increased and more specific avidity of C1q binding, complement activation, CDC, oligomer formation, oligomer stability and/or other actuator-related function such as ADCC, receptor binding gamma Fc, protein A binding, protein G binding, ADCP, CDCC, complement-enhanced cytotoxicity, antibody-mediated phagocytosis, internalization, apoptosis, binding to the complement receptor of an opsonized antibody and/or combinations thereof.

[00367] The “mixed mutant” aspect may also comprise two variants that each comprise more than one mutation listed in Table 2A, in the Fc region of the human IgG1 heavy chain, such as a first variant comprising the S440K/mutations. K447E and a second variant comprising the K439E/K447/P448 mutation; such as a first variant comprising the K439E/K447E mutation and a second variant comprising the S440K/K447/P448 mutation.

[00368] The variants in a “mixed mutant” pair as described herein may derive from the same or different antibody precursors. Furthermore, the “mixed mutant” aspect can also be used in bispecific or asymmetric antibodies. Furthermore, the first, second and third antibodies can bind different epitopes, on the same or different targets.

[00369] Furthermore, the “mixed mutant” aspect can provide a CDC or other effective response that is more specifically targeted to tumor cells that express two specific tumor antigens, by utilizing a first antibody against the first antigen with a mutation of one K439E and a second antibody against the second antigen with an S440K or S440R mutation. By utilizing the “mixed mutant” aspect comprising three variants, optionally being bispecific antibodies, can provide a CDC or other effective response that is more specifically targeted to tumor cells that express at least two, such as, two, three, four, five or six, tumor-specific antigens.

[00370] In an embodiment of any of the “single mutant”, “double mutant” and “mixed mutant” aspects, the variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.

[00371] In any embodiment of the “mixed mutant” aspect, the first, second and/or third variant may comprise the same or different mutation as any of the amino acid substitutions listed in Table 1.

Multispecific Antibodies

[00372] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptide comprising an Fc domain of an immunoglobulin and a link region.

[00373] It should be understood that any embodiment of the “single mutant”, “double mutant” and “mixed mutant” aspects described here can be used in the multispecific antibody aspect described below.

[00374] Thus in one embodiment the variant is an antibody selected from a monospecific antibody, bispecific antibody or multispecific antibody.

[00375] In a particular embodiment, the bispecific antibody has the format described in WO 2011/131746.

[00376] In a main aspect, the invention relates to a variant of a precursor antibody which is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen binding region and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same or different antigens and wherein the first and /or the 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 a human IgG1 heavy chain and in that the first polypeptide comprises the additional mutation in an amino acid residue selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of a human IgG1 heavy chain and the second polypeptide comprises the additional mutation in a amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain and wherein the additional mutation in the first polypeptide is different from the additional mutation in the second polypeptide.

[00377] In one embodiment, the mutation is a deletion, insertion or substitution. Such amino acid substitution can occur with any naturally or non-naturally occurring acids.

[00378] The bispecific antibody of the present invention is not limited to a particular format and this can be any of those described above and here.

[00379] In a particular embodiment of the present invention, (i) the first polypeptide comprises an additional mutation in the amino acid residue corresponding to K409, such as K409R, in the Fc region of a human IgG1 heavy chain and (ii) the second polypeptide comprises the additional mutation in the amino acid residue corresponding to F405, such as F405L, in the Fc region of a human IgG1 heavy chain; or wherein alternatively (iii) the first polypeptide comprises the additional mutation at the amino acid residue corresponding to F405, such as F405L, in the Fc region of a human IgG1 heavy chain and (iv) the second polypeptide comprises the additional mutation at the residue of amino acid corresponding to K409, such as K409R, in the Fc region of a human IgG1 heavy chain.

[00380] In a particular embodiment, the mutation in one or more amino acid residues are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00381] Such bispecific antibodies according to the invention can be generated as described in example 22. Furthermore, the effect on CDC killing by the generated heterodimeric proteins can be tested by using an assay as used in example 23.

[00382] The bispecific antibody may, for example, comprise an antigen-binding region of a CD20 antibody and an antigen-binding region of a CD38 antibody and an amino acid substitution in one or more amino acids listed in Tables 1 and/or 2A/B. Exemplary CD20 binding regions include those of ofatumumab (2F2), 7D8 and 11B8, described in WO2004/035607, which is incorporated herein by reference in its entirety, and rituximab (WO 2005/103081). Exemplary CD38 binding regions include those of 003 and daratumumab (005), described in WO2006/099875, which is incorporated herein by reference in its entirety.

[00383] In one embodiment, the bispecific antibody binds to different epitopes of the same or different target.

[00384] In another embodiment, the first mutation in the first and second polypeptides may be the same or different.

[00385] In one embodiment of the “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody aspect, the variant is an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or Human IgE, optionally a human full-length antibody, such as a human full-length IgG1 antibody.

[00386] In any aspect of "single mutant", "double mutant", "mixed mutant" and the multispecific antibody aspects the C1q binding of the antibody is determined according to the assays described in example 4, the CDC is determined according with the assays described in example 5, 6 or 10, the mutation is not in an amino acid residue directly involved in C1q binding, optionally as determined by comparing C1q binding in an ELISA assay according to example 3 with the C1q binding in a cell-based assay according to example 4 and the ADCC is determined according to the assays described in example 12.

[00387] Additionally, the invention provides for a preparation of a variant of any “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody aspect or embodiment described above. The invention also provided by the composition comprises a variant of any of the “double mutant” aspect and embodiment described above, for example, pharmaceutical compositions. The invention also provides the use of any such variant, preparation or composition as a medicament.

[00388] The above “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody aspects of the invention are particularly applicable to human antibody molecules having an IgG1 heavy chain comprising the relevant segment, P247 to K447, corresponding to underlined residues 130 to 330 of the human IgG1 heavy chain constant region (UniProt Accession No. P01857; SEQ ID No.: 1): 1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv 51 htfpavlqss glyslssvvt vpssslgtqt yicnv nhkps ntkvdkkvep 101 kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs 151 hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk 201 eykckvsnka Ipapiektis kakgqprepq vytlppsrde Itknqvsltc 251 Ivkgfypsdi avewesngqp ennykttppv Idsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk

[00389] The present invention can also be applied to antibody molecules having a human IgG2 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 126 to 326 of the IgG2 heavy chain constant region (accession number P01859; SEQ ID NO: 2) vt vpssnfgtqt ytcnvdhkps ntkvdktver 101 kccvecppcp appvagpsvf Ifppkpkdtl misrtpevtc vvvdvshedp 151 evqfnwyvdg vevhnaktkp reeqfnstfr vvsvltvvhq dwlngkeykc 201 kvsnkglpap iektisktkUUUg qprepqvytl ppsreemtkn qvsltclv kg 251 UUUfypsdiavew esngqpenny kttppmldsd gsfflysklt vdksrwqqgn 301 UUUvfscsvmhea Ihnhytqksl slspgk

[00390] The present invention can also be applied to antibody molecules having a human IgG3 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to residues 177 to 377 of the IgG3 heavy chain constant region (UniProt Accession No. P01860, SEQ ID No.: 3), underlined in the following: 1 astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs wnsgaltsgv 50 vshed 201 pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh qdwlngkeyk 251 ckvsnkalpa piektisktk gqprepqvyt lppsreemtk nqvsltclvk 301 gfypsdiave wessgqpenn ynttppmlds dgsfflyskl tvdksrwqqg 351 nifscsvmhe alhnrftqks lslspgk

[00391] The present invention can also be applied to antibody molecules having a human IgG4 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to underlined residues 127 to 327 of the IgG4 heavy chain constant region (accession number P01859, SEQ ID NO: 4) 1 astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv 51 htfpavlqss glyslssvv t vpssslgtkt ytcnvdhkps ntkvdkrves 100 251 gfypsdiave wesngqpenn ykttppvlds dgsfflysrl tvdksrwqeg 301 nvfscsvmhe alhnhytqks lslslgk

[00392] The present invention can also be applied as an antibody having a human IgG1m(f) allotype heavy chain portion. The amino acid sequence of the IgG1m(f) allotype (the CH3 sequence is underlined) - SEQ ID NO: 5 1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv 51 htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep 10 1 kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs 151 hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk 201 eykckvsnka lpapiektis kakgqprepq vytlppsree mtknqvsltc 251 lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk

[00393] An alignment of the respective segments of the constant regions IgG1, IgG2, IgG3, IgG4 and IgG1m(f) is shown in Figure 2. Consequently, any mutation in an amino acid described in Table 1 or Table 2A and B can be introduced into its equivalent position in IgG2, IgG3, IgG4, and/or IgG1m(f) as defined by the alignment to obtain a variant according to the invention.

[00394] In one embodiment, the invention provides a variant of a full-length IgG1, IgG2, IgG3 or IgG4 antibody, which comprises one or more amino acid substitutions according to an aspect described above.

[00395] In any aspect of “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody, the Fc region of an IgG1 heavy chain may comprise a sequence from residues 130 to 330 of SEQ ID NO: 1 , residues 126 to 326 of SEQ ID N°: 2, residues 177 to 377 of SEQ ID N°: 3 or residues 127 to 327 of SEQ ID N°: 4.

[00396] In one embodiment, a precursor 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.

[00397] In one embodiment, the Fc region of an IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO: 1.

[00398] The precursor antibody can be any precursor antibody as described herein. The precursor antibody in this context is also intended to be the first precursor and the second precursor antibodies.

[00399] In one embodiment, the precursor antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

[00400] In one embodiment the precursor antibody is a human full-length antibody, such as a human full-length IgG1 antibody.

[00401] In one embodiment, the precursor antibody, first precursor antibody and second precursor antibody is a human IgG1 antibody, for example, the IgG1m(za) or IgG1m(f) allotype, optionally comprising an Fc region that comprises SEQ ID NO: 1 or 5.

[00402] In one embodiment, the precursor antibody is a human IgG2 antibody, optionally comprising an Fc region comprising SEQ ID NO: 2.

[00403] In one embodiment, the precursor antibody is a human IgG3 antibody, optionally comprising an Fc region comprising SEQ ID NO: 3.

[00404] In one embodiment, the precursor antibody is a human IgG4 antibody, optionally comprising an Fc region comprising SEQ ID NO: 4.

[00405] In particular embodiments of any of the “single mutant”, “double mutant”, “mixed mutant” and multispecific antibody aspects, the variant comprises an amino acid sequence in which it has a degree of identity to amino acids P247 to K447 of SEQ ID NO: 1, 2, 3, 4 and 5 of at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99%, except by the mutations introduced in accordance with the present invention.

[00406] Thus, the variant may comprise a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 except for any mutation defined herein.

[00407] In any of the above “single mutant”, “double mutant”, “mixed mutant” and multispecific aspects according to the present invention can be understood to include the following embodiments.

[00408] In one embodiment, the first and/or second precursor antibody is an antibody fragment, optionally selected from the group consisting of a monovalent antibody, a heavy chain antibody, a strand exchange engineered domain (SEED) , a triomab, a dual variable domain immunoglobulin (DVD-Ig), a knob-into-holes antibody, a mini-antibody, a dual affinity retargeting molecule (Fc-DART or Ig-DART); a LIGHT-Y antibody, a Biclonic antibody, a dual-targeting (DT)-Ig antibody, a two-in-one antibody, a cross-linked Mab, a mAb2, a CovX body, an IgG-like bispecific antibody, a Ts2Ab , a BsAb, a HERCULES antibody, a TvAb, a ScFv/Fc fusion antibody, a SCORPION, a scFv fragment fused to an Fc domain, and a dual scFv fragment fused to an Fc domain.

[00409] In yet another embodiment, both the first and second precursor antibodies bind to an antigen expressed on the surface of a human tumor cell.

[00410] In yet another embodiment, the antigens for the first and second precursor antibodies are separately selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV envelope protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.

[00411] In yet another embodiment, the first and second precursor antibodies are fully human.

[00412] In yet another embodiment, the antigens for the first and second precursor antibodies are, in any order, selected from CD20 and CD38, optionally wherein the first and second precursor antibodies are, in any order, selected from 7D8 and 005.

[00413] In yet another embodiment, both the first antibody and the second antibody bind to antigens expressed on the surface of a bacterial cell or a virion.

[00414] In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia , S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis.

[00415] In yet another embodiment, the first and second precursor antibodies bind to the same antigen.

[00416] In another embodiment, the first and second precursor antibodies are the same antibody.

[00417] In another embodiment, the precursor antibody is selected from 7D8 and 005.

Compositions

[00418] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00419] The invention also relates to compositions comprising variants and precursor antibodies which may be any variant and the precursor antibody as described herein. The specific aspects and embodiments will be described below. Furthermore, such variants can be obtained according to any method described herein.

[00420] In one aspect the present invention relates to a composition comprising a first and a second variant of a precursor polypeptide each comprising an Fc domain of an immunoglobulin and a binding region, wherein the first 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 a human IgG1 heavy chain.

[00421] In one embodiment, the first and/or second variant comprises 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 a human IgG1 heavy chain.

[00422] In a preferred embodiment, the first and/or second variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.

[00423] In one embodiment, both the first and second variants comprise one or more mutations that may be the same or different.

[00424] In another embodiment, the first variant comprises one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain and wherein the second variant does not comprise one or more mutations in an amino acid residue selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain.

[00425] In one embodiment, the composition comprises at least one molecule comprising at least one CH2-CH3 domain of an immunoglobulin and a variant according to the invention, wherein the molecule comprises a mutation in one or more residues of amino acids selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S, E345K and E345Q, in the Fc region of a human IgG1 heavy chain.

[00426] The molecule described in the embodiment can be referred to as an “Fc-only molecule” and can further comprise, for example, a joint region. However, such a joint region cannot be included.

[00427] A composition comprising the Fc-only molecule and any variant according to the invention can be applied for use in image-forming diagnostic methods or to modulate the avidity of the variants once bound to the cell surface.

[00428] The Fc-only molecule may further comprise the additional mutation at an amino acid residue corresponding to K439 and/or S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is at S440 and not S440Y or S440W and if the first mutation is S440Y or S440W the additional mutation is in the amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain.

[00429] In another embodiment, (i) the first variant further comprises a mutation in a position corresponding to K439 in the Fc region of a human IgG1 heavy chain and (ii) the second variant further comprises a mutation in a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is not S440Y or S440W; or wherein (i) and (ii) it may be, alternatively (iii) the first variant further comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is not S440Y or S440W; and (iv) the second variant further comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain.

[00430] In one embodiment, the mutation at position K439 in the Fc region of a human IgG1 heavy chain is K439D/E, and/or the mutation at position S440 in the Fc region of a human IgG1 heavy chain is S440K/ R..

[00431] In yet another embodiment, the present invention relates to a composition as defined herein, wherein (i) the first variant further comprises a pro-drug and (ii) the second variant comprises an activator for the pro-drug. - medicine in a first variant; or wherein (i) and (ii) may alternatively be (iii) the second variant comprises a prodrug and (iv) the first variant comprises an activator for the prodrug in a second variant.

[00432] The term “prodrug” should be understood according to the present invention, as a relatively non-cytotoxic drug precursor that must undergo chemical conversion, for example, by metabolic processes, before becoming a pharmacological agent active (anti-cancer). Examples of prodrugs and methods of preparation thereof are well known in the art. An example is an antibody combination comprising a prodrug-enzyme in which drug release is provided by binding an antibody conjugated to a prodrug and binding an antibody conjugated to an activator to said prodrug. to their antigen targets present on the same cell. This brings the prodrug and its activator close to each other and the drug is therefore released locally, capable of, for example, penetrating surrounding cells and killing these 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).

[00433] The term “activator of a prodrug” should be understood according to the present invention, as a molecule capable of converting a prodrug into an active medicine. Examples of activators of a prodrug and methods of preparing them are well known in the art. An example of an activator may be enzymes that behave as a catalyst for the conversion of the prodrug into an active drug. (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).

[00434] In one embodiment the first and/or second precursor polypeptide is a first and a second precursor antibody each comprising an Fc domain of an immunoglobulin and an antigen binding region.

[00435] In one embodiment, the first and second antibodies are each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each human full-length antibody, such as each Human full-length IgG1.

[00436] In one embodiment, the first and second antibodies are each selected from a monospecific, bispecific or multispecific antibody.

[00437] In yet another embodiment, the first and/or second precursor antibody is each bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen binding region and a second polypeptide comprising a second CH2-CH3 region 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 said first region of CH2-CH3 comprises an additional amino acid mutation at a position selected from that corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of a human IgG1 heavy chain and in which the second region of CH2-CH3 comprises an additional amino acid mutation at a position selected from that corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain and wherein the additional amino acid mutation in the first region of CH2- CH3 is different from the additional amino acid mutation in the second region of CH2-CH3.

[00438] In a preferred embodiment, the additional amino acid mutation of the first CH2-CH3 region is in the position corresponding to K409, such as K409R, in the Fc region of a human IgG1 heavy chain and in which the amino acid mutation The additional second CH2-CH3 region is at the position corresponding to F405, such as F405L, in the Fc region of a human IgG1 heavy chain.

[00439] In one embodiment, the first and second variant of the composition bind to different epitopes on the same or different antigens.

[00440] In one embodiment, one or both of the first and second variants are conjugated to a drug, toxin or radiolabel, such as one or both of the first and second variants are conjugated to a toxin via a connector.

[00441] In one embodiment, one or both of the first and second variants are parts of a fusion protein.

[00442] In a particular embodiment, the first and/or second variant of the composition comprises only one mutation.

[00443] In embodiments, wherein the second variant does not comprise any of the mutations listed herein and described, such second variant may include any of the examples of suitable second antibodies listed above in relation to CDC augmentation methods.

[00444] In one embodiment, the at least one first mutation in the first and second variants are different.

[00445] In one embodiment, the first variant and second variant is each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each human full-length antibody, such as each Human full-length IgG1.

[00446] In one embodiment, the first variant and second variant are each selected from a monospecific antibody, bispecific antibody or multispecific antibody.

[00447] In yet another embodiment, the first and second variants bind to different epitopes on the same antigen or different antigens. Thus, in the embodiment where the first and second antibodies are bispecific antibodies can bind to each of the different epitopes. The at least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the composition thus comprises targeting four different epitopes on the same or different targets.

[00448] In another aspect, the invention relates to a composition comprising any variant, any bispecific antibody or any composition described herein and a pharmaceutically acceptable carrier.

[00449] It is considered that any of the embodiments according to a “mixed mutant” aspect can be comprised in any of the embodiments of the composition.

[00450] In one embodiment, variants of the first and second precursor antibodies bind to antigens expressed on the same cell.

[00451] In another embodiment, the variant of the first precursor antibody comprises an amino acid substitution of K439 in an amino acid selected from E and D.

[00452] In another embodiment, the amino acid substitution in a variant of the first precursor antibody is K439E.

[00453] In another embodiment, the variant of the second precursor antibody comprises an amino acid substitution of S440 in an amino acid selected from K and R.

[00454] In another embodiment, the amino acid substitution in a variant of the second variant of the precursor antibody is S440K.

[00455] In another aspect, the invention relates to a pharmaceutical composition comprising a variant of the first precursor polypeptide or precursor antibody and a variant of the second precursor polypeptide or precursor antibody of any of the embodiments listed above.

[00456] Pharmaceutical compositions can be formulated according to conventional techniques such as those described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutical composition of the present invention may, for example, include diluents, fillers, salts, buffers, detergents (e.g., a non-ionic detergent such as Tween-20 or Tween-80), stabilizers (e.g., sugars or free amino acids). protein), preservatives, isotonicity 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, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol).

[00457] The pharmaceutical composition can be administered by any suitable route and method. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. The term "parenterally administered" as used herein means modes of administration other than topical and enteral administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous injection. , transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachinoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal and infusion.

Parts kit

[00458] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00459] The invention also relates to kit of parts for simultaneous, separate or sequential use in therapy comprising variants of the precursor polypeptides and precursor antibodies, wherein any variant of the precursor polypeptide and the precursor antibody may be as described herein. The specific aspects and embodiments will be described below. Furthermore, such variants can be obtained according to a method described herein.

[00460] In one aspect the present invention relates to a kit of parts for simultaneous, separate or sequential use in therapy comprising a first variant of a precursor polypeptide and a second variant of a precursor polypeptide, wherein the first variant comprises one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor (FcRn) and wherein (i) said first variant comprises a mutation at a position corresponding to K439 in the Fc region of the chain of human IgG1 heavy chain and said second variant comprises a mutation at a position corresponding to S440 in the Fc region of the human IgG1 heavy chain, with the proviso that the mutation at S440 is not S440Y or S440W, (ii) said first variant comprises a mutation in a position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain and said second variant comprises a mutation in a position corresponding to K447K/R/H and 448P in the Fc region of the human IgG1 heavy chain or (iii) said first variant comprises a mutation in a position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain and said second variant comprises a mutation in a position corresponding to K447K/R/H, 448K/R/H and 449P in Fc region of the human IgG1 heavy chain.

[00461] In one embodiment, the first or both variants of a precursor polypeptide and the second variant of a precursor polypeptide may be an antibody comprising an Fc domain of an immunoglobulin and an antigen-binding region.

[00462] In one embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D/E, and/or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K/R.

[00463] In another aspect, the present invention relates to a kit of parts for simultaneous, separate or sequential use in therapy, which comprises a first variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a region of binding and a second variant of a precursor polypeptide comprising an Fc domain of an immunoglobulin and a binding region, wherein the variant comprises one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S , E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain and provided that the variant does not contain any additional mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor (FcRn) and wherein the second variant does not comprise a mutation in an amino acid residue selected from the group corresponding to E430X, E345X, S440Y and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain.

[00464] In embodiments, wherein the second variant does not comprise any of the above-described listed mutations, such second variant may include any of the examples of the suitable second antibody listed above in relation to methods of effective functions.

[00465] In one embodiment, the at least one first mutation in the first and second variants are different.

[00466] In one embodiment, the first variant and second variant is each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each human full-length antibody, such as each Human full-length IgG1.

[00467] In one embodiment, the first variant and second variant are each selected from a monospecific antibody, bispecific antibody or multispecific antibody.

[00468] In yet another embodiment, the first and second variants bind to different epitopes on the same or different antigens. Thus, in the embodiment where the first and second antibodies are bispecific antibodies can bind to each two different epitopes. The at least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the kit of parts for simultaneous, separate or sequential use in therapy thus comprises targeting up to four different epitopes on the same or different targets.

[00469] In yet another embodiment, one or both of the first variant and second variant is conjugated to a drug, toxin or radiolabel, such as wherein one or both of the first variant and second variant is conjugated to a toxin via a connector.

[00470] In yet another embodiment, one or both of the first variant and second variant is part of a fusion protein.

[00471] It is considered that any of the embodiments according to a “mixed mutant” aspect can also be comprised in any of the kit of parts for simultaneous, separate or sequential use in therapy, embodiments.

[00472] In one embodiment, variants of the first and second precursor antibodies bind to antigens expressed on the same cell.

[00473] In another embodiment, the variant of the first precursor antibody comprises an amino acid substitution of K439 in an amino acid selected from E and D.

[00474] In another embodiment, the amino acid substitution in a variant of the first precursor antibody is K439E.

[00475] In another embodiment, the variant of the second precursor antibody comprises an amino acid substitution of S440 in an amino acid selected from K and R.

[00476] In another embodiment, the amino acid substitution in a variant of the second precursor antibody variant is S440K.

[00477] In another aspect, the invention relates to a kit of pharmaceutical parts for simultaneous, separate or sequential use in therapy, which comprises the variant of the first precursor polypeptide or precursor antibody and a variant of the second precursor polypeptide or antibody precursor to any of the embodiments listed above.

[00478] Pharmaceutical parts kits for simultaneous, separate or sequential use in therapy can be administered by any suitable route and manner. In one embodiment, a kit of pharmaceutical parts for simultaneous, separate or sequential use in therapy, of the present invention is administered parenterally. The term "parenterally administered" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous injection. , transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachinoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal and infusion.

Combinations

[00479] Additionally, the invention provides for a preparation of an aspect of the variant of any "single mutant" or embodiment described above, that is, preparations comprising multiple copies of the variant. The invention also provides for a composition comprising an aspect of the variant of any "single mutant" and embodiment described above, for example, the pharmaceutical composition. The invention also provides for the use of any such “single mutant” variant, preparation or composition as a medicament.

[00480] The invention also provides for combinations of variants, wherein a variant comprises at least one mutation according to the invention and a variant comprises at least one other mutation according to the invention, as well as preparations and pharmaceutical compositions of such variant combinations and their use as a medicine. Preferably, the two variants bind to the same antigen or to different antigens typically expressed on the surface of the same cell, cell membrane, virion and/or other particle.

Conjugates

[00481] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00482] In one aspect, the present invention relates to a variant, wherein said variant is conjugated to a drug, toxin or radiolabel, such as wherein the variant is conjugated to a toxin via a linker.

[00483] In one embodiment said variant is part of a fusion protein.

[00484] In another aspect, the variant of the invention is not conjugated at the C terminus to another molecule, such as the toxin or label. In one embodiment, the variant is conjugated to another molecule at another site, typically at a site that does not interfere with oligomer formation. For example, the antibody variant may, at another location, be linked to a compound selected from the group consisting of a toxin (including a radioisotope), a prodrug or a medicament. Such a compound can cause the death of the most effective target cells, for example, in cancer therapy. The resulting variant is therefore an immunoconjugate.

[00485] Thus, in still one aspect, the present invention provides an antibody linked or conjugated to one or more therapeutic moieties, such as a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, and/or a radioisotope. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates”. Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins”.

[00486] A cytotoxin or cytotoxic agent includes any agent that is harmful to cells (e.g., kills). 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, dihydroxy anthracindione, maytansine or a analogue or derivative thereof, antitumor enedyene antibiotics including neocarzinostatin, calicheamicins, esperamycins, dynemycins, lidamycin, kedarcidin or analogues or derivatives thereof, anthracyclines, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC1065 (also known as rachelmycin) or analogues or derivatives of CC-1065), dolastatin, pyrrole [2,1-c] [1,4] benzodiazepines (PDBs) or analogues thereof, antibiotics ( such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g. tubulin inhibitors) such as monomethyl auriestatin E, monomethyl auriestatin F or other analogues or derivatives of dolastatin 10; Histone deacetylase inhibitors such as trichostatin A hydroxamic acids, vorinostat (SAHA), belinostat, LAQ824 and panobinostat as well as benzamides, entinostat, CI994, mocetinostat and aliphatic acid compounds such as phenylbutyrate and valproic acid, proteasome inhibitors such as Danoprevir, bortezomib, amatoxins such as -amantine, diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules); ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin , pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modecin, gelanin, Abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, gerarnin, mitogelin, restrictocin, fenomycin and enomycin toxins. Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, melittin, Cecropin and P18; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin A, mousegrape antiviral protein, diphtheria toxin and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with an antibody of the present invention as described elsewhere herein, such as, for example, anti-cancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to an antibody of the present invention. present invention.

[00487] In one embodiment, the drug conjugates of the present invention comprise an antibody as described in this conjugate to auriestatins or auriestatin peptide analogs and derivatives (U.S.5635483; U.S.5780588). Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and cell and nuclear division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer activity (US5663149) and anti-fungal (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965. The drug moiety of auriestatin can be linked to the antibody via a linker, through the N (amino) terminus or the C (terminus) of the peptide drug moiety.

[00488] Exemplary auriestatin embodiments include the N-terminally linked monomethyl auriestatin drug moieties DE and DF, described in Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623, filed March 28, 2004 and described in U.S. 2005/0238649).

[00489] An exemplary auriestatin embodiment is MMAE (monomethyl auriestatin E). Another exemplary auriestatin embodiment is MMAF (monomethyl auriestatin F).

[00490] In one embodiment, an antibody of the present invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., a DNA molecule containing the immunostimulatory CpG motif). In another embodiment, an antibody of the present invention is conjugated to an aptamer or a ribozyme.

[00491] In one embodiment, antibodies comprising one or more radiolabeled amino acids are provided. A radiolabeled variant can be used for both diagnostic and therapeutic purposes (radiolabeled molecules conjugation is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. 4,681,581, U.S. 4,735,210, U.S. 5,101,827, U.S. 5,102,990 (US RE35,500), U.S. 5,648,471 and U.S. 5,697,902. conjugated by the chloramine T method.

[00492] In one embodiment, the variant of the present invention is conjugated to a radioisotope or a radioisotope-containing chelate. For example, the variant may be conjugated to a chelating linker, for example, DOTA, DTPA or thiuxetane, which allows the antibody to be complexed with a radioisotope. The variant may also or alternatively comprise or is conjugated to one or more radiolabeled amino acids or another radiolabeled molecule. A radiolabeled variant can be used for both diagnostic and therapeutic purposes. In one embodiment the variant of the present invention is coupled to an alpha-emitter. Examples 3 14 15 35 90 99 125 11 -m 1 TQDT3C pr\c i-r»/¦» 111 ra-tvi ^1—I 1~' x -'-'x. y/ >> ¦ >-» non-limiting radioisotopes include H, C, N, S, Y, Tc, I, 111Ina, 131I, 186Re, 213Bs, 225Ac, and 227Th.

[00493] In one embodiment the variant of the present invention can be conjugated to a cytokine 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, IFNß, IFNy, GM-CSF, CD40L, ligand Flt3, stem cell factor, ancestry and TNFa.

[00494] The variants of the present invention can also be chemically modified by covalent conjugation to a polymer to increase, for example, the circulation of their half-life. Exemplary polymers and methods for linking them to peptides are illustrated in, for example, U.S. 4,766,106, U.S. 4,179,337, U.S. 4,495,285 and U.S. 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG having a molecular weight between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).

[00495] Any method known in the art for conjugating the variant of the present invention to conjugated molecules, such as that described above, can be used, including the methods described by Hunter et al., Nature 144, 945 (1962), David et al. al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Met. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such variants can be produced chemically by conjugating another moiety to the N-terminal site or C-terminal site of the variant or fragment thereof (e.g., an H- or L-chain antibody) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated variant derivatives can also be generated by conjugation to internal residues or sugars, where appropriate.

[00496] Agents can be linked directly or indirectly to a variant of the present invention. An example of indirect binding of a second agent is binding through a spacer or linker moiety to cysteine or lysine residues in the bispecific antibody. In one embodiment, a variant is conjugated to a prodrug molecule that can be activated in vivo to a therapeutic drug via a spacer or linker. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug moiety of the antibody into the intracellular environment. In some embodiments, the linker is cleaved by a cleavable agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveola). For example, the spacers or linkers may be cleaved by enzymes associated with the tumor cell or other tumor-specific conditions, whereby the active drug is formed. Examples of such prodrug and linker technologies are described in WO20083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 by Syntarga BV, et al. Suitable antibody prodrug technology and duocarmycin analogs can also be seen in U.S. Patent No. 6,989,452 (Medarex), incorporated herein by reference. The linker may also or alternatively be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, lysosomal or endosomal protease. And in some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug within target cells (see e.g., DubowchiK and Walker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see for example, US6214345, which describes the synthesis of doxorubicin with the Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of a Val-Cit and Phe-Lys linker include but are not limited to MC-vc-PAB described below, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA, where MC is an abbreviation for caproyl maleimido, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid. An advantage of using proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[00497] In yet another embodiment, the linker moiety is not cleavable and the drug is released by antibody degradation (see US 2005/0238649). Typically, such a linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment" in the context of the linker means no more than 20%, typically no more than about 15%, more typically no more than about 10%, and most typically no More than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of the variable antibody drug conjugate compound are cleaved when a variable antibody drug conjugate compound antibody variable presents in an extracellular environment (e.g., plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating an antibody variable drug conjugate compound with plasma for a predetermined period of time (e.g., 2, 4, 8, 16 or 24 hours) and then quantification of the amount of free medicine present in plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures (where Ab stands for the antibody and p, representing drug loading (or average number of cytostatic or cytotoxic drugs per antibody molecule), is 1 to about 8, for example, p can be from 4-6, such as from 3-5 or p can be 1, 2, 3, 4, 5, 6, 7 or 8).

[00498] Examples where a cleavable linker is combined with an auriestatin include MC-vc-PAB-MMAF (also denoted as vcMMAF) and MC-vc-PAB-MMAF (also denoted as vcMMAE), where MC is an abbreviation for caproyl maleimido, vc is an abbreviation for Val-Cit (valine-citrulline) based linker and PAB is an abbreviation for p-aminobenzylcarbamate.

[00499] Other examples include auristatins combined with a non-cleavable linker, such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation for caproyl maleimide).

[00500] In one embodiment, the drug linker portion is vcMMAE. The vcMMAE drug linker portion and conjugation methods are described in WO2004010957, US7659241, US7829531, US7851437 and US 11/833,028 (Seattle Genetics, Inc.), (which are incorporated herein by reference) and the vcMMAE drug linker portion is linked to antibodies on cysteines using a method similar to that described herein.

[00501] In one embodiment, the drug linker portion is mcMMAF. The mcMMAF drug linker portion and methods of conjugation are described in US7498298, US 11/833,954 and WO2005081711 (Seattle Genetics, Inc.), (which are incorporated herein by reference) and the mcMMAF drug linker portion is linked to variants on cysteines using a method similar to that described herein.

[00502] In one embodiment, the variant of the present invention is linked to a chelating linker, for example, tiuxethane, which allows the bispecific antibody to be conjugated to a radioisotope.

[00503] In one embodiment, each member (or Fab member) of the variant is linked directly or indirectly to the same one or more therapeutic moieties.

[00504] In one embodiment, only one member of the variant is linked directly or indirectly to one or more therapeutic moieties.

[00505] In one embodiment, each member of the variant is linked directly or indirectly to different therapeutic moieties. For example, in embodiments where the variant is a bispecific antibody and is prepared by exchanging the controlled Fab member of two different monospecific antibodies, e.g., a first and a second antibody, as described herein, such bispecific antibodies can be obtained. through the use of monospecific antibodies that are conjugated or associated with different therapeutic moieties.

Additional uses

[00506] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00507] Still in one aspect, the invention relates to a variant of the invention as described above for use as a medicine, in particular for use as a medicine for the treatment of diseases or disorders, in which death mediated by CDC of a target cell (e.g., a tumor, bacterial or fungal cell) or target organism (e.g., a virus) is desired or a cell infected by the virus or bacteria. Examples of such diseases and disorders include, without limitation, cancer or bacterial, fungal or viral infections.

[00508] In another aspect, the present invention relates to the variants, bispecific antibodies, compositions and kit of parts described here, for treating a disease, such as cancer.

[00509] In another aspect, the present invention relates to a method for treating a human comprising administering a variant, a composition or a kit of parts described herein.

[00510] In another aspect, the present invention relates to a method for treating cancer in a human that comprises administering a variant, a composition or a kit of parts.

[00511] “Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of facilitating, improving, preventing or eradicating symptoms (cure) or disease status.

[00512] An "effective amount" or “therapeutically effective amount” refers to an effective amount, in the dosages and for the periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount of an antibody may vary depending on factors such as the disease status, age, sex and weight of the individual and the ability of the antibody to elicit a desired response in the individual. The therapeutically effective amount is also one in which any toxic or harmful effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[00513] In another aspect, the present invention relates to the use of a variant, a composition or kit of parts according to any of the embodiments described herein for use in a diagnostic method.

[00514] In another aspect, the present invention relates to a diagnostic method comprising administering a variant, a composition or a kit of parts according to any embodiments described herein to at least the body of a human or other mammal.

[00515] In another aspect, the present invention relates to the use of a variant, a composition or kit of parts according to any of the embodiments described herein formed an image of at least one part of the body of a human or another mammal.

[00516] In another aspect, the present invention relates to a method for imaging at least one part of the body of a human or other mammal, which comprises administering a variant, a composition or a kit of parts in accordance with any embodiments described herein.

[00517] Without being bound by theory, the effective amount of the therapeutically active compound can be decreased when any “single mutant” aspect or embodiment according to the present invention is introduced into such a therapeutically active compound.

[00518] The antigens for cancer antibodies may be the same as described herein. Examples 15 to 18 describe specific applications for providing enhanced and/or more specific complement activation or CDC of tumor cells. For example, an anti-tumor antibody according to a “single mutant” aspect, comprising, for example, an E345R mutation, can provide for an enhanced CDC or ADCC, ADCP response of tumor cells. Further, in a variant of this method, a mutation according to a “single mutant” aspect, such as, for example, E345R, E430, or S440S/Wor any other mutation listed in Table 1, can be added to each antibody, thereby providing for an enhanced CDC and/or ADCC response specifically targeting tumor cells expressing at least two antigens.

[00519] Suitable antibodies for bacterial infections include, without limitation, those that target S. aureus, such as chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting Lipoteichoic acid (LTA) that is incorporated into the cell wall of staphylococci 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 by reference in their entirety. Example 14 describes a specific embodiment using S. aureus antibody variants that comprise an 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-mediating ability of an antibody against a bacterial antigen.

[00520] Suitable antigens for viral or fungal infections may be any of those described herein.

[00521] In one embodiment, the antigen to which a variant binds is not human EphA2. In another embodiment, the variant is not derived from human EphA2 mAb 12G3H11 (described in Dall'Acqua et al., supra, which is incorporated herein by reference in its entirety). In another embodiment, the antigen to which a variant binds is not IL-9. In another embodiment, the variant is not derived from the Fa-hG1 or Fa-hG4 antibody described in WO2007005612, therefore incorporated by reference in its entirety or any variant thereof. In one embodiment, the antigen to which the variant binds is not HIV-1 gp120. In another embodiment, the variant is not derived from the human b12 IgG1K antibody directed against gp120.

[00522] In a particular embodiment, the variant is derived from a bispecific antibody precursor. The bispecific antibody may be of any isotype, such as, for example, IgG1, IgG2, IgG3 or IgG4, and may be a full-length antibody or an Fc-containing fragment thereof. An exemplary method for preparing a bispecific antibody is described in WO 2008/119353 (Genmab).

Dosages

[00523] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region. Effective dosages and dosage regimens for the antibody depend on the disease or condition being treated and can be determined by those skilled in the art. An exemplary non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg/kg, such as about 0.1 to 50 mg/kg, for example, about 0.1 to 20 mg/kg, such as about 0.1 to 10 mg/kg, for example, about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg/kg.

[00524] 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 variant and/or to compensate for complement consumption. 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. Combined administration can be simultaneous, separate or sequential. In a particular embodiment, the invention provides for a kit comprising pharmaceutical composition comprising a variant of the invention and at least one complement factor or related component in the same or different pharmaceutical composition, together with instructions for use.

[00525] The antibody variants of the present invention can also be administered in combination therapy, that is, combined with other therapeutic agents relevant to the disease or condition being treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more additional therapeutic agents, such as cytotoxic, chemotherapeutic or anti-angiogenic agents. Such combined administration may be simultaneous, separate or sequential.

[00526] In a further embodiment, the present invention provides a method for treating or preventing disease, such as cancer, which method comprises administering to a patient in need thereof the therapeutically effective amount of a pharmaceutical variant or composition of the present invention, in combination with radiotherapy and/or surgery.

Preparation method

[00527] It should be understood that all embodiments described herein with reference to a precursor antibody, first precursor antibody or second precursor antibody may also be applicable to another precursor, first precursor or second precursor polypeptides comprising an Fc domain of an immunoglobulin and a linker region.

[00528] The invention also provides isolated nucleic acids and vectors encoding a variant according to any of the aspects described above, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors and expression systems for the antibodies and variants thereof are known in the art and described in the examples. In embodiments where the variant comprises not only a heavy chain (or Fc-containing fragment thereof) but also a light chain, the nucleotide sequences encoding the light and heavy chain portions may be present in the same or different nucleic acids or vectors. .

[00529] The invention also provides a method for producing, in a host cell, an antibody variant according to any of the aspects described above, wherein said variant comprises at least the Fc region of a heavy chain, said method which comprises the following steps: a) providing a nucleotide construct encoding said Fc region of said variant, b) expressing said nucleotide construct in a host cell, and c) recovering said antibody variant from the cell culture of the said host cell.

[00530] And in some embodiments, the antibody is a heavy chain antibody. In more embodiments, however, the antibody will also contain a light chain and thus said host cell still expresses a construct encoding the light chain, in the same or a different vector.

[00531] Suitable host cells for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, PER-C6, NS/0 and Sp2/0 cells. In one embodiment, said host cell is a cell that is capable of linked glycosylation by Asda proteins, for example, a eukaryotic cell, such as a mammalian cell, for example, a human cell. In yet another embodiment, said host cell is a non-human cell that is genetically engineered to produce glycoproteins having human or human-like glycosylation. Examples of such cells are genetically modified Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325) and genetically modified Lemna minor (Cox et al ., Nature Biotechnology 12 (2006) 1591-1597).

[00532] In one embodiment, said host cell is a host cell that is not capable of efficiently removing C-terminal K447 lysine residues from antibody heavy chains. For example, table 2 in Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists a number of such antibody production systems, e.g. Sp2/0, NS/0 or transgenic mammary gland (goat), in which only partial removal of C-terminal lysines is obtained. In one embodiment, the host cell is a host cell with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells in which to express the variants of the invention and therefore to produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03/035835; and WO99/54342. Additional methods for generating engineered glycoforms are known in the art and include but are not limited to those described in 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, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[00533] The invention also relates to an antibody obtained or obtainable by the method of the invention described above.

[00534] Still in one aspect, the invention relates to a host cell capable of producing an antibody variant of the invention. In one embodiment, the host cell has been transformed and transfected with a nucleotide construct of the invention.

[00535] The present invention is further illustrated by the following examples that should not be construed as further limiting.

EXAMPLES Example 1 Design and generation of 7D8 mutants

[00536] The human monoclonal antibody HuMab-7D8 (described in WO 2004/035607) was used as a model antibody. This belongs to a group of human IgG1 anti-CD20 antibodies, including atumumab (HuMax-CD20, 2F2). These antibodies target a single membrane-proximal epitope on the CD20 molecule and showed strong CDC.

[00537] To test the functional relevance of oligomeric Fc-Fc interactions in complement and CDC activation, amino acids in the hydrophobic pathway at the Fc:Fc interface were mutated to potentially disrupt the interaction on the Fc-Fc side and the CDC efficacy of 7D8. In a first series of mutants (Table 3), mutations were introduced to change charges at positions that were chosen based on the 1HZH crystal structure and described to be exposed in hydrophobic pathways in the CH2 domain-CH3 domain (Burton Mol Immunol 1985 Mar;22(3):161-206)).

[00538] From the first series of mutations, I253D and H433A were observed to induce the strongest effect on CDC loss by 7D8 (e.g., Example 5). The crystal structure of 1HZH shows that I253 and H433 bind two different pockets in the Fc opening positions of the association antibody. Based on these data, a second series of mutations was synthesized around positions I253 and H433 in the crystal structure to further study the importance of residues at the Fc:Fc-side interface for CDC. The second series of mutations around positions I253 and H433 that potentially destabilize the Fc:Fc interface and consequently CDC are listed in Table 4.

[00539] To exclude the possibility that disruption of direct binding sites for C1q was the cause of the effects observed in CDC, a double mutant was generated based on two single mutants that showed loss of CDC, to test its ability to restore the loss of CDC by the simple mutants. This principle is chemically represented in Figure 1D. The double mutant is listed in Table 5 and a structural representation is shown in Figure 4 and Figure 5.

[00540] Mutants were prepared using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, a forward and a reverse primer encoding the desired mutation were used to replicate the full-length plasmid DNA template encoding the 7D8 heavy chain with the IgG1m(f) allotype. The resulting DNA mixture was digested by sweating DpnI to remove plasmid DNA from the source DNA and used to transform E. coli. Mutant plasmid DNA isolated from the resulting colonies was verified by DNA sequencing (Agowa, Germany). Plasmid DNA mixtures encoding both the heavy and light chains of antibodies were transiently affected into Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer. Table 3: Series 1 mutations introduced into the CH2-CH3 domain of 7D8. Table 4: Series 2 Mutations introduced into the CH2-CH3 Domain of 7D8. Table 5: Double mutations introduced into the CH2-CH3 domain of 7D8 to combine two single mutations each showing loss of CDC.

Example 2 Binding of CD20 on cells by 7D8 mutants

[00541] Binding of purified antibody samples to CD20 positive cells was analyzed by FACS analysis. The 1st set of mutations (Table 3) was tested in Daudi cells and the second set of mutations (Table 4) was tested in Raji cells. 105 cells were incubated in 50 µl polystyrene 96-well round-bottom plates (Greiner bio-one 650101) with serial dilutions of antibody preparations (range 0.04 to 10 µg/ml in triple dilutions for the 1st series in Daudi and range from 0.003 to 10 µg/ml in triple dilutions for the 2nd series in Raji) in RPMI1640/0.1% BSA at 4° C for 30 minutes. after washing twice in RPMI1640/0.1% BSA, cells were incubated in 100 µl with secondary antibody at 4°C for 30 minutes. As a secondary antibody, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG (F0056, Dako, Glostrup, Denmark; 1/100) was used for all experiments in Daudi cells and for the experiments with 7D8 antibodies in Daudi cells. Raji. For the 7D8 antibody experiments in Raji cells, goat anti-human F(ab')2 coat light chain conjugated to R-phycoerythrin (R-PE) (2062-09, SouthernBiotech; 1/500) was used as a secondary antibody. . Next, the 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 in a Cantoll FACS (BD Biosciences). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V5.01 software (GraphPad Software, San Diego, CA, USA).

[00542] Binding of the 7D8 antibody to Daudi cells was not affected by the introduction of point mutations in the CH2-CH3 domain and was identical to all tested mutants and wild-type 7D8. Furthermore, 7D8 antibody binding to Raji cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain compared to wild-type 7D8, except for E345R. Decreased binding of IgG1-7D8-E345R was detected on CD20 positive Raji cells at tested concentrations above 0.3 µg/ml. Also for H433D and H433R, decreased binding was detected at the highest antibody concentration tested (10 µg/ml). The decreased binding by IgG1-7D8-E345R, H433D, and H433R may be explained by secondary antibody protection, as direct labeling of E345R and H433R resulted in similar binding or even increased binding to Daudi cells. The increased avidity can be explained by Fc-Fc side binding by E345R and H433R compared to wild-type IgG1-7D8.

[00543] The combination of the K439E and S440K mutations did not affect the binding of the 7D8 antibody to Raji cells and was identical to that of the single mutants and wild-type 7D8.

Example 3 C1q ELISA binding by 7D8 mutants

[00544] C1q binding by 7D8 mutants was tested in an ELISA, in which purified antibodies were coated on the plastic surface, performing random antibody multimerization. Pooled human serum was used as a source of C1q.

[00545] 96-well Microlon ELISA plates (Greiner, Germany) were coated overnight at 4°C with a serial dilution of the antibodies in PBS (range 0.58 to 10.0 µg/ml in 1.5 dilutions). turn). Plates were washed and blocked with 200 µl/well of 0.5x PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. With washes between incubations, plates were incubated sequentially with 3% bound human serum (Sanquin, product# M0008) for 1 hour at 37°C, with 100 µl/well of rabbit anti-human C1q (DAKO, product # A0136, 1/4,000) for 1 hour at room temperature and with 100 µl/reservoir of swine anti-rabbit IgG-HRP (DAKO, P0399, 1:10,000) when the antibody is detected for 1 hour at room temperature. Development was carried out for approximately 30 minutes with 1 mg/ml of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped by adding 100 µl of 2% oxalic acid. Absorbance was measured at 405 nm on a microplate reader (Biotek, Winooski, VT). Log-transformed data were analyzed by fitting sigmoidal dose response curves with variable slope using GraphPad Prism software. The EC50 values of the mutants were normalized per plate against wild-type IgG1-7D8 and multiplied by the average of all wild-type IgG1-7D8 data.

[00546] As shown in Figure 6 and Table 6, the point mutations tested have minimal effect on C1q binding as measured by ELISA. For the IgG1-7D8-I253D mutant, slightly less efficient binding of C1q was measured in ELISA (higher EC50 value). Coating efficacy was tested for all antibodies and was observed to be similar for all antibodies. Table 6: EC50 for C1q binding in ELISA

Example 4 Binding of C1q in cells by 7D8 mutants

[00547] Coating antibodies on a plastic surface results in an artificial static system of antibody binding and Fc tail presentation. Therefore, complement binding was also tested in a cell-based assay, in which C1q binding to antibody-opsonized CD20-positive B cells was measured by FACS analysis. In experiments with series 1 mutants, Daudi or Raji cells were suspended on ice in 90 µl of RPMI 1640 medium with 10% FBS (2 x 106 cells/ml). 10 µl of a concentration series of C1q (Complement Technologies, Tyler, TX) was added (final concentration range varies between 0-60 µg/ml and 0-140 µg/ml depending on maximum binding). Then, 10 µl of purified antibody (final concentration of 10 µg/ml, i.e., saturation conditions) was added and the reaction mixtures were immediately transferred to a 37° C water bath and incubated for 1 hour. In experiments with series 2 mutants, test mAb was added to Daudi cells in bulk, then varying concentrations of C1q were added to aliquots and the mixtures incubated as above. Cells were washed three times with PBS/1% BSA and incubated for 30 minutes at room temperature with rabbit FITC-labeled anti-C1q antibody (DakoCytomation, 10 ug/ml). Cells were washed with PBS/1%BSA and resuspended in PBS or fixed in 2% formaldehyde in PBS. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and average fluorescence intensities were converted to molecule equivalent soluble fluorescence (MESF) using calibrated beads (Spherotech).

[00548] Dissociation constants (KD values) for binding of C1q to CD20 positive cells opsonized with the indicated 7D8 antibodies were calculated using SigmaPlot® software (Systat Software Inc., Washington). Average KD values were calculated from the repeated binding experiments (4-fold in Daudi cells, 3-fold in Raji cells) and compared with the KD value for C1q binding in cells opsonized with wild-type 7D8 (Table 7 and Table 8).

[00549] Series 1 mutants were tested in both Daudi and Raji cells and gave the same results. In contrast to the C1q ELISA results, most of the mutants tested showed decreased C1q binding avidity (increased KD) in both antibody-opsonized Daudi (Table 7A) and Raji (Table 8) cells. Compared to wild-type 7D8, IgG1-7D8-Q311A and H435A showed little to no decrease, I253A, I253Y and N434A a more pronounced decrease, and I253D and H433A a very drastic decrease in C1q binding avidity in Daudi or Raji cells. opsonized. IgG1-7D8-H435R showed a slightly higher avidity (lower KD) for C1q binding than wild-type 7D8 in both cell types, which, however, was not significant.

[00550] Series 2 mutants were tested in Daudi cells. Compared to wild-type 7D8, IgG1-7D8-E345R, E382R, and H433R showed increased binding avidity in opsonized Daudi cells, reflected by lower KD values (Table 7B). all other Series 2 mutants showed decreased binding avidity to wild-type 7D8, with G385D, Y436D, Q438D, K439E, and S440K showing drastically increased KD values (Table 7B) and H433D and Y436C showing such drastically reduced binding that neither reliable KD value can be measured.

[00551] The double mutant IgG1-7D8-K439E/S440K showed restored C1q binding in antibody-opsonized Daudi cells, while both single mutants showed decreased C1q binding compared to wild-type 7D8. The binding avidity of the K439E/S440K double mutant was even slightly increased compared to wild-type 7D8 (Table 7C). Mixtures of IgG1-7D8-K439E and IgG1-7D8-K440E single mutants were able to completely restore C1q binding that was comparable to the C1q binding of wild-type 7D8 (Table 7C).

[00552] The discrepancy between the unchanged C1q binding in the ELISA (Example 3) and the affected C1q binding in the cell-based assay by the 7D8 IgG1-mutants, shows that the tested CH3 positions are involved in the Fc interaction: Fc between antibody molecules do not influence C1q binding directly, but are important determinants that affect the positioning of antibody Fc tails when bound in cells and thereby also the strength of C1q binding. Table 7A: KD values for C1q binding to antibody-opsonized Daudi cells (series 1 mutants) Table 7B: KD values for C1q binding to antibody-opsonized Daudi cells (series 2 mutants) Table 7C: KD values for C1q binding to antibody-opsonized Daudi cells (double mutant)

Example 5 Efficacy of C1q for 7D8 mutants in a CDC assay in CD20 positive Raji cells

[00553] The efficacy of C1q using cells opsonized with the IgG1 7D8 mutants was tested in a CDC assay to investigate the impact of the observed changes in C1q binding avidity on CDC activity. Therefore, a CDC assay was performed using normal human serum devoid of C1q that was supplemented with a defined concentration series of C1q. 0.1 x 106 Raji cells were preincubated in round-bottom 96-well plates (Nunc, Rochester, NY) with purified antibody at 10 µg/ml and a concentration range of human C1q (0.005, 0.025, 0.1 , 0.3, 1.0, 5.0, 30.0 µg/ml) at room temperature for 15 minutes in a total volume of 100 µL of RPMI1640 medium, supplemented with 0.1% BSA. Next, 25 µL of C1q-depleted serum (Quidel, San Diego, CA) was added and incubated at 37°C in a water bath for 30 minutes or in an incubator for 45 minutes. After incubation, the reaction was stopped by placing the samples on ice, cell lysis was determined in FACS using the propidium iodide viable cell exclusion assay (PI, Sigma Aldrich, Zwijndrecht, the Netherlands). % lysis was determined as follows: % lysis = (number of PI pos cells / total number of cells) x 100%.

[00554] Wild-type 7D8 pory lysis in the presence of 30 µg/ml C1q minus lysis when no C1q was added was set at 100%. CH50 values (the concentration of C1q resulting in 50% lysis) were calculated from fitting sigmoidal dose response curves to log-transformed data GraphPad Prism Software. The CH50 values of the mutants were normalized to wild-type 7D8 (Table 9).

[00555] The data in Table 9 shows that, according to C1q binding avidity measurements, IgG1-7D8-Q311A, E382R and H435A showed no decrease in C1q efficacy; I253A, I253Y, G385D, N434A and Y436C a significant decrease in the efficacy of C1q and I253D, H310K, K322A, H433A, H433D, Y436D, Q438D, K439E and S440K almost completely lose the ability to induce CDC with all concentrations of C1q tested.

[00556] IgG1-7D8-H435R and H433R used more mildly efficient C1q resulting in more efficient CDC than wild-type 7D8. IgG1-7D8-E345R showed a drastic decrease in C1q efficacy, which resulted in significantly higher CDC lysis compared to wild-type 7D8 (Table 9).

[00557] Figure 7 shows that the combination of mutation of K439E and S440K, both resulting in the loss of CDC as a single mutant, restored CDC in the C1q efficacy assay when both mutations were combined into one molecule (double mutant K439E/ S440K) or when both single mutants were combined (K439E + S440K mixture). Table 9: CH50 for C1q efficacy in a CDC assay in Raji cells

Example 6 CDC for 7D8 mutants in a CDC assay in CD20 positive cells

[00558] 0.1 x 106 cells were preincubated in round-bottom 96-well plates (Nunc, Rochester, NY) with antibody concentration series (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 for 15 minutes on a shaker at room temperature. Next, 20 µL of normal human serum was added as a source of C1q (final concentration 20%) and incubated in an incubator at 37°C for 45 minutes. the reaction was stopped by adding 30 µl of ice-cold RPMI medium, supplemented with 0.1% BSA. Cell lysis was determined in FACS using propidium iodide.

[00559] For the CDC assays on Daudi cells, EC50 values (the antibody concentration resulting in 50% lysis) were calculated from fitting sigmoidal dose response curves to log-transformed data using the GraphPad Prism Software. The EC50 values of the mutants were normalized to wild-type 7D8 (Table 10 and Table 11).

[00560] 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; a significant worsening of CDC (higher EC50) than wild-type 7D8 was observed for IgG1-7D8-I253D, I253Y, H310K, G385D, H433A, H433D, N434A, Y436C, Y436D, Q438D, K439E, S440K and I253D/H433A, which only induced CDC at higher antibody concentrations; binding of C1q-deficient mutant IgG1-7D8- K322A, which was included as the control, almost completely loses the ability to induce CDC and does not reach EC50 at the concentrations tested; IgG1-7D8-H435R showed more efficient CDC than wild-type 7D8 in Daudi cells. Importantly, according to a C1q efficacy CDC assay, E345R showed dramatically better CDC than wild-type 7D8 with a 10-fold lower EC50 value of Daudi cells (Table 10). Figure 8 shows that the combination mutation of K439E and S440K, both resulting in the loss of CDC as a single mutant, restored CDC when both mutations were combined into one molecule (K439E/S440K double mutant) or when both single mutants were combined (mixture of K439E + S440K).

[00561] Table 11 shows that similar data were observed for IgG1 7D8 mutants in Raji cells. Table 10: EC50 calculated from the CDC assay on Daudi cells Table 11: EC50 calculated from the CDC assay on Raji cells

Example 7 Classification of 7D8 mutants according to their ability to induce CDC

[00562] For the 7D8 mutants tested 7D8, a correlation was observed between C1q binding in Daudi cells (described in Example 4) and C1q efficacy assays in Raji cells (described in Example 5) and between C1q binding in Daudi cells in CDC assays on Daudi and Raji cells (described in Example 6) (correlation data Table 13). Therefore, KD values from C1q binding assays in Daudi cells were used to classify all tested 7D8 mutant mutants according to their ability to induce CDC, as shown in Table 12. Table 12: Classification of all tested 7D8 mutants by according to the descending KD values for C1q binding in Daudi cells, which serves as a proxy for its ability to induce CDC. Table 13: Correlation between C1q binding in Daudi cells (Example 4) and C1q efficacy assays in Raji cells (Example 5) and between C1q binding in Daudi cells and CDC assays in Daudi and Raji cells (Example 06) . Data were log-transformed before correlation was analyzed.

Example 8 Design and generation of CD38 antibody 005 mutants

[00563] The human monoclonal antibody HuMab 005 is a fully human IgG1,K9 antibody described in WO/2006/099875. Here, it was used as a model antibody for validation of Fc mutations identified to enhance CDC activity. The mutations tested are listed in Table 14.

[00564] DNA constructs for the different mutants were prepared and transiently transfected as described in Example 1, using the heavy chain of HuMab 005 with the IgG1m(f) allotype as a standard for the mutagenesis reactions. Table 14: Series of mutations that were introduced into the CH2-CH3 Domain of 005 (HuMax-CD38).

Example 9 Binding of CD38 on cells by HuMab-005 mutants

[00565] Binding of unpurified antibody samples to CD38-positive Daudi and Raji cells was analyzed by FACS analysis. 105 cells were incubated in 100 µl polystyrene 96 well round bottom plates with serial dilutions of antibody preparations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10 ,0, 30.0 µg/ml) in RPMI1640/0.1% BSA at 4° C for 30 minutes. After washing twice in RPMI1640/0.1% BSA, cells were incubated in 50 µl with rabbit anti-human IgG F(ab')2 conjugated to FITC (cat. no. F0056; DAKO; 1:150 ) at 4° C for 30 minutes. Next, the 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 in a Cantoll FACS (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.01 software. as a negative control, supernatant from mock-transfected cells was used.

[00566] The binding of HuMab 005 to Daudi cells was not greatly affected by the introduction of point mutations in the CH2-CH3 domain. All antibodies tested bound Daudi cells in a dose-dependent manner. Binding was similar to wild-type HuMab-005 for all mutants tested, with the exception of 005-E345R, which showed slightly reduced binding. However, without being bound by any theory, the lower binding should be a result of decreased binding by the secondary antibody, analogous to IgG1-7D8-E345 in Example 2. The actual binding avidity for 005-E345R should be similar or even increased in compared to 005-WT, however we cannot confirm this because of the loss of directly labeled antibodies.

[00567] Binding of HuMab-005 to Raji cells was also not greatly affected by the introduction of point mutations in the CH2-CH3 domain. All Raji cells bound to the tested antibodies in a dose-dependent manner. Maximum binding was similar to that of wild-type 005 for the 005-I253D and H433A mutants and lower for the 005-E435R, K439E, S440K mutants and the combination of 005-K439E + 005-S440K. However, without being bound by any theory, the lower binding must be a result of decreased binding by the secondary antibody, analogous to IgG1-7D8-E345R in example 2 (epitope protection).

Example 10 CDC assay on CD38 positive cells by CD38 005 antibody mutants

[00568] 0.1 x 106 Daudi or Raji cells were pre-incubated in round bottom 96-well plates with a range of concentrations of unpurified antibodies (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 for 15 minutes on a shaker at room temperature. Next, 25 µL of normal human serum was added as a source of C1q (final concentration 20%) and incubated in an incubator at 37°C for 45 minutes. the reaction was stopped by placing the plates on ice. 10 µl of propidium iodide was added and cell lysis as determined by FACS.

[00569] The CDC enhancing capacity of the E435R mutation, which has been shown to enhance CDC activity in both 7D8 and 005 antibodies in Daudi or Raji cells, was further analyzed in Wien133 cells with different concentration of normal human serum (NHS). 0.1 x 106 of Wien133 cells were preincubated for 15 minutes on a shaker at room temperature in round-bottom 96-well plates with a concentration range of unpurified antibodies (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 total volume of 100 µL. The reaction mixture was incubated in an incubator at 37°C for 45 minutes. The reaction was stopped by placing the plates on ice. 10 µl of propidium iodide was added and cell lysis as determined by FACS.

[00570] Mutations identified in the CH2-CH3 region that resulted in loss or increased CDC activity for the CD20 antibody 7D8, were observed to have the same effect on the recognition of the 005 CD38 antibody. Figure 9 showed that 005-I253D, H443A, K439E and S440K showed complete loss of CDC activity in both Daudi (Figure 9A) and Raji (Figure 9B) cells, whereas strongly enhanced CDC activity showed in the 005- E345R in both cell lines. Comparable to the 7D8 data, a combination of 005-K439E + 005-S440K, both resulting in the loss of CDC as a single mutant, resulted in restored CDC. Surprisingly, 005-E435R still strongly induced CDC in Wien133 cells, whereas wild-type 005 is not capable of inducing CDC death (Figure 9C). Killing of CDC by 005-E345R in Wien133 cells was observed at both 20% and 50% serum concentrations (Figure 9C). Also in Raji cells, both 7D8-E345R and 005-E345R showed enhanced CDC in vitro in 50% serum, with similar efficacy as in 20% serum (Figure 9D).

[00571] As the E345R mutation in the CH2-CH3 region resulted in enhanced CDC activity in both the CD20 antibody 7D8 and CD38 antibody 005 tested, the E345R mutation is considered to be a general antibody modification that can be applied to induce or enhance CDC.

Example 11 IgG1 antibodies containing the CDC E345R enhancer mutation are less sensitive to CDC inhibition by the Fc-binding peptide DCAWHLGELVWCT than wild-type antibodies

[00572] By mutating amino acid positions in the hydrophobic pathway at the IgG Fc:Fc interface, the effectiveness of CDC was observed to be disrupted or enhanced. The involvement of interactions at the Fc-Fc interface, and thus possibly the formation of an oligomeric structure (e.g., hexameric ring) as observed in the crystal structure of b12, in CDC efficacy has yet to be explored. therefore, a 13-residue peptide (DCAWHLGELVWCT (SEQ ID NO: 7)) was used targeting a consensus binding site in the hydrophobic pathway region on the surface of wild-type IgG Fc (Delano et al., Science 2000 Feb 18 ;287(5456):1279-83). Indeed, the identification of the consensus binding site on the surface of IgG Fc as a tailoring region that is primed for interaction with a variety of distinct molecules (Delano et al., Science 2000 Feb 18;287(5456):1279 -83), is consistent with the identification of the core amino acids in the hydrophobic pathway that are involved in the Fc-Fc interaction in the crystal structure of IgG1 b12 (Saphire et al., Science 2001 Aug 10;293(5532):1155-9 ). The interactions that are present at all binding interfaces are mediated by a divided series of six amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435 and Tyr-436), as counted from split main chain (Delano et al., Science 2000 Feb 18;287(5456):1279-83). Consequently, the Fc-binding peptide is expected to affect the Fc-Fc interaction and consequently the CDC efficacy.

[00573] 0.1 x 106 Daudi cells were pre-incubated in 75 µL with 1.0 µg/ml unpurified antibody in round-bottom 96-well plates for 10 minutes at room temperature on a shaker. 25 µL of a concentration range (range 0.06 to 60 µg/ml final concentration) of the Fc-binding peptide DCAWHLGELVWCT was added to the opsonized cells and incubated for 10 minutes on a shaker at room temperature. Next, 25 µL of NHS was added as a source of complement (final concentration 20%) and incubated in a 37°C incubator for 45 minutes. The reaction was stopped by adding 25 µl of ice-cold RPMI medium, supplemented with 0.1% BSA. 15 µl of propidium iodide was added and cell lysis as determined by FACS analysis.

[00574] CDC mediated by wild-type 005 (Figure 10A) or 7D8 (Figure 10B) was observed to be inhibited by the Fc-binding peptide DCAWHLGELVWCT in a dose-dependent manner. These competition data again suggest the involvement of Fc-Fc interactions in the IgG hydrophobic pathway in CDC efficacy. The CDC-enhanced IgG1-005-E345R and IgG1-7D8-E345R mutants were both less sensitive to competition for the Fc-binding peptide compared to their corresponding wild-type antibodies, suggesting that the E345R mutation results in increased stability of the Fc-Fc interaction and CDC consequently increased.

Example 12 Antibody-dependent cell-mediated cytotoxicity (ADCC) of CD38-expressing cells by CD38 HuMAb 005 antibody variants

[00575] Daudi cells were collected (5 x 106 cells/ml), washed (twice in PBS, 1200 rpm, 5 minutes) and collected in 1 ml of RPMI 1640 medium supplemented with 10% cosmic bovine serum (CCS) (HyClone, Logan, UT, USA), to which 200 µCi 51Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. The mixture was incubated in a stirring water bath for 1 hour at 37° C. After washing the cells (twice in PBS, 1200 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10 % CCS, counted by triptan blue exclusion and diluted to a concentration of 1 x 105 cells/ml.

[00576] Meanwhile, peripheral blood molecular cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium ; Lonza, Verviers, France). After resuspension of cells in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue exclusion and concentrated to 1 x 107 cells/ml.

[00577] For the ADCC experiment, 50 µL of 51Cr-labeled Daudi cells (5,000 cells) were pre-incubated with 15 µg/ml CD38 antibody IgG1-005 or mutant IgG1-005-E345R in a total volume of 100 µL medium RPMI supplemented with 10% CCS in a 96-well microtiter plate. After 10 minutes at room temperature, 50 µL of PBMCs (500,000 cells) were added, resulting in an actuator to target ratio of 100:1. The maximum amount of cell lysis as determined by incubating 50 µL of 51Cr-labeled Daudi cells (5,000 cells) with 100 µL of 5% Triton-X100. The amount of spontaneous lysis as determined by incubating 5,000 51Cr-labeled Daudi cells in 150 µL of medium without any antibodies or actuator cells. The level of antibody-dependent cell lysis as determined by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. Subsequently, cells were incubated 4 hours at 37°C, 5% CO2. To determine the amount of cell lysis, cells were centrifuged (1200 rpm, 3 min) and 75 µL of supernatant was transferred to micron tubes, after which the released 51Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows: (cpm sample - cpm Ab-independent lysis)/(cpm lysis max. - cpm spontaneous lysis) x 100%

[00578] Table 15 shows the calculated EC50 values of IgG1-005-wt and IgG1-005-E345R in the ADCC assay performed. Four samples were tested. IgG1-005-E345R shows a significantly lower EC50 value than IgG1-005-wt of all four samples tested. Table 15 EC50 values calculated from the four experiments carried out.

[00579] Figure 11 shows that, compared to the wild-type HuMab-005 antibody, the mutant IgG1-005-E345R demonstrated enhanced efficacy of ADCC capacity, being able to induce ADCC at lower concentrations.

Example 13 FcRn binding and pharmacokinetic analysis of 7D8 mutants compared to wild-type 7D8

[00580] The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting IgG from degradation. After internalization, FcRn binds to the Fc regions of the antibody in endosomes, where the interaction is stable in the moderately acidic environment (pH 6.0). Upon recycling to the plasma membrane, where the environment is neutral (pH 7.4), the interaction is not lost and the antibody is released back into circulation. This influences the half-life of plasma IgG.

[00581] The ability of the 7D8 IgG1-7D8-E354R mutant to interact with mouse, cynomolgus monkey and human FcRn was tested in an ELISA. All incubations were carried out at room temperature. 96-well plates were coated with 5 µg/ml (100 µL/well) recombinantly produced biotinylated extacellular domain of FcRn (mouse, human, cynomolgus) (FcRnECDHis-B2M-BIO), diluted in PBST plus 0.2% from BSA; 1 hours. Plates were washed 3 times with PBST and serially diluted 3 times (in PBST/0.2% BSA, pH 6.0) wild-type IgG1-7D8 or IgG1-7D8-E354R were added and plates were incubated for 1 hour . Plates were washed with PBST/0.2% BSA, pH 6.0. Goat anti-human IgG (Fab'2)-HRP (Jackson Immuno Research, cat no.:109-035-097) diluted in PBST/0.2% BSA, pH 6.0 was added and the plates were incubated for 1 hour. After washing, ABTS was added as the substrate and the plates were incubated in the dark for 30 minutes. Absorbance was read at 405 using an EL808 ELISA reader.

[00582] The mice in this study were housed in a barrier unit at the Central Laboratory Animal Facility (Utrecht, The Netherlands) and maintained in filter-top cages with water and food provided ad libitum. All experiments were approved by the animal ethics committee of Utrecht University.

[00583] To analyze the pharmacokinetics of 7D8 mutants in vivo, SCID mice (C.B-17/IcrCrl-scid-BR, Charles-River) were injected intravenously with 100 µg (5 mg/kg) 7D8 wild-type, IgG1- 7D8- E354R, -S440K or K322A; 3 mice per group.

[00584] 50 µL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 24 hours, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in vials containing heparin and centrifuged for 5 minutes at 10,000 g. Plasma was stored at −20°C until determination of mAb concentrations.

[00585] Human IgG concentrations were determined using a sandwich ELISA. The mouse mAB anti-human IgG-kappa clone MH16 (#M1268, CLB Sanquin, The Netherlands), coated on 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 µg/ml was used as capture antibody. After blocking the plates with PBS supplemented with 2% chicken serum, samples were added, serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 2% chicken serum), and incubated on a shaker. plate for 1 h at room temperature (RT). The plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA) and developed with 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). Absorbance was measured on a microplate reader (Biotek, Winooski, VT) at 405 nm.

[00586] SCID mice were chosen because they have low plasma IgG concentrations and therefore relatively low IgG release. This provides a PK model that is very sensitive to detect changes in release due to decreased binding of the FcY-part to the neonatal Fc receptor (FcRn).

[00587] Statistical tests were performed using GraphPad PRISM version 4 (Graphpad Software).

[00588] Figure 12 shows that both HuMab-7D8 and wild-type IgG1-7D8-E345R bound well to mouse, human and cynomolgus FcRn. Binding of IgG1-7D8-E345R was slightly better than that of wild-type 7D8.

[00589] Figure 13 shows plasma concentrations over time. There was no difference in the change in plasma concentrations (release) over time of wild-type HuMab-7D8 versus one of IgG1-7D8-E345R, -S440K, or K322A.

Example 14 Use of the Fc-Fc stabilizing mutation E345R against increased bactericidal activity of IgG antibodies against bacteria expressing Fc-binding surface proteins

[00590] The complement cascade system is an important host defense mechanism against pathogens and can be divided into three different activation pathways to recognize pathogens: i) the classical antibody-mediated pathway, which is activated upon C1q binding to the pathogen-bound antibody, ii) the lectin and iii) the alternative pathway, in which the complement system that directly recognizes and is triggered by the pathogen in the absence of antibody. The three pathways converge at the stage of C3 cleavage and C3b deposition. Microorganisms have developed multiple 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). The Protein 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). Until now, the antiphagocytic effect of Protein A and its role in the pathogenesis of S. aureus has been explained by the interaction between Protein A and IgG, which results in an incorrect antibody orientation being recognized by the neutrophil Fc receptor (Foster Nat Rev Microbiol (2005) Dec;3(12):948-58).

[00591] Example 11 shows that CDC mediated by B cell-specific IgG1 antibodies was inhibited by competition from the Fc-binding peptide DCAWHLGELVWCT. The peptide targets the consensus binding site on IgG Fc that coincides with the binding site for 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 evasion mechanism may work by competition with Fc binding, resulting in destabilization of the Fc-Fc interaction of a microbe-specific antibody and consequently inhibition of activation. antibody-mediated complement. Furthermore, Example 11 also shows that B cell-specific IgG1 antibodies containing the CDC-enhancing E345 mutation were less sensitive to CDC inhibition by competition from the Fc-binding peptide DCAWHLGELVWCT than wild-type antibodies. By extrapolation of these results to Fc-binding proteins expressed in microbes, increased stabilization of IgG1 Fc-Fc interactions by the E345R mutation should make microbe-specific antibodies less prone to complement inhibition by a pathogen escape strategy through intermediary from competition for Fc binding by microbial surface proteins such as Protein A. Consequently, the introduction of the E345R mutation into IgG antibodies directed against a bacterium should result in increased C3b deposition on the bacteria and increased bacterial activity in comparison with the precursor wild-type antibodies.

[00592] As an in vitro measure for complement-mediated bacterial killing, both phagocytosis by neutrophils and the generation of C3a in plasma, which coincides with the deposition of C3b in bacteria, can be determined as described below. Indeed, it has been described that C3b deposition in S. aureus results in enhanced phagocytosis and correlates with bacterial killing (Rooijakkers et. al., Nature Immunology 2005: 6,920-927).

[00593] S. aureus will be labeled with FITC by incubating an exponentially growing bacterial culture with 100 µg/ml FITC for 1 hour at 37° C in 0.1 M carbonate buffer (pH 9.6) . Human polymorphic nuclear cells (PMN) will be isolated using a Ficoll gradient. FITC-labeled bacteria will be opsonized with a range of concentrations of specific antibodies with or without the E345R mutation. Phagocytosis will be performed in vitro by incubating 1 x 108 FITC-labeled bacteria opsonized with human PMN in the presence of 25% serum devoid of IgG as a source of complement for 25 minutes at 37° C in a total volume of 200 µL under vigorous shaking. . The cells will be fixed and the erythrocytes will be lysed by incubation with FACS lysis solution for 15 minutes at room temperature. After washing, phagocytosis will be measured by FACS. The neutrophil population will be selected from forward and side scatter gating and phagocytosis will be expressed as the average fluorescence in the neutrophil population. Alternatively, C3a generation will be measured in samples by ELISA as a measure for complement activation and C3b deposition.

[00594] It is expected that antibodies containing S. Aureus-specific antibodies containing the E345R mutation will induce more complement activation and phagocytosis by neutrophils than the precursor wild-type antibodies. An example of an antibody that can be used in such experiments is the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting lipoteichoic acid (LTA) that is included in the cell wall of staphylococci (Baker, Nat Biotechnol. 2006 Dec; 24(12):1491-3; Weisman et al., Int Immunopharmacol. 2009 May;9(5):639-44).

Example 15 Use of CDC-inhibiting mutations that restrict CDC activation to target cells in a simultaneous manner linked by a mixture of two different therapeutic monoclonal antibodies

[00595] As described in Example 6, CD20 K439E and S440K antibody 7D8 mutations decreased the effectiveness of CDC as monoclonal antibodies. The 7D8 antibody mixture containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously. As described in Example 10, the CD38005 K439E and S440K antibody mutations decreased the effectiveness of CDC as monoclonal antibodies. The 005 antibody mixture containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously.

[00596] It may be advantageous to restrict efficient CDC induction to target cells that express two specific antigens simultaneously, which exploits their combined expression to improve CDC induction selectivity. For the induction of CDC to cells bound by both CD20 and CD38 antibodies simultaneously, the pair of 7D8-K439E and 005-S440K or the pair of 7D8-S440K and 005-K439E will be added or mixed 1:1 in CDC experiments as follows. . 0.1 x 106 Daudi or Raji cells will be pre-incubated in round bottom 96-well plates with a concentration range of unpurified antibodies 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 for 15 minutes on a shaker at room temperature. Next, 25 µL normal human serum will be added as a source of complement (final concentration 20%) and incubated in an incubator at 37° C for 45 minutes. The reaction will be stopped by placing the plates on ice. 10 µl of propidium iodide will be added and cell lysis will be determined by FACS. It will be expected that 7D8-K439E, 005-S440K, 7D8-S440K and 005-K439E will exhibit improved CDC efficacy. It is expected that the simultaneous addition of 7D8-K439E and 005-S440K will restore efficient CDC specifically in cells expressing both CD20 and CD38. Likewise, it is expected that the mixture of 7D8-S440K and 005-K439E will restore efficient CDC in cells expressing both CD20 and CD38.

Example 16

[00597] CDC specificity enhanced by combining E345R complementary inhibition mutations with K439E and S440K complementary inhibition mutations in a mixture of two different monoclonal antibodies

[00598] As described in Example 6, the CD20 7D8 K439E and S440K antibody mutations decreased the efficiency of CDC as monoclonal antibodies. The 7D8 antibody mixture containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously. As described in Example 10, CD38 K439E and S440K antibody 005 mutations decreased the effectiveness of CDC as monoclonal antibodies. The 005 antibody mixture containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by simultaneously mutant antibodies.

[00599] It may be advantageous to restrict the enhancement of CDC induction to target cells that express two specific antigens simultaneously, which exploit their combined expression to improve the selectivity of enhanced CDC induction. It may also be advantageous to restrict the enhancement of CDC induction to target cells that are bound by mixtures of at least two different antibodies simultaneously, said antibodies that bind an identical cell surface antigen at two different epitopes simultaneously or in two cross-competitions. , similar or identical epitopes.

[00600] Therefore, to restrict the induction of enhanced CDC to cells bound by both CD20 and CD38 antibodies simultaneously, the CDC-enhancing mutation E345R was combined with the CDC-inhibiting mutations in antibodies 7D8-E345R/K439E, 7D8-E345R /S440K, 005-E345R/S440K and 005-E345R/K439E. These antibodies were added separately or mixed 1:1 in CDC experiments as follows. 0.1 x 106 Wien133 cells (other cell types such as Daudi or Raji cells can also be used) were pre-incubated in round-bottom 96-well plates with a concentration range of unpurified antibodies (final concentration from 0.056 to 10,000 ng/ml in triple dilutions for 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K or 005-E345R/K439E) or antibody mixtures (0.01 µg/ml final concentrations and CD20 antibody mixed with 0 to 333 ng/ml triple dilutions of CD38 antibody or 3.3 µg/ml CD38 antibody mixed with 0.0056 to 1,000 ng/ml triple dilutions of CD20 antibodies) in a total volume of 100 µL for 15 minutes in a shaker at room temperature. Next, 25 µL normal human serum was added as a source of complement (final concentration 20%) and incubated in an incubator at 37°C for 45 minutes. The reaction was stopped by placing the plates on ice. 10 µl of propidium iodide was added and cell lysis as determined by FACS.

[00601] A concentration series of antibody 005-E345R/K439E or 005-E345R/S440K was mixed with a fixed concentration of 0.01 µg/ml 7D8 double mutant antibodies (maximum concentration with minimal CDC in Wien133 cells as an agent simple as determined from Figure 14A) to realize the complementary combinations 005-E345R/K439E + 7D8-E345R/S440K or 005-E345R/S440K + 7D8-E345R/K439E. Figure 14C shows that double mutant CD38 antibody 005 induced CDC dose dependent on the presence of fixed concentration of complementary 7D8-E345R/K439E or 7D8-E345R/S440K CD20 antibody, respectively. The efficacy of CDC by these complementary combinations (Figure 14C) was comparable to the 005-E345R single mutant antibody (enhancer) as a single agent (Figure 14B). In contrast, in the presence of irrelevant b12 antibody, both 005-E345R/K439E and 005-E345R/S440K showed hardly any CDC in the concentration series tested (comparable to 005-E345R/K439E or 005-E345R/S440K as single agents shown in Figure 14B).

[00602] A concentration range of 7D8-E345R/K439E or 7D8-E345R/S440K antibody was mixed with a fixed concentration of 3.3 µg/ml double mutant antibody 005 (which shows small but limited CDC in Wien133 cells as a simple agent as determined from Figure 14B) to realize the complementary combinations 7D8-E345R/K439E + 005-E345R/S440K or 7D8-E345R/S440K + 005-E345R/K439E. Figure 14D shows that the 7D8 double mutant CD20 antibodies induced CDC very efficiently in the presence of the complementary 005-E345R/K439E or 005-E345R/S440K CD38 antibody respectively, even at the lowest concentrations tested, similar to no more than a few double mutant 7D8 antibody molecules per cell. To eliminate the contribution of increased Fc tail density on the cell membrane to the CDC observed by mixing antibodies 7D8 and 005 with complementary K439E and S440K mutations, also, antibody combinations with noncomplementary mutations were tested. Figure 14D shows that non-complementary combinations showed much lower CDC efficacy than complementary combinations, as a result of less efficient Fc-Fc interaction than complementary combinations.

[00603] These data suggest that CDC induction (enhanced) by therapeutic antibodies may be limited to cells that bind a simultaneous mixture of two complementary antibodies, in this case with different antigen specificities, thereby increasing target cell specificity requiring the co-expression of both antigens.

[00604] As can be seen in Figure 14A and 14B, 7D8-E345R/K439E, 005-E345R/S440K, 7D8-E345R/S440K and 005-E345R/K439E showed limited CDC efficiency compared to 7D8-E345R alone. It is further seen, that the mixture of 7D8-E345R/K439E and 7D8-E345R/S440K allowed CDC with enhanced efficiency compared to wild-type 7D8 antibodies as the single agent. Likewise, it was observed that the mixture of 005-E345R/K439E and 005-E345R/S440K allowed CDC with enhanced efficiency compared to the wild-type 005 antibody as the single agent (data not shown).

Example 17 Use of CDC inhibitory mutations that restrict efficient CDC activation to antibody complexes consisting exclusively of therapeutically administered antibodies

[00605] As described in Example 6, the CD20 K439E/S440K antibody 7D8 double mutant restored CDC efficiency diminished by K439E or S440K single point mutants. As described in Example 10, the CD38 antibody K439E/S440K double mutant 005 restored CDC efficiency inhibited by K439E or S440K single point mutants. As noted, single point mutations disrupt the Fc:Fc interaction with the unmutated amino acid on the facing side of the Fc:Fc interface. Introduction of the compensatory mutation on the facing side of the Fc:Fc interface restored CDC efficiency. CDC was thus apparently restricted to antibody complexes consisting exclusively of antibodies containing both mutations.

[00606] In another example, CDC induction is restricted to antibody complexes consisting exclusively of therapeutically administered antibodies. To restrict CDC induction to cells therapeutically bound by CD20 or CD38 antibodies exclusively, CDC inhibition mutations K439E and S440K will be combined into antibodies 7D8-K439E/S440K or 005-K439E/S440K. These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG as follows. 0.1 x 106 Daudi or Raji cells will be pre-embedded in round bottom 96-well plates with a concentration range of unpurified antibodies 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 for 15 minutes on a shaker at room temperature. Next, 25 µL normal human serum will be added as a source of complement (final concentration 20%) and incubated in an incubator at 37° C for 45 minutes. The reaction will be stopped by placing the plates on ice. 10 µl of propidium iodide will be added and cell lysis will be determined by FACS.

[00607] 7D8-K439E/S440K is expected to induce CDC with similar efficiency to the wild-type 7D8 antibody. Other than non-specific IgG, 7D8-K439E/S440K is expected not to affect the efficiency of CDC induction for this antibody. Likewise, it is expected that 005-K439E/S440K will enable CDC with similar efficiency to wild-type HuMAb 005. In addition, non-specific IgG to 005-K439E/S440K is expected not to affect the efficiency of CDC induction for this antibody.

Example 18 Use of CDC inhibitory mutations that restrict enhanced CDC activation to antibody complexes consisting exclusively of therapeutically administered antibodies

[00608] As described in Example 6, the K439E/S440K double CD20 antibody mutant 7D8 restored the CDC efficiency diminished by the K439E or S440K single point mutants. As described in Example 10, the CD38 antibody HuMAb 005 double mutant K439E/S440K restored CDC efficiency inhibited by single point mutants K439E or S440K. As noted, single point mutations disrupt the interaction of Fc:Fc with the unmutated amino acid on the facing side of the Fc:Fc interface. Introduction of the compensatory mutation on the facing side of the Fc:Fc interface restored CDC efficiency. Efficient CDC was thus apparently restricted to complex antibodies consisting exclusively of antibodies containing both mutations.

[00609] In another example, the enhancement of CDC induction is restricted to antibody complexes that consist exclusively of therapeutically administered antibodies. By evaluating and selecting mutations that stimulate the Fc:Fc interaction exploited for CDC stimulation, one can identify mutations that can form CDC-inducing antibody complexes with serum antibodies nonspecific to the target antigen of interest. . To restrict enhanced CDC induction to cells bound by the CD20 complex or CD38 antibodies exclusively, the CDC E345R enhancing mutation will be combined with the CDC inhibitory mutations in antibodies 7D8-E345R/K439E/S440K or 005-E345R/K439E/ S440K. These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG. 0.1 x 106 Daudi or Raji cells will be pre-incubated in round bottom 96-well plates with a concentration range of unpurified antibodies 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 for 15 minutes on a shaker at room temperature. Next, 25 µL human serum will typically be added as a source of complement (20% final concentration) and incubated in an incubator at 37°C for 45 minutes. The reaction will be stopped by placing the plates on ice. 10 µl of propidium iodide will be added and cell lysis will be determined by FACS.

[00610] It is expected that 7D8-E345R/K439E/S440K will induce CDC with enhanced efficiency compared to wild-type HuMAb 7D8. The addition of non-specific IgG to 7D8-E345R/K439E/S440K is expected not to affect the efficiency of CDC induction compared to the wild-type 7D8 antibody. Likewise, it is expected that 005-E345R/K439E/S440K will enable CDC with enhanced efficiency compared to the wild-type 005 antibody. The addition of non-specific IgG to 005-E345R/K439E/S440K is expected not to affect the efficiency of CDC induction relative to the wild-type 005 antibody.

Example 19 Use of a Mutant Assessment Method to Identify Mutations that Stimulate Antibody Oligomerization Mediated by Fc:Fc Interaction Detected by the CDC Assay

[00611] As described in Examples 6 and 10, amino acid mutations were identified by stimulating CDC for antibodies that recognize two different target antigens, CD20 and CD38, in multiple cell lines that express varying levels of said antigens. Surprisingly, the E345R single point mutation was found to be sufficient to impart CDC-dependent cell lysis of Wien133 cells to the anti-CD38 antibody 005, which failed to lyse these cells by CDC in the wild-type IgG1 format.

[00612] Other mutations at or on the periphery of the Fc:Fc interface should stimulate oligomerization and CDC in an analogous manner. Alternatively, mutations must indirectly stimulate oligomerization, for example, through the Fc:Fc interactions it induces allosterically.

[00613] To determine whether other amino acid mutations should stimulate Fc-measured antibody oligomerization, a library of anti-CD38 IgG1-005 mutants was evaluated using the CDC assays, both individually and mixed in a pairwise manner to select for e.g. , pairs of amino acids that interact across the Fc:Fc interface. However, the same strategy can be applied to other antibodies, such as another IgG1 or IgG3 antibody.

[00614] A library focused on mutations in the positions indicated in table 16 was generated. Mutations were introduced into the IgG1-005 Fc region using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, for each desired mutation position, a reverse and forward primer encoding a degenerate codon at the desired location were used to replicate the 005 heavy chain full-length plasmid DNA template with IgG1m(f) allotype. The resulting DNA mixtures were digested using DpnI to remove the source plasmid DNA and used to transform E. coli. The resulting colonies were pooled and cultured and plasmid DNA was isolated from the pools and transformed back into E. coli to obtain the clonal colonies. Mutant plasmid DNA isolated from the resulting colonies was checked by DNA sequencing (LGC genomics, Berlin, Germany). Expression cassettes were amplified from plasmid DNA by PCR and DNA mixtures containing both the wild-type light and heavy chains of IgG1-005 were randomly transfected into Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US). essentially as described by the manufacturer. Supernatants from transfected cells containing the antibody mutants were collected. Mutant antibody supernatants were evaluated in CDC assays in both individual and paired mixtures as follows.

[00615] 0.1 x 106 Daudi or Wien-133 cells (other cell types such as Raji cells can be used) were pre-incubated in the round bottom 96-well plates with 1.0 ug/ml of the antibodies not purified in a total volume of 100 µL for 15 min on a shaker at room temperature. Then, 30 µL of normal human serum was added as a source of complement (30% of the final concentration) and incubated in a 37°C incubator for 45 minutes. The reaction was stopped by placing the plates on ice. 10 µl of propidium iodide was added and cell lysis was determined by FACS.

[00616] The mutations described in table 16, table 17 and table 18 were selected for their ability to enhance oligomerization as detected by CDC efficiency, as a single mutant or when mixed with other mutants, for example, in the face of a mutation through Fc:Fc interface. Mutations may optionally be further assessed for their ability to not comprise FcRn, protein-A or protein-G binding, ADCC, ADCP or other effective functions mediated by the Fc domain. The combination of such stimulation of point mutations in an Fc domain can stimulate oligomerization and CDC efficiency further.

[00617] Mutations in the CH2-CH3 region incorporated into the CD38 005 antibody were tested for their ability to inhibit oligomerization as determined by the CDC in Daudi cells. Lysis of the mutant antibody was compared to wild-type 005, whereby lysis was shown at 100%. The cutoff for inhibition was <66% lysis. Measured in this way, most of the tested mutations inhibit CDC (see Table 16).

[00618] Mutations in the CH2-CH3 region incorporated into the CD38 005 antibody were tested for their ability to enhance oligomerization as determined by CDC in Wien133 cells (Table 17). The wild-type CD38 005 antibody is not able to induce CDC in Wien133 cells. Mutants showing >39% cell lysis were counted as enhancement. Completely unexpected, virtually all substitutions obtained for amino acids E345 and E430 stimulate cell lysis by CDC. To verify this result, amino acids E345, E430 and S440 were replaced with each possible mutation by site-directed mutagenesis and tested for their ability to enhance oligomerization as determined by CDC of Wien133 cells using a batch of human serum, producing efficient lysis. slower (Table 18). Again, all E345 and E430 substitutions induce efficient CDC of Wien133 cells.

[00619] The following preferred mutations caused >39% cell lysis of Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E3 45N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430 H, E430I, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W. Table 16 Percentage of Daudi cell lysis in the presence of 1.0 µg/ml IgGl-005 antibody point mutations. Wild-type IgGl-005 lyses 66% of cells under these conditions. For each of the positions in which they were replaced by another amino acid are given in the outer left column. The substituted amino acid for each particular position is given followed by the percentage of measured lysis indicated in the parentheses () in the horizontal lines of the individual positions. Table 17 Percentage of lysis of Wien-133 cells in the presence of 1.0 µg/ml IgGl-005 antibody point mutants. Wild-type IgGl-005 lyses 3% of cells under these conditions. For each of the individual positions that have been replaced by another amino acid are given in the left outer column. The substituted amino acid for each particular position is given followed by the percentage of measured lysis indicated in the parentheses () on the horizontal line of the individual positions. Table 18 Percentage of lysis of Wien-133 cells in the presence of 1.0 µg/ml IgG1-005 antibody point mutants. Wild-type IgGl-005 lyses 12% of cells under these conditions. Each of the individual positions that have been replaced by another amino acid are given in the outer left column. The substituted amino acid for each particular position is given followed by the percentage of measured lysis indicated in the parentheses () on the horizontal line of the individual positions.

Example 20 In vivo efficacy of IgG1-7D8-E345R in a subcutaneous B-cell lymphoma xenograft model

[00620] The in vivo anti-tumor efficacy of the IgG1-7D8-E345R antibody was evaluated in a subcutaneous model with Raji-luc #2D1 cells. These cells show ~300,000 of the CD20 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. Cells were cultured in RPMI with 10% cosmic calf serum (HyClone, Logan, UT), penicillin and streptomycin, 1% sodium pyruvate (v/v), and 1 µg/mL puromycin (P-8833, Sigma , Zwijndrecht). Cells were harvested in log phase (approximately 70% confluency). Six- to eleven-week-old female SCID mice (CB-17/IcrPrkdc-scid/CRL) were used (Charles-River). On day 0.5 x 106 Raji-luc #2D1 cells in 200 µL of PBS were subcutaneously injected into the right flank of each mouse. Tumor development was monitored by measuring with a caliper. When the average tumor volume was 100 mm3 (around day 7), mice were classified into groups (n=9) and treated by intraperitoneal (ip) injection of a single dosage of 50 µg of antibody per mouse (2 .5 mg/kg). All antibody samples were supplemented with irrelevant antibody b12 to obtain a total antibody concentration of 0.5 mg/mL. Treatment groups are shown in Table 18. Seven days after treatment, blood samples were obtained to determine human IgG serum levels to verify administration of the correct antibody. Tumors were measured at least twice a week using the caliper (PLEXX) until an endpoint tumor volume of 1500 mm3, tumors showed ulcerations or until serious clinical signs were observed. Table 18: Treatment and dosage groups.

[00621] Figure 15A shows average tumor development on day 22, when all groups were still complete. The wild-type IgG1-7D8 antibody slowly inhibited tumor development compared to the negative control IgG1-b12 antibody, although this was not statistically significant. Only IgG1-7D8-E345R inhibited tumor development significantly compared to the negative control IgG1-b12 antibody (one-way ANOVA analysis p< 0.01).

[00622] Figure 15B shows a Kaplan-Meier plot of the percentage of mice with tumor sizes less than 700 mm3. Compared to mice treated with negative control IgG1-b12 antibody, tumor formation was significantly delayed in mice treated with IgG1-7D8-E345R antibody (Mantel-Cox analysis p< 0.01), but not in mice treated with IgG1- 7D8 wild type.

[00623] These data show that the E345R mutation enhances the in vivo anti-tumor efficacy of the CD20 7D8 antibody.

Example 21 In vivo efficacy of IgG1-005-E345R in a subcutaneous B-cell lymphoma xenograft model

[00624] The in vivo anti-tumor efficacy of the IgG1-005-E345R antibody was evaluated in a subcutaneous model with Raji-luc #2D1 cells. These cells show ~150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The protocol for tumor inoculation and mediation is basically the same as described in Example 20. On day 0.5x106 of Raji-luc #2D1 cells in 200 µL of PBS were injected s.c. into the right flank of SCID mice. When the average tumor volume was 100 mm3 (around day 7), mice were classified into groups (n=7) and treated by i.p. of a simple dosage of 500 µg of antibody per mouse (25 mg/kg). Treatment groups are shown in Table 19. Tumors were measured up to an endpoint tumor volume of 1500 mm3 or until tumors showed ulcerations or serious clinical signs were observed to avoid further discomfort.

[00625] Figure 16A shows average tumor development on day 21, when all groups were still completed. The wild-type IgG1-005 antibody slowly inhibits tumor development, although this is not statistically significant. Only IgG1-005-E345R significantly inhibits tumor development compared to the irrelevant antibody control on day 21 (one-way ANOVA p< 0.05).

[00626] Figure 16B shows a Kaplan-Meier plot of the percentage of mice with tumor sizes less than 500 mm3. Tumor formation was significantly delayed in mice treated with IgG1-005-E345R antibody compared to mice treated with negative control antibody IgG1-b12 (Mantel-Cox analysis p<0.001) or wild-type IgG1-005 (p<0.001). 05).

[00627] These data show that the introduction of the E345R mutation into the CD38 005 antibody results in enhanced in vivo anti-tumor activity. Table 19: Treatment and dosage groups.

Example 22 Monovalent target binding further enhances CDC efficacy of E345R antibodies

[00628] A molecular surface of the IgG1 hexameric ring observed in the b12 crystal structure demonstrates that for each IgG in the hexameric ring, one of the two C1q binding sites faces up and the other site faces down the ring structure and also one Fab member of each antibody is oriented up and one is oriented down, resulting in only one Fab member per antibody to take part in antigen binding, suggesting monovalent binding per antibody molecule in the hexameric antibody ring. Monovalency can bring antibodies upon antigen binding into a hexamerization compatible orientation. To test this hypothesis, the CDC efficacy of a CD38/EGFR bispecific antibody with the E345R mutation was tested on CD38-positive, EGFR-negative Wien133 cells, in which this bispecific antibody can bind only monovalently through CD38 and compared to CDC efficacy of the bivalent binding CD38 antibody, also with the E345R mutation. The human monoclonal antibody HuMax-EGFr (2F8, described in WO 2004/056847) was used as a basis for the EGFR antibodies described in this example.

[00629] Bispecific antibodies were generated in vitro according to the DuoBody™ platform, i.e. 2-MEA-induced Fab member exchanger as described in WO 2011/147986. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by this method, IgG1 molecules carrying certain mutations in the CH3 domain were generated: in one of the parenteral IgG1 antibodies the F405L mutation, in the other parenteral IgG1 antibody the K409R mutation. To generate the bispecific antibodies, these two parenteral antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 µL of TE. at 37° C for 90 min. The reduction reaction is stopped when the 2-MEA reducing agent is removed by use of spin columns (Microcon, 30k, Millipore centrifuge filters) according to a manufacturers' protocol.

[00630] For the CDC assay, 0.1 x 106 of the Wien133 cells were pre-incubated in the round-bottom 96-well plates with a range of antibody concentrations (0.01 to 10.0 µg/mL) in a volume total of 100 µL for 15 min on a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00631] Figure 17 shows that, as expected, CD38 antibodies without the E345R mutation (wild type IgG1-005 and IgG-b12-K409R x IgG1-005-F405L) do not induce the death of Wien133 cells. Also the EGFR antibody IgG1-2F8-E345R/F405L, which does not bind to EGFR-negative Wien133 cells (data not shown), does not induce CDC, as expected. The introduction of the K409R mutation does not influence the ability of the IgG1-005-E345R antibody to induce ~60% death in Wien133 cells (described in Example 10). Interestingly, the CD38/EGFR bispecific antibody IgG1-005-E345R/K409R x IgG1-2F8-E345R/F405L, which can only bind monovalently to CD38-positive, EGFR-negative Wien133 cells, showed enhanced maximal CDC killing. (from ~60% to ~100% death).

[00632] These data show that monovalent targeting can still enhance the maximum killing capacity of antibodies containing the E345R mutation that enhances CDC. Furthermore, these data show that the mutation that enhances E345R oligomerization, as measured by enhancement of CDC activity, can be applied to other antibody formats, such as DuoBody.

Example 23 Oligomerization that enhances the E345R mutation can be applied to other antibody formats such as DuoBody™

[00633] The effect of the E345R mutation was tested on a bispecific antibody in the DuoBody format. CDC assays were performed with CD20/CD38 bispecific antibodies on CD20-positive, CD38-positive Wien133 and Raji cells.

[00634] Bispecific antibodies were generated as described in Example 22. For the CDC assay, 0.1 x 106 of Wien133 or Raji cells were pre-incubated in round-bottom 96-well plates with a range of antibody concentrations (0 .01 to 30.0µg/mL) in a total volume of 100 µL for 15 min on a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00635] Figure 18 shows that the introduction of the CDC-enhancing E345R mutation of the IgG1-005-F405L x IgG1-7D8-K409R bispecific antibody into Wien 133 (Figure 18A) and Raji (Figure 18B) cells. These data show that the E345R oligomerization-enhancing mutation can be applied to other antibody formats to enhance CDC activity.

Example 24 E345R rescues CDC by EGFR antibody 2F8, which can be further enhanced by monovalent target binding

[00636] As described in Examples 6, 10 and 26, CDC enhanced or rescued E345R for antibodies that recognize different hematological tumor targets (CD20 and CD38). To extend the analysis to a solid tumor antigen, the effect of E345R on the CDC capacity of the EGFR antibody 2F8 was tested in A431 squamous cell carcinoma cells. Furthermore, the effect of monovalent EGFR targeting on E345R-mediated CDC induction was tested using a bispecific EGFRxCD20 antibody (IgG1-2F8-E345R/F405L x IgG1-7D8-E345R/K409R) on EGFR-positive, CD20-negative A431 cells.

[00637] Bispecific antibodies were generated as described in Example 22. For the CDC assay, 5 x 106 of A431 cells/mL were labeled with 100 µCi 51Cr for 1h at 37° C. The cells were washed three times with PBS and replaced suspended in the medium at a concentration of 1 x 105 cells/mL. 25,000 of the labeled cells were incubated in the round-bottom 96-well plates with a concentration range of the unpurified antibodies (0-30 µg/mL at 3-fold dilution) in a total volume of 100 µL for 15 min at room temperature. Then, 50 µL of the normal human serum dilution was added as a source of complement (25% of the final concentration) and incubated in a 37°C incubator for 1 h. Cells were centrifuged (3 min at 300 x g) and 25 µL of supernatant was added to 100 µL of microscint in a white 96-well optiplate (PerkinElmer) for incubation on a shaker (750 rpm) for 15 min. 51Cr release was determined as counts per minute (cpm) in a scintillation counter. Maximal lysis (100%) was determined by the 51Cr level measured in the supernatant of cells treated with Triton X-100. Spontaneous lysis was determined by the 51Cr level measured in the supernatant of cells incubated without antibody. Specific cell lysis was calculated according to the formula: specific lysis = 100 x (sample cpm - cpm spont) / (cpm max - cpm spont).

[00638] Figure 19 shows that IgG1-2F8-E345R/F405L is capable of lysing A431 cells by CDC, whereas wild type 2F8 is not capable of killing A431 cells. These data show that CDC activity can be rescued in the EGFR 2F8 antibody by introducing the E345R mutation. This potentially extends the applicability of CDC enhancing E345R mutation to antibody targeting solid tumor antigens.

[00639] The bispecific EGFRxCD20 antibody IgG-2F8-E345R/F405L x IgG1-7D8-E345R/K409R further showed CDC enhancement in EGFR-positive, CD20-negative A431 cells.

[00640] These data further support the hypothesis that monovalency facilitates the formation of Fc-Fc interactions and subsequent CDC induction as postulated by a CD38-binding antibody described in Example 22.

Example 25 CDC enhances or rescues E345R by CD38 antibody 003 and CD20 antibodies 11B8 and rituximab

[00641] As described in Examples 6, 10 and 24, CDC activity enhances or induces E345R of several antibodies with different target specificities (CD20, CD38 and EGFR), as tested in multiple cell lines expressing variable levels of said antigens . Therefore, the introduction of the E345R mutation was considered to be the general mechanism to enhance or rescue CDC for existing antibodies. Further supporting this, the effect of the E345R mutation on CDC was tested by many antibodies with variable intrinsic CDC efficacy in Daudi and Wien133 cells: CD38 003 antibody, described in WO 2006/099875, and CD20 antibodies rituximab (type I) and 11B8 (type II), described in WO 2005/103081. CD20 antibodies can be divided into two subgroups (Beers et al. Seminars in Hematology 47, (2) 2010, 107-114). Type I CD20 antibodies displayed a remarkable ability to activate complement and extract CDC by redistributing CD20 molecules on the plasma membrane into lipid clusters, which cluster antibody Fc regions and enable enhanced C1q binding. Type II CD20 antibodies do not appreciably change CD20 distribution and without concomitant clustering, they are relatively inefficient in CDC.

[00642] 0.1 x 106 of the Daudi or Raji cells were pre-incubated in the round bottom 96 well plates with a range of concentrations of the unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1 , 0.3, 1.0, 3.0, 10.0 µg/mL) in a total volume of 70 µL for 15 min on a shaker at room temperature. Then, 30 µL of normal human serum was added as a source of C1q (30% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00643] Figure 20 shows that the CDC that enhances the E345R mutation for all antibodies tested in both (A) Daudi cells and (B) Wien133 cells. Interestingly, at the concentrations used all antibodies that do not induce CDC in wild-type format, CDC efficiently induces after introduction of the E345R mutation: CD38 mAb 003 and CD20 type II mAb 11B8 in Daudi cells and CD38 mAbs 005 and 003 and CD20 type II mAb 11B8 in Wien133 cells. These data suggest that enhancement of antibody oligomerization, more specifically by introduction of an E345R mutation, is a general mechanism for enhancing or rescuing CDC by existing antibodies.

Example 26 Internalization enhancing E345R donates tissue factor antibodies

[00644] To test whether enhanced oligomerization can induce increased antibody internalization, colocalization studies of wild-type mutated tissue factor (TF) antibodies and E345R on the lysomal marker LAMP1 were performed by confocal microscopy.

[00645] SK-OV-3 cells were grown on glass coverslips (thickness 1.5 microns, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue culture medium at 37° C for 1 day. Cells were pre-incubated for 1 hour with 50 µg/mL leupeptin (Sigma) to block lysosomal activity, after which 10 µg/mL tissue factor (TF) antibody (WO 2010/066803) was added. Cells were incubated for an additional 1, 3, or 16 hours at 37°C. Then, cells were washed with PBS and incubated for 30 minutes at room temperature (RT) with 4% formaldehyde (Klinipath). Slides were washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 minutes with blocking buffer containing 20 mM NH4Cl to quench formaldehyde. Slides were washed again with blocking buffer and incubated for 45 minutes at room temperature with a mouse-anti-human CD107a-APC cocktail (BD Pharmingen) to identify lysosomal LAMP1 and goat-anti-human IgG-FITC (Jackson) for identify TF antibodies. Slides were washed again with blocking buffer and mounted overnight on microscope slides using 20 µL of mounting medium (6 grams of glycerol [Sigma] and 2.4 grams of Mowiol 4-88 [Omnilabo] was dissolved in 6 mL of distilled water to which 12 mL of 0.2M Tris [Sigma] pH8.5 was added followed by incubation for 10 minutes at 50-60°C; mounting medium was aliquoted and stored at -20°C) . Slides were imaged with a Leica SPE-II confocal microscope (Leica Microsystems) equipped with 63x 1.32-0.6 oil immersion objective lenses and LAS-AF software.

[00646] The 12-bit grayscale TIFF images were analyzed by collocation using MetaMorph® software (Meta Series 6.1 version, Molecular Devices Inc, Sunnyvale California, USA). The images were imported as stacks and the background was subtracted. Identical threshold settings were used (manually presented) for all FITC images and all APC images. Co-localization was described as the pixel intensity of FITC in the region of interest (ROI), where the ROI is composed of all APC-positive regions. To compare different slides stained with different TF antibodies, images were normalized using APC pixel intensity. Mouse-anti-human CD107a-APC was used to stain the lysosomal marker LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ between various image-forming TF antibodies.

[00647] Normalized values for the colocalization of FITC and APC are expressed as arbitrary units according to the formula [(TPI FITC x percentage of colocalization)/100] x [1/TPI APC] Percentage of colocalization = TPI FITC that colocalizes with an APC pixel/TPI APC TPI, full pixel intensity

[00648] Figure 21 depicts the amount of FITC pixel intensity of the wild-type and E345R mutated TF antibodies that overlap with the APC-labeled lysosomal marker. For each antibody or condition tested, three different images were analyzed from a slide containing ~1, 3, or >5 cells. Variation was observed between different images within each slide. It was still evident that the E345R mutation for antibodies 011 and 098 resulted in increased lysosomal colocalization after 1 hour of incubation when compared to wild type 011 and 98. These results indicate that the E345R mutation induces faster internalization and lysosomal colocalization and it should therefore potentiate antibody drug conjugates.

Example 27 CDC enhanced by the E345R mutation in rituximab in different B cell lines with similar CD20 expression but different levels of membrane-bound complement regulatory proteins

[00649] Examples 25 and 28 show that the CDC efficacy of wild-type rituximab in Daudi and Wien133 cells was enhanced to introduce the E345R mutation. This enhanced CDC efficacy results from E345R-mediated stabilization of Fc-Fc interactions. The concomitantly formed hexameric antibody ring structure on the target cell membrane can then promote the efficient generation of the membrane attack complex to facilitate the capture and concentration of active complement components near the cell membrane. As a result of this efficient complement activation, the inhibition of the effects of membrane-binding complement regulatory proteins (mCRP) should be partially overcome. Overexpression of mCRPs, such as CD55, CD46 and CD59, is considered as a barrier to successful immunotherapy with monoclonal anti-tumor antibodies (Jurianz et al., Mol Immunol 1999 36:929-39; Fishelson et al. Mol Immunol 2003 40:109-23, Gorter et al., Immunol Today 1999 20:576-82, Zell et al., Clin Exp Immunol. 2007 Dec 150(3):576-84). Therefore, the efficacy of rituximab-E345R was compared to that of wild-type rituximab in a series of B cell lines with different levels of the mCRPs CD46, CD55, and CD59, but comparable levels of the target CD20 expression.

[00650] The B cell lines Daudi, WIL2-S, WSU-NHL, MEC-2 and ARH-77 express comparable amounts of CD20 molecules (~250,000 specific antibody binding capacity - sABC) as determined by QIFIKIT analysis ( data not shown). To compare the expression levels of complement regulatory proteins between these cell lines, QIFIKIT analysis was performed to determine the levels of CD46 (mouse anti-human CD46, CBL488, J4.48 Chemicon clone), CD55 (mouse anti-human mouse CD55, CBL511, Clone BRIC216, Chemicon) and CD59 (mouse anti-human CD59, MCA1054x, clone MEM-43, Serotec).

[00651] For the CDC assay, 0.1 x 106 of the cells were pre-incubated in the round bottom 96-well plates with a saturation antibody concentration range (0.002-40.0 µg/mL at 4-fold dilution ) in a total volume of 100 µL for 15 min on a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS. Maximum CDC-mediated killing was calculated from two independent experiments using the upper part of the best fit values from a nonlinear fit in GraphPad PRISM 5.

[00652] Figure 22A-D shows that the introduction of E345R into wild-type rituximab resulted in enhanced CDC efficacy as seen by increased maximum lysis and decreased EC50 for all B cell lines tested.

[00653] Figure 22E shows that the maximal CDC-mediated killing induced by the rituximab-E345R mutant was always greater than that by wild-type rituximab, regardless of the expression levels of membrane-binding complement regulatory proteins. These data indicate that the introduction of E345R enhances the therapeutic potential of monoclonal antibodies as tumor cells are less effective in evading antibody-mediated complement attack by E345R-containing antibodies.

Example 28 Comparison of CDC kinetics for E345R and wild-type antibodies

[00654] Introduction of the mutation that stabilizes the Fc:Fc E345R interaction was shown to enhance or rescue CDC as seen by the decreased EC50 values and increased maximum lysis for the different antibodies in the different cell lines described in Example 6 (CD20 7D8 antibody in Daudi and Raji), Example 10 (antibody CD38 005 in Daudi, Raji and Wien133) and Example 25 (antibody CD38 003 and antibodies CD20 rituximab and 11B8 in Daudi and Wien133). Next, the kinetics of CDC reactions were further analyzed to clarify the difference in CDC efficacy between wild-type and E345R antibodies.

[00655] 0.1 x 106 of the Raji cells were pre-incubated in the round bottom 96-well plates with antibody at a saturating concentration (10.0 µg/mL) in a total volume of 100 µL for 15 min in a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for different periods of time, ranging between 0 and 60 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00656] Figure 23A shows that the wild-type CD20 IgG1-7D8 antibody showed maximum CDC-mediated killing of 80% of Raji cells, which already reached after 5 min under the conditions tested. However, for IgG-7D8-E345R, 80% Raji cell death was observed even faster, after 3 min. Maximum lysis by IgG-7D8-E345R (95%) was also achieved after 5 minutes.

[00657] Figure 23B shows that also for the wild-type CD20 antibody rituximab, which is less potent than 7D8 induces CDC in the Raji cells used, introduction of the E345R mutation resulted in faster killing of target cells. Wild-type rituximab showed a maximum CDC-mediated killing of 32%, achieved after 20 minutes. Rituximab-E345R achieved 32% of death already after approximately 3 minutes and notably, maximum lysis by rituximab-E345R (85%) was also achieved after 20 minutes.

[00658] Figure 23C+D shows that the Raji cells used, which are resistant to CDC-mediated killing for wild-type CD38 IgG1-003 and IgG1-005 antibodies, must be killed quickly to introduce the E345R mutation. IgG1-003-E345R and IgG1-005-E345R showed maximum CDC (50% and 60%, respectively) already after 5 min.

[00659] In summary, E345R antibodies are more potent than two wild-type counterparts, which result from a combination of higher efficacy (lower EC50), increased maximum lysis, and faster CDC reaction kinetics.

Example 29 Comparison of CDC kinetics for bispecific antibodies with or without the E345R mutation

[00660] In example 23 it is described that the E345R mutation can be applied to the bispecific antibody CD38xCD20 IgG1-005-F405L x IgG1-7D8-K409R that was generated by the DuoBody platform, resulting in an enhanced killing capacity as observed by a decreased EC50 in CDC assays in Raji and Wien133 cells. Next, CDC reaction kinetics were further analyzed to clarify the difference in CDC efficacy between CD38xCD20 bispecific antibodies with and without E345R.

[00661] 0.1 x 106 of the Raji cells were pre-incubated in the round bottom 96-well plates with antibody at a saturating concentration (10.0 µg/mL) in a total volume of 100 µL for 15 min in a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for different periods of time, ranging between 0 and 60 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00662] Figure 24 shows that the IgG1-005-F405L x IgG1-7D8-K409R bispecific antibody induces a maximum CDC-mediated killing of 83%, which was achieved after 10 minutes. Introduction of E345R resulted in increased maximum killing by IgG1-005-E345R-F405L x IgG1-7D8-E345R-K409R (98%), which was already reached after 2 minutes. These data indicate that introducing the mutation that stabilizes Fc-Fc E345R into the bispecific antibody results in accelerated CDC-mediated killing of target cells.

Example 30 Comparison of CDC kinetics for monovalent binding antibodies with and without E345R

[00663] Example 22 shows that monovalent target binding further enhances the CDC efficacy of the E345R antibodies as seen by enhanced maximal lysis with a CD38xEGFR bispecific antibody in CD38-positive, EGFR-negative Wien133 cells. Next, CDC reaction kinetics were further analyzed to clarify the difference in CDC-mediated killing ability between monovalent binding antibodies with and without E345R.

[00664] Bispecific CD38xEGFR and CD20xEGFR antibodies, with or without the E345R mutation, were generated in vitro according to a DuoBody platform as described in Example 22. The CDC efficacy of the CD38xEGFR bispecific antibodies was tested on CD38-positive, EGFR-positive cells. negative Raji, in which bispecific antibodies can only bind monovalently through CD38. 0.1 x 106 of the Raji cells were pre-incubated in the round bottom 96-well plates with antibody at a saturating concentration (10.0 µg/mL) in a total volume of 100 µL for 15 min on a shaker at temperature environment. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for different periods of time, ranging between 0 and 60 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00665] Figure 25 shows that the CD38xEGFR bispecific antibody (IgG1-005-K409R x IgG1-2F8-F405L) induces a maximum CDC-mediated killing of 55%, which was achieved after approximately 10 minutes. The introduction of E345R resulted in increased maximum kill (96%), which already reached within 5 minutes.

[00666] Figure 25 shows that the CD20xEGFR bispecific antibody (IgG1-7D8-K409R x IgG1-2F8-F405L) induces a maximum CDC-mediated killing of 85%, which was achieved after approximately 5 minutes. However, with the CD20xEGFR antibody with E345R introduced, 85% lysis was observed faster, after 2 minutes. Maximum lysis by the E345R CD20xEGFR antibody (97%) was also reached after 5 minutes.

[00667] In summary, the introduction of the E345R mutation into these monovalent binding antibodies resulted in the most potent antibodies, which resulted from the combination of increased maximum lysis and faster CDC reaction kinetics.

Example 31 CDC for a Combination of Therapeutics and E345R/Q386K Antibodies

[00668] As described in Example 19, mutant CD38 antibodies derived from IgG1-005 should induce efficient CDC in Wien133 cells when the E345 position of the wild-type antibody was substituted at any amino acid other than Glutamate (E). This suggests that oligomerization, as a prerequisite of CDC, is prevented by the presence of the glutamate side chain at position 345 of the antibody. Since E345 in one Fc is close to Q386 in the second Fc coat portion in the hexameric antibody ring structure, the E345-mediated hindrance of oligomerization in a first antibody should possibly be removed by substitutions at position Q386 of a second antibody. This would then enable E345 in the first antibody to interact better with the mutated position 386 in the second antibody in case both antibodies are combined. To test this hypothesis, CDC assays were performed on Wien133, in which wild-type antibodies (IgG1-003, IgG1-005, or IgG1-11B8) were mixed with IgG1-005-E345R/Q386K or IgG1-005-E345R/ Q386K/E430G as an example.

[00669] 0.1 x 106 of the Wien133 cells were pre-incubated in the round bottom 96-well plates with a concentration range of unpurified IgG1-005-E345R/Q386K, IgG1-005-E345R/Q386K/E430G or antibody control (0.0001-20.0 µg/mL at 3.33-fold dilution) in the presence or absence of 1.0 or 10.0 µg/mL IgG1-003, IgG1-005 or IgG1-11B8 antibody of type wild type in a total volume of 100 µL for 15 min on a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00670] Figure 26A/B/C shows that the CD38 IgG1-005-E345R/Q386K antibody induces CDC-mediated lysis of Wien133 cells in a dosage-dependent manner (dashed line). The IgG1-005-E345R/Q386K combination with 1 or 10 µg/mL of wild-type CD38 IgG1-003 antibody (Figure 26A) or wild-type CD20 IgG1-11B8 antibody (Figure 26B) resulted in increased maximum cell lysis. The combination of IgG1-005-E345R/Q386K with wild-type IgG1-005 inhibited CDC in a dose-dependent manner, possibly to compete for the binding site (Figure 26C).

[00671] Figure 26D/E/F shows similar results for the CD38 IgG1-005-E345R/Q386K/E430G antibody.

[00672] These data indicate that wild-type IgG1-003 and IgG1-11B8 antibodies participated in antibody oligomerization and CDC activation when combined with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G. In such combinations, the hindrance of oligomerization by position E345 that is present in the wild-type antibody must be at least partially removed by the Q386K substitution in the mutant antibody. This application is of particular interest to improve therapies with antibodies that are wild type at position E345, such as rituximab, ofatumumab, daratumumab or trastuzumab. Also, such antibodies that induce oligomerization must promote the formation of cellular binding complexes with the patient's own antibodies directed against target cells similar to tumor cells or bacteria.

[00673] Example 19 describes multiple amino acids in addition to E345 that enhance CDC upon mutation, for example, E430 and S440, of which specific mutations induce efficient CDC in Wien133 cells when incorporated into the CD38 IgG1-005 antibody. With the exception of mutants I253 and Y436, the identified oligomerization-enhancing mutations contact the unmutated amino acids in the second Fc coat portion in the hexameric ring structure. Therefore, the identified oligomerization-enhancing mutations, both alone and in combination, can also be expected to promote oligomerization with the unmutated antibodies and further optimization of such mutants should be achieved by the selection strategy similar to that applied in example 19.

Example 32 CDC induced by E345R in IgG2, IgG3 and IgG4 antibody isotypes

[00674] To test whether the introduction of mutations that promote oligomerization can stimulate the CDC activity of non-IgG1 antibody isotypes, isotypic variants of the CD38 antibody IgG1-005 were generated with the constant domains of human IgG2, IgG3 or IgG4 that produce IgG2-005, IgG3-005 and IgG4-005 by methods known in the art. Furthermore, the E345R oligomerization-enhancing mutation was introduced into all of these antibodies, producing IgG2-005-E345R, IgG3-005-E345R, and IgG4-005-E345R. In a similar manner, also IgG2-003 and IgG2-003-E345R were generated from the CD38 antibody IgG1-003. The CDC efficacy of the different isotypes was compared in an in vitro CDC assay.

[00675] 0.1 x 106 of the Wien133 cells were pre-incubated in the round-bottom 96-well plates with 10 µg/mL of the unpurified antibodies in a total volume of 100 µL for 15 min on a shaker at room temperature. IgG1-005-E345R was added at 3.0 µg/mL. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS.

[00676] Figure 27 shows that IgG2-005, IgG2-003, IgG3-005 and IgG4-005 were unable to lyse (A) Daudi or (B) Wien133 cells efficiently under the conditions tested (observed ~20% lysis was considered as grounds). Introduction of the E345R mutation enables potent CDC in Daudi cells across all IgG isotypes tested. These results were confirmed using CDC on Wien133 cells, although IgG3-005-E345R showed limited CDC activity relative to other isotypic variants. These data indicate that in addition to IgG1, a mutation-enhancing oligomerization such as E345R can also be applied to promote the CDC activity of IgG2, IgG3, and IgG4 antibodies.

Example 33 CDC by IgG1-005 and IgG1-005-E345R in an EX VIVO CDC assay on patient-derived CD38-positive B-cell chronic lymphocytic leukemia cells (CLL).

[00677] The cryopreserved primary cells from the CLL patient samples were obtained from the hematopathology biobank of the CDB-IDIBAPS-Hospital Clínic (Dr. Elias Campo, Hematopathology Unit, Department of Pathology, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain), or from clinical studies by the National Heart, Lung and Blood Institute (NHLBI) (Dr. Adrian Wiestner, NHLBI, Hematology Branch of the National Institutes of Health (NIH), Bethesda). Informed consent was obtained from patients in accordance with the Institutional Ethics Committee of the Hospital Clinic (Barcelona, Spain) or the Institutional Review Board of the NIH and the Declaration of Helsinki. All samples were genetically and immunophenotypically characterized.

[00678] The CLL samples were categorized into two groups according to air CD38 expression as determined by FACS: five samples were included in the high CD38 group (between 50% and 98% of CD38 expression in Daudi cells) and four samples were included in the low CD38 group (between 0.5% and 3% of CD38 expression in Daudi cells).

[00679] Fluorescently labeled CLL cells (labeling with 5 µM calcein AM) were incubated with a series of antibody concentrations (0.01-10 µg/mL in 10-fold dilutions). Next, normal human serum was added to the antibody-opsonized cells (100,000 cells/well) as a source of complement (10% of the final concentration) and incubated for 45 min at 37°C. The supernatants were recovered and the fluorescence was read. in a Synergy™ HT fluorometer as a cell lysis medication. Cell death was calculated as follows:

[00680] Specific lysis = 100 x (spontaneous sample lysis)/(maximum lysis- spontaneous lysis) where maximum lysis is determined by a sample of cells treated with 1% Triton and spontaneous lysis is determined from the sample where the Cells were incubated in the presence of 10% NHS without antibody.

[00681] Figure 28 shows that CDC IgG1-005-E345R strongly enhanced efficacy compared to wild-type IgG1-005 in both primary CLL cells with high CD38 expression and primary CLL cells with low CD38 expression.

Example 34 FcRn binding of IgG1-005 mutants compared to IgG1-005

[00682] The neonatal Fc receptor (FcRn) is responsible for the long half-life of plasma IgG by protecting IgG from degradation. After antibody internalization, FcRn binds to the antibody Fc region in the endosomes, where the interaction is stable in the mildly acidic environment (pH 6.0). When recycling the plasma membrane, where the environment is neutral (pH 7.4), the interaction is lost and the antibody is released back into circulation. This influences the half-life of IgG plasma.

[00683] The ability of the IgG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, K439E and S440K to interact with FcRn from the mouse, cynomolgus monkey and human was tested in an ELISA. In mouse FcRn ELISA mutants P247G and I253D were also tested. I253D was used as a negative control for FcRn binding. All incubations were carried out at room temperature. 96-well plates were coated with 5 µg/mL (100 µL/well) recombinantly produces the biotinylated extracellular domain of FcRn (mouse, human or cynomolgus) (FcRnECDHis-B2M-BIO), diluted in PBST plus 0.2% BSA and incubated for 1 hour. Plates were washed 3 times with PBST and 3 times periodically diluted (in PBST/0.2% BSA, pH 6.0) wild-type IgG1-005 or 005 mutants were added and plates were incubated for 1 hour. Plates were washed with PBST/0.2% BSA, pH 6.0. Goat anti-human IgG(Fab'2)-HRP (Jackson Immuno Research, cat no.:109-035-097) diluted in PBST/0.2% BSA, pH 6.0 was added and the plates were incubated for 1 hour. After washing, ABTS was added as substrate and plates were incubated in the dark for 30 minutes. Absorbance was read at 405 nm using an EL808 ELISA reader. Data generated in the mouse FcRn ELISA were analyzed using best fit values from a non-linear agonist dosage response fit using GraphPad PRISM 5 log-transformed concentrations and the evident affinity (EC50) was calculated (Table 20). The experiment shows that FcRn binding was not altered by any of the IgG1-005 mutants compared to wild-type IgG1-005. Table 20 Evident affinity (EC50) in µg/ml of IgG1-005 and mouse FcRn mutants

[00684] Figure 29 shows that wild-type IgG1-005 and all tested reservoir IgG1-005 mutants bound to mouse, human and cynomolgus FcRn at pH 6.0. No significant binding to FcRn was detected at pH 7.4 (data not shown).

Example 35 CDC enhanced by different mutations in rituximab in Ramos and SU-DHL-4 B cell cells

[00685] As described in Example 19, the oligomerization and CDC activity of the anti-CD38 antibody IgG1-005 can be estimated by simple mutations in specific residues in or on the periphery of the Fc:Fc interface. Oligomerization can also be indirectly stimulated by other types of mutations in residues outside the Fc:Fc interface that allosterically strengthen Fc:Fc interactions. This was also tested by the anti-CD20 IgG1 antibody rituximab in two B cell lines (Ramos and SU-DHL-4). The following mutations were tested: E345K, E345Q, E345R, E345Y, E430G, E430S, E430T and S440Y (essentially as described in Example 19).

[00686] For the CDC assay, 0.1 x 106 of the cells (Ramos or SU-DHL-4) were pre-incubated in the round bottom 96-well plates with a range of saturation antibody concentrations (0.0001- 10.0 µg/mL at 3-fold dilution) in a total volume of 100 µL for 15 min on a shaker at room temperature. Then, 25 µL of normal human serum was added as a source of complement (20% of the final concentration) and incubated in a 37°C incubator for 45 min. The reaction was stopped by placing the plates on ice. 10 µL of propidium iodide was added and cell lysis was determined by FACS. Data were analyzed using best fit values from a nonlinear agonist dose response fit using log-transformed concentrations in GraphPad PRISM 5.

[00687] Figure 30 shows that all rituximab mutants tested were able to increase CDC efficacy in both B cell lines.

Example 36 Target-independent liquid-phase complement activation: IgG1-005 mutants compared to wild-type IgG1-005

[00688] Target-independent complement activation may constitute a safety issue when an antibody activates complement in, for example, the bloodstream or organ tissue. This may result in the products of unwanted complement activation or unwanted complement deposition. To test target-independent liquid-phase complement activation 100 µg/ml of igG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, wild-type IgG1-005, or heat-aggregated IgG (HAG, positive control) were incubated in 90% normal human serum for 1 hour at 37° C. Samples were then transferred to an ELISA kit to measure C4d generation (Micro Vue C4d-fragment, Quidel, San Diego, CA , USA). C4d is an activation fragment of C4 that is a marker for activation of the classical component pathway.

[00689] Figure 31 shows that wild-type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S and IgG1-005-S440Y shows minimal C4 activation, considering IgG1-005-E345R, IgG1-005-E430F and IgG1-005-E430T which show increased C4d generation (C4 activation) compared to wild-type IgG1-005.

Example 37 Plasma release rates of IgG1-005 mutants compared to wild-type IgG1-005

[00690] The mice in this study were housed in a barrier unit at the Central Laboratory Animal Facility (Utrecht, The Netherlands) and maintained in the cages with superior filters and food provided ad libitum. All experiments were approved by Utrecht University animal ethics committee. SCID mice (C.B-17/Icr-Prkdc<Scid>/IcrIcoCrl, Charles-River) were injected intravenously with 500 µg of antibody using 3 mice per group.

[00691] 50 µL of blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in the heparin containing vials and centrifuged for 5 minutes at 10,000 g. Plasma was stored at -20°C until determination of antibody concentrations.

[00692] Specific human IgG concentrations were determined using a total hIgG and CD38-specific sandwich ELISA.

[00693] For the total hIgG ELISA, mouse anti-human IgG mAb - clone MH16 cover (#M1268, CLB Sanquin, The Netherlands), coated by Microlon ELISA 96-well plates (Greiner, Germany) at a concentration of 2 µg /mL was used as capture antibody. After blocking the plates with PBS supplemented with 0.2% bovine serum albumin, samples were added, periodically diluted to ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin ) and incubated on a plate shaker for 1 h at room temperature (RT). Plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA) and developed with 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) ( ABTS; Roche, Mannheim, Germany). Absorbance was measured on a microplate reader (Biotek, Winooski, VT) at 405 nm.

[00694] For the specific CD38 ELISA, His-labeled CD38 extracellular domain was coated on Microlon ELISA 96-well plates (Greiner, Germany) at a concentration of 2 µg/mL. After blocking the plates with ELISA buffer, samples periodically diluted with ELISA buffer were added and incubated on a plate shaker for 1 h at room temperature (RT). Plates were subsequently incubated with 30 ng/ml mouse anti-human IgG1-HRP, (Sanquin M1328, clone MH161-1) and developed with 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). Absorbance was measured on a microplate reader (Biotek, Winooski, VT) at 405 nm

[00695] Figure 32A shows IgG release rates from the wild-type reference antibody IgG1-005 and the 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, IgG1-005-S440Y. Mutants IgG1-005-E430S, IgG1-005-E430G and IgG1-005-S440Y, IgG1-005-E430T, IgG1-005-E345K, IgG1-005-E345Q and IgG1-005-E345Y showed release rates similar to those of wild-type IgG1-005. The mutants IgG1-005-E430F and IgG1-005-E345R showed a faster release rate. The plasma release rate was calculated as dose/AUC (mL/day/kg). The AUC value (area under the curve) was determined from the concentration time curves.

[00696] Figure 32B shows IgG release rates as determined by the CD38-specific ELISA of the wild-type reference antibody IgG1-005 and the antibody variants IgG1-005-E345K, IgG1-005-E345R, IgG1-005- E430G, IgG1-005-E430S and IgG1-005-S440Y when injected intravenously one day after intraperitoneal administration of 8.0 mg of the unrevealing IgG1-B12 control antibody. The IgG1 wild-type reference antibody in the absence of the irrelevant b12 control was included as a control. Mutants IgG1-005-E430S, IgG1-005-E430G, IgG1-005-S440Y and IgG1-005-E345K showed release rates similar to those of the wild type. The IgG1-005-E345R mutant showed faster release.

EQUIVALENTS

[00697] That person skilled in the art will recognize, or be able to determine using no more than routine experiment, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims. Any and all combinations of embodiments described in the dependent claims are also considered to be within the scope of the invention.

Claims (2)

1. Method for enhancing complement-dependent cytotoxicity (CDC) IN VITRO without altering target-independent fluid-phase complement activation of a precursor antibody comprising an Fc domain of an immunoglobulin and a binding region, characterized by the fact that the method comprises introducing a mutation to the precursor antibody at an amino acid residue corresponding to E345K or E345R in the Fc region of a human IgG1 heavy chain, wherein the Fc region comprises the sequence set forth in SEQ ID NO 1 or 5, evaluating the response CDC induced by the variant antibody when bound to the surface of an antigen-expressing cell in the presence of effector cells or complement compared to the precursor antibody; evaluating the target-independent fluid phase complement activity of the variant antibody compared to the precursor antibody by incubating the antibody in normal human serum and determining the generation of C4d compared to the precursor antibody; and selecting a variant antibody with an enhanced CDC response and unchanged target-independent phase complement activation. 2. Method according to claim 1, characterized by the fact that the precursor antibody is a monospecific, bispecific or multispecific antibody.