Polypeptide variants and uses thereof

Abstract

To provide Fc region-containing polypeptides, such as antibodies, that have decreased Fc effector functions such as, decreased binding to C1q, decreased complement-dependent cytotoxicity (CDC) and may also have decreased activation of other effector functions resulting from one or more amino acid modifications in the Fc-region.SOLUTION: A mutant Fc region results in stabilized Fc-Fc interactions when one or more polypeptides or one or more antibodies bind to their target, one or more antigens on a cell surface. At the same time, the mutant Fc region may also reduce complement-dependent cytotoxicity (CDC) due to one or more amino acid modifications in the Fc region, as well as activation of other effector functions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to polypeptides, e.g., antibodies, containing an Fc region that have reduced Fc effector functions, e.g., reduced binding to C1q, reduced complement-dependent cytotoxicity (CDC), and optionally reduced activation of other effector functions due to one or more amino acid modifications within the Fc region. [Background technology]

[0002] Background of the Invention Fc-mediated effector functions of monoclonal antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP), contribute to a therapeutic window defined by efficacy and toxicity. CDC is initiated by the binding of C1q to the Fc region of an antibody. C1q is a multimeric protein consisting of six binding globular heads attached to a stalk. Because each binding globular head has low affinity for IgG, C1q must gain avidity by binding to many IgG1 molecules on the cell surface to trigger the classical complement pathway. ADCC and ADCP are initiated by the binding of the IgG Fc region to Fcγ receptors (FcγRs) on effector cells.

[0003] IgG hexamerization upon target binding on the cell surface has been shown to support strong C1q binding. This hexamerization is mediated by intermolecular noncovalent Fc-Fc interactions, which can be enhanced by point mutations within the CH3 domain, such as E345R and E430G.

[0004] WO2013 / 004842 (Patent Document 1) discloses antibodies or polypeptides comprising a mutant Fc region with one or more amino acid modifications that result in modified effector functions, such as complement-dependent cytotoxicity (CDC).

[0005] WO2014 / 108198 (Patent Document 2) discloses polypeptides, such as antibodies, comprising a mutant Fc region with one or more amino acid modifications that result in increased complement-dependent cytotoxicity (CDC).

[0006] WO2012 / 130831 (Patent Document 3) relates to Fc region-containing polypeptides having altered effector functions as a result of one or more amino acid substitutions within the Fc region of the polypeptide. Such polypeptides exhibit reduced affinity for human FcyRIIIa and / or FcyRIIa and / or FcyRI compared to polypeptides comprising a wild-type IgG Fc region, and exhibit a reduction in ADCC induced by the polypeptides of at least 20% compared to ADCC induced by polypeptides comprising a wild-type human IgG Fc region. WO2012 / 130831 (Patent Document 3) does not disclose Fc region-containing polypeptides with enhanced Fc-Fc interactions and / or enhanced hexamer formation ability.

[0007] As noted above, previous efforts at enhancing Fc-Fc interactions between polypeptides and / or antibodies have had the effect of enhancing effector function, e.g., enhancing CDC and / or ADCC, which results in cell death of target cells to which the antibody or polypeptide binds.

[0008] Although enhancing Fc-Fc interactions between antibodies can be used to amplify the effect of antibody binding to a target on the cell surface, in cases where the target cell is an effector cell, such as a T cell, NK cell, or other effector cell whose mechanism of action involves binding to an effector cell (such as in bispecific antibodies), interaction with C1q or Fc gamma R and / or activation of Fc effector function, such as CDC and / or ADCC, may be unnecessary. Thus, there is a need for antibodies that have enhanced Fc-Fc interactions but that do not engage in C1q binding and / or do not have Fc gamma R interactions, thereby activating Fc effector function, such as CDC and / or ADCC.

[0009] It is therefore an object of the present invention to provide a variant polypeptide or antibody comprising an Fc region and an antigen-binding region of human IgG, which has enhanced Fc-Fc interaction and reduced effector function, such as CDC and / or ADCC, compared to a parent polypeptide, wherein the parent polypeptide is a human IgG of the same isotype and has the same antigen-binding region, and which has a first mutation, which is an Fc-Fc-enhancing mutation, at an amino acid position corresponding to E345, E430, or S440 in human IgG1 (with the proviso that the mutation at position S440 is S440Y or S440W).

[0010] Another object of the present invention is to provide a polypeptide or antibody having enhanced Fc-Fc interaction properties without inducing effector functions such as CDC. Another object of the present invention is to provide a polypeptide or antibody having enhanced Fc-Fc interaction properties without inducing effector functions such as CDC or ADCC. Another object of the present invention is to provide a polypeptide or antibody having enhanced Fc-Fc interaction properties without inducing effector functions such as CDC or ADCC. A further object of the present invention is to provide a polypeptide or antibody having enhanced Fc-Fc interaction but reduced Fc effector function, e.g., reduced CDC and / or ADCC, compared to the parent polypeptide, and having only a first mutation that results in enhanced Fc-Fc interaction. Yet another object of the present invention is to provide a polypeptide or antibody that activates signaling without activating Fc effector function, e.g., CDC and / or ADCC, when the antigen-binding region of the polypeptide or antibody is bound to a corresponding antigen, and optionally induces enhanced signaling. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2013 / 004842 [Patent Document 2] WO2014 / 108198 [Patent Document 3] WO2012 / 130831 Summary of the Invention

[0012] In a first aspect, the present invention provides a polypeptide or antibody having an Fc region and an antigen-binding region, wherein the Fc region has a first mutation that is an Fc-Fc enhancing mutation and a second mutation that reduces C1q binding and / or Fc gamma R binding and / or Fc effector function, such as CDC and / or ADCC activity.

[0013] The inventors of the present invention have surprisingly found that by introducing a second mutation in the Fc region corresponding to amino acid position E322 or P329 in the Fc region of human IgG, it is possible to reduce effector function, such as CDC and / or ADCC activity, while retaining the oligomerization ability of the first mutation.

[0014] Without being limited by theory, it is believed that when the polypeptides or antibodies of the present invention bind to a target on a cell surface, more stable binding interactions are possible between the Fc regions of the two polypeptide or antibody molecules, resulting in enhanced oligomerization, such as hexamer formation, without enhancing Fc-mediated effector function. The polypeptides or antibodies of the present invention further have reduced C1q binding and / or reduced Fc gamma R binding compared to their parent polypeptides or parent antibodies that contain the first mutation but not the second mutation. The polypeptides or antibodies of the present invention have reduced Fc effector function compared to their parent polypeptides or parent antibodies that contain the first mutation but not the second mutation. Some polypeptides or antibodies of the present invention have reduced Fc effector function, such as CDC, compared to their parent polypeptides or parent antibodies. Some polypeptides or antibodies of the present invention have reduced Fc effector function, such as ADCC, compared to their parent polypeptides or parent antibodies. Some polypeptides or antibodies of the present invention have reduced Fc effector function, such as CDC and ADCC, compared to their parent polypeptides or parent antibodies. Some polypeptides or antibodies of the present invention further have a reduced Fc effector response compared to the same polypeptide or antibody not comprising the first and second mutations, i.e., comprising a wild-type Fc region. Some polypeptides of the present invention have low C1q binding and / or low Fc gamma R binding. Some polypeptides or antibodies of the present invention have a low CDC response. Some polypeptides or antibodies of the present invention have a low ADCC response. Some polypeptides or antibodies of the present invention are characterized by having both a low ADCC response and a low CDC response and / or other low effector responses.

[0015] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region and an antigen-binding region of human IgG, wherein the Fc region comprises a CH2 and a CH3 domain, and wherein the Fc region comprises (i) a first mutation and (ii) a second mutation corresponding to the following amino acid positions in human IgG1 according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No.91-3242): i. a first mutation at E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; and ii. A second mutation at K322 or P329 The present invention provides a polypeptide or antibody comprising:

[0016] That is, in a first aspect of the present invention, the inventors have found that introducing a second mutation at one of the amino acid positions corresponding to K322 or P329 in the Fc region of a polypeptide or antibody having a first mutation that enhances Fc-Fc interactions and thus oligomerization after target binding can reduce Fc effector function. The mutation corresponding to amino acid position K322 or P329 in the Fc region of the polypeptide or antibody has the effect of reducing one or more Fc effector functions to a reduced level compared to a parent polypeptide or antibody having the same first mutation but without the second mutation. Thus, in one aspect of the invention, the polypeptide or antibody has at least one first mutation which may be selected from one of positions E430, E345 or S440 (provided that the mutation at S440 is S440Y or S440W), and the polypeptide or antibody has at least one second mutation which may be selected from one of positions K322 or P329.

[0017] In one embodiment of the present invention, the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. In one embodiment of the present invention, the first mutation is selected from E430G or E345K. In a preferred embodiment, the first mutation is E430G.

[0018] In one aspect of the invention, the second mutation is selected from the group consisting of K322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, P329A, and P329Y.

[0019] In one aspect of the invention, the second mutation is at amino acid position P329, with the proviso that the second mutation is not P329A.

[0020] In one aspect of the invention, the second mutation is at amino acid position P329, with the proviso that the second mutation is not P329A or P329G.

[0021] In one aspect of the invention, the Fc region does not comprise mutations at amino acid positions corresponding to L234 and L235. That is, in one aspect of the invention, the Fc region comprises wild-type amino acids L and L at positions corresponding to L234 and L235 in human IgG1, where the positions are according to EU numbering.

[0022] In a further aspect, the present invention relates to a method for reducing an Fc effector function of a polypeptide or antibody comprising an Fc region and an antigen-binding region of human IgG, wherein the Fc region, together with the CH2 and CH3 domains, comprises: introducing a first mutation corresponding to (i) amino acid position E430, E345, or S440 in human IgG1 according to EU numbering (with the proviso that the mutation at S440 is S440Y or S440W); and (ii) a second mutation corresponding to amino acid position K322 or P329 in human IgG1 according to EU numbering.

[0023] That is, the present inventors have found that one or more of the effector functions can be reduced by introducing a second mutation at one of amino acid positions K322 or P329 of a polypeptide or antibody having a first mutation corresponding to one of amino acid positions E430, E345, or S440 (provided that the mutation at S440 is S440Y or S440W), which results in enhanced oligomerization after target binding on the cell surface and thus enhanced Fc effector function. Thus, the second mutation can reduce the Fc effector function of the polypeptide or antibody to a level equivalent to or lower than that of a parent polypeptide having a first mutation at a position corresponding to E430, E345, or S440 (provided that the mutation at S440 is S440Y or S440W).

[0024] In another aspect, the present invention relates to compositions comprising at least one polypeptide or antibody described herein.

[0025] In another aspect, the present invention relates to a polypeptide, antibody, or composition described herein for use as a medicament.

[0026] In another aspect, the invention relates to a polypeptide, antibody, or composition described herein for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0027] In another aspect, the present invention relates to a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of a polypeptide, antibody, or composition described herein.

[0028] [The present invention 1001] A polypeptide comprising a human IgG Fc region and an antigen-binding region, the Fc region comprises CH2 and CH3 domains; the Fc region comprising (i) a first mutation and (ii) a second mutation corresponding to the following amino acid positions in human IgG1 according to EU numbering: i. a first mutation at E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; and ii. A second mutation at K322 or P329 Including, The polypeptide. [The present invention 1002] 1001. The polypeptide of claim 10, wherein the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [The present invention 1003] 1001. The polypeptide of the present invention, wherein the first mutation is selected from E430G or E345K. [The present invention 1004] Any of the aforementioned polypeptides of the present invention, wherein the second mutation is selected from the group consisting of K322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, P329A, and P329Y. [The present invention 1005] Any of the aforementioned polypeptides of the present invention, wherein the second mutation is K322E. [The present invention 1006] The polypeptide of any of claims 1001 to 1004, wherein the second mutation is selected from the group of P329R, P329K, and P329D. [The present invention 1007] Any of the preceding polypeptides of the invention, wherein the Fc region comprises one or more additional mutations. [The present invention 1008] Any of the preceding polypeptides of the invention, wherein the Fc region comprises one or more additional mutations within the CH2 or CH3 domain. [The present invention 1009] The polypeptide of the invention 1007 or 1008, wherein the Fc region comprises a further mutation corresponding to position K439 in the CH3 domain, or the further mutation may be present at position S440 if the first mutation is not present at position S440. [The present invention 1010] The polypeptide of the present invention, wherein said further mutation is selected from S440K or K439E. [The present invention 1011] Any of the polypeptides of the present invention, wherein the Fc region comprises up to 10 mutations, such as 9 mutations, for example 8 mutations, for example 7 mutations, for example 6 mutations, for example 5 mutations, for example 4 mutations, for example 3 mutations, or for example 2 mutations. [The present invention 1012] Any of the polypeptides of the present invention having an Fc effector function that is reduced by at least 20%, for example at least 30% or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% compared to an identical parent polypeptide having the same first mutation but not the second mutation. [The present invention 1013] Any of the polypeptides of the present invention which do not induce Fc effector function. [The present invention 1014] 10. The polypeptide of claim 10, wherein the Fc effector function is selected from the group consisting of complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding. [The present invention 1015] Any of the aforementioned polypeptides of the present invention which is an antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody. [The present invention 1016] Any of the aforementioned polypeptides of the present invention, wherein the Fc region is of human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or mixed isotype. [The present invention 1017] Any of the aforementioned polypeptides of the present invention, wherein the Fc region is of human IgG1 isotype. [The present invention 1018] Any of the aforementioned polypeptides of the present invention, which is a human antibody, a humanized antibody, or a chimeric antibody. [The present invention 1019] Any of the aforementioned polypeptides of the present invention, wherein the antigen-binding region binds to a member of the TNFR-SF. [The present invention 1020] The polypeptide of the present invention, wherein the TNFR-SF does not contain an intracellular death domain. [The present invention 1021] The polypeptide of the present invention 1019, wherein the member of the TNFR-SF is selected from the group of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR. [The present invention 1022] 1020. The polypeptide of the present invention, wherein the TNFR-SF is selected from the group consisting of OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. [The present invention 1023] A method for reducing an Fc effector function of a polypeptide comprising an Fc region and an antigen-binding region of a human immunoglobulin, comprising: the Fc region comprises CH2 and CH3 domains; the Fc region comprises a first mutation corresponding to (i) position E430, E345, or S440 in human IgG1 according to EU numbering; the method comprising introducing a second mutation corresponding to (ii) position K322 or P329 in human IgG1 according to EU numbering; The method. [The present invention 1024] 1025. The method of claim 1023, wherein the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [The present invention 1025] 1025. The method of claim 1023 or 1024, wherein the first mutation is selected from E430G or E345K. [The present invention 1026] 1026. The method of any of claims 1023 to 1025, wherein the second mutation is selected from the group consisting of K322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, P329A, and P329Y. [The present invention 1027] 1027. The method of any of claims 1023 to 1026, wherein the second mutation is selected from the group of K322E, P329R, P329K, and P329D. [The present invention 1028] 1028. The method of any of claims 1023 to 1027, wherein the Fc region comprises one or more additional mutations within the CH3 domain. [The present invention 1029] 1029. The method of claim 1028, wherein the Fc region comprises an additional mutation in the CH3 domain corresponding to one of positions S440 or K439 in human IgG1 according to EU numbering. [The present invention 1030] 1029. The method of claim 1029, wherein said further mutation is selected from S440K or K439E. [The present invention 1031] Any of the methods of inventions 1023 to 1030, wherein the Fc effector function is reduced by at least 20%, such as at least 30% or at least 40%, or at least 50% or at least 60% or at least 70%, or at least 80% or at least 90% compared to an identical parent polypeptide having the same first mutation but not the second mutation. [The present invention 1032] 1032. The method of any of claims 1023 or 1031, wherein the Fc effector function is selected from the group of complement dependent cytotoxicity (CDC), complement dependent cell-mediated cytotoxicity (CDCC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding. [The present invention 1033] The method of claim 1032, wherein ADCC is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a comparison antibody that is identical to the antibody except that it does not contain the second mutation. [The present invention 1034] A composition comprising at least one polypeptide of any one of 1001 to 1022 of the present invention. [This invention 1035] A composition of the invention 1034 comprising any one or more polypeptides of the invention. [The present invention 1036] 1036. The composition of any of claims 1034 to 1035, comprising a first polypeptide and a second polypeptide as defined in any of claims 1001 to 1022. [This invention 1037] The antibody or antibody comprises a first polypeptide comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation at K439 or S440, provided that if the additional mutation is present at S440, the first mutation is not present at S440, and the first and second Fc regions do not contain additional mutations at the same amino acid position; Composition of the present invention 1036. [The present invention 1038] Any of the compositions of claims 1036 to 1037, wherein the first polypeptide and the second polypeptide bind to different epitopes on one or more members of the TNFR-SF having an intracellular death domain selected from the group consisting of TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, and EDAR. [This invention 1039] 8. The composition of any of claims 1036 to 1037, wherein the first polypeptide and the second polypeptide bind to different epitopes on one or more members of the TNFR-SF that do not have an intracellular death domain, e.g., OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. [The present invention 1040] Any of the compositions of claims 1036 to 1037, wherein the first polypeptide that binds to a member of the TNFR-SF that does not have an intracellular death domain, such as OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR, does not block binding of a second antibody that binds to a member of the TNFR-SF that does not have an intracellular death domain, such as OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. [The present invention 1041] The first polypeptide and the second polypeptide are present in the composition at a molar ratio of 1:49 to 49:1, for example, a 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio, a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio, Any of compositions 1034 to 1040 of the present invention, wherein the molar ratios are 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. [The present invention 1042] 1041. The composition of any of claims 1034 to 1040, wherein the first polypeptide and the second polypeptide and / or any further polypeptide are present in the composition in an equimolar ratio. [This invention 1043] Any of the compositions of 1034 to 1042 of the present invention, which is a pharmaceutical composition. [This invention 1044] A polypeptide of any one of 1001 to 1022 or a composition of any one of 1034 to 1043 for use as a pharmaceutical. [This invention 1045] A polypeptide of any of claims 1001 to 1022 or a composition of any of claims 1034 to 1044 for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. [The present invention 1046] A method of treating an individual having a disease, comprising the step of administering to the individual an effective amount of any of the antibodies or compositions of the invention. [This invention 1047] The method of claim 1046, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. [This invention 1048] The method of any of claims 1046 to 1047, further comprising the step of administering an additional therapeutic agent. [This invention 1049] the additional therapeutic agent is a chemotherapeutic agent (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), a kinase inhibitor (including but not limited to sorafenib, sunitinib, or everolimus), an apoptosis modulator (including but not limited to recombinant human TRAIL or birinapant), a RAS inhibitor, a proteasome inhibitor (including but not limited to bortezomib), 1048. The method of claim 1048, wherein the anti-cancer agent is one or more selected from the group consisting of a histone deacetylase inhibitor (including but not limited to vorinostat), a dietary supplement, a cytokine (including but not limited to IFN-γ), an antibody or antibody mimetic (including but not limited to anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR antibodies and antibody mimetics), and an antibody-drug conjugate. [The present invention 1050] A kit of parts comprising any of the polypeptides or compositions of the present invention, wherein the polypeptides or compositions are present in one or more containers, for example vials. [This invention 1051] The kit of parts of the present invention 1050, wherein any of said polypeptides or compositions of the present invention are for simultaneous, separate or sequential use in therapy. [This invention 1052] Use of a polypeptide or composition of any of claims 1001 to 1043 for the manufacture of a medicament for the treatment of a disease. [This invention 1053] The use of the present invention 1052, wherein the disease is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. These and other aspects of the invention, particularly the various uses and therapeutic applications of the above polypeptides or antibodies, are described in further detail below. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 shows the effect of a C1q binding-inhibiting mutation (D270A / K322A, denoted as AA) in the Fc domain of IgG1 on the CDC efficacy of IgG-005 mutants with and without one or more mutations for enhanced Fc-Fc interactions (E430G, denoted as G; E345R, denoted as R; E345R / E430G / S440Y, denoted as RGY). CD38-positive Daudi cells were incubated with a range of concentrations of CD38 (mutant) antibodies in the presence of pooled 20% normal human serum (NHS). CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. IgG1-b12 mAb against HIV gp120 and its mutants were used as non-binding isotype control mAbs. Representative examples are shown. [Figure 2] Figure 1 shows the effect of a single amino acid substitution in the human IgG1 C1q binding site on the CDC efficacy of IgG1-005 mutants with the E430G mutation for enhanced Fc-Fc interactions. (A) For CDC assays, Daudi cells were incubated with a range of concentrations of IgG1-005-E430G with the D270R, K322E, P329D, or P329R mutations in the presence of pooled 20% NHS. CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. A sample without antibody was used as a negative control for CDC efficacy. A representative example of two experiments is shown. (B) Binding of C1q to cell-bound IgG1-005-E430G antibodies with the K322E, P329D, or P329R mutations was analyzed by FACS flow cytometry analysis and is shown by the mean fluorescence intensity (MFI) of FITC-labeled rabbit anti-HuC1q antibody. [Figure 3]Figure 1 shows the effect of substitution of amino acid K322 on the CDC efficacy of IgG-005-E430G with enhanced Fc-Fc interactions. Daudi cells were incubated with a range of concentrations of CD38 antibody mutants in the presence of pooled 20% NHS. CDC efficacy is shown as percentage lysis, determined by the percentage of propidium iodide (PI)-positive cells. IgG1-b12-E430G, an antibody against HIV gp120, was used as a non-binding isotype control with Fc-Fc-enhancing mutations. [Figure 4A] Biophysical characterization of IgG1-005-E430G antibody variants with additional mutations K322D, K322E, or K322N by (A) capillary electrophoresis / sodium dodecyl sulfate (CE-SDS) and (B) high-performance size-exclusion chromatography (HP-SEC). (A) Left panel: non-reducing conditions; right panel: reducing conditions. [Figure 4B] Biophysical characterization of IgG1-005-E430G antibody variants with additional mutations K322D, K322E, or K322N by (A) capillary electrophoresis / sodium dodecyl sulfate (CE-SDS) and (B) high-performance size-exclusion chromatography (HP-SEC). (B) HP-SEC profiles of individual antibodies are plotted with a y-axis offset of 0.1 A280 units. [Figure 5] Figure 1 shows the effect of substitution of amino acid P329 on the CDC efficacy of IgG-005-E430G with enhanced Fc-Fc interactions. Daudi cells were incubated with a range of concentrations of CD38 antibodies in the presence of pooled 20% normal human serum (NHS). CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. IgG1-b12-E430G, an antibody against HIV gp120, was used as a non-binding isotype control with Fc-Fc-enhancing mutations. [Figure 6]Figure 1 shows the effect of substitution of amino acid P329 on FcγRIIIa activation by IgG-005-E430G, which has enhanced Fc-Fc interactions, as measured by the Bioluminescent ADCC Reporter BioAssay. FcγRIIIa activation by antibody binding to Daudi cells was quantified using FcγRIIIa-expressing Jurkat reporter cells, which express luciferase upon FcγRIIIa binding. Luciferase production is expressed in relative light units (RLU). For each data point, the mean and standard deviation of duplicates are shown. A representative example of two experiments is shown. [Figure 7] Figure 1 shows the effect of mutations K322E, P329A, P329D, P329K, and P329R on ADCC-mediated killing by IgG1-005-E430G. ADCC of Daudi cells was examined in an in vitro 51Cr-release assay using freshly isolated PBMCs from healthy human donors at an E:T ratio of 100:1. Antibody IgG1-b12 against HIV gp120 was used as a non-binding isotype control. For each data point, the mean and standard deviation of quintuplicate samples are shown. A representative example of PBMCs from one donor is shown. [Figure 8] The effect of introducing the P329D mutation on C1q binding or CDC efficacy of different mutants of IgG-005 with enhanced Fc-Fc interactions (E345K, E345R, and E345R / E430G / S440Y, denoted as RGY) is shown. (A) C1q binding to cell-bound antibodies was analyzed by FACS flow cytometry analysis and is shown by the mean fluorescence intensity (MFI) of FITC-labeled rabbit anti-HuC1q antibodies. (B) In vitro CDC assays were performed on Daudi cells in the presence of 20% pooled normal human serum (NHS). CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. The antibody IgG1-b12 against HIV gp120 was used as a non-binding isotype control. [Figure 9A]1 shows biophysical characterization of IgG1-005-RGY antibody variants with additional mutations K322E or P329D by (A) HP-SEC, (B) CE-SDS, and (C) native MS. [Figure 9B] 1 shows biophysical characterization of IgG1-005-RGY antibody variants with additional mutations K322E or P329D by (A) HP-SEC, (B) CE-SDS, and (C) native MS. [Figure 9C] 1 shows biophysical characterization of IgG1-005-RGY antibody variants with additional mutations K322E or P329D by (A) HP-SEC, (B) CE-SDS, and (C) native MS. [Figure 10A] The effects of P329D and K322E mutations on Fc-Fc interaction and clustering of saturating concentrations of an agonistic DR5 antibody with the E430G mutation for enhanced Fc-Fc interaction are shown. (A) The involvement of Fc-Fc interaction in the induction of apoptosis by an agonistic DR5 antibody with the E430G mutation is shown in a 3-day viability assay in BxPC-3 human cancer cells, with inhibition of killing in the presence of the Fc-binding peptide DCAWHLGELVWCT. Introduction of the P329D (B) or K322E (C) mutation reduced the IC50 for killing by an agonistic DR5 antibody with the E430G mutation, but maximal killing was still achieved, as shown in a 3-day viability assay in BxPC-3 human cancer cells at saturating antibody concentrations of 5 μg / mL (B) and 10 μg / mL (C). Error bars indicate standard deviation. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11]Figure 1 shows the clearance rate of antibody administered intravenously at 500 μg to SCID mice. (A) Total human IgG in serum samples was determined by ELISA and plotted as a concentration versus time curve. Each data point represents the mean + / - standard deviation of triplicate samples. (B) Clearance up to 21 days after antibody administration was determined by the injected dose, D, and the area under the curve, AUC, of ​​the concentration-time curve according to the formula D*1.000 / AUC. A representative example of two independent ELISA experiments is shown. [Figure 12] Figure 1 shows the effect of substitution of amino acid P329 on the CDC efficacy of IgG1-005-E430G, which has enhanced Fc-Fc interactions. Daudi cells were incubated with a range of concentrations of CD38 antibodies in the presence of pooled 20% normal human serum (NHS). CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. IgG1-b12, an antibody against HIV gp120, was used as a non-binding isotype control. [Figure 13] Figure 1 shows the effect of substitution of amino acid P329 on the CDC efficacy of different IgG isotype variants of Campath-E430G with enhanced Fc-Fc interactions. Wien 133 cells were incubated with a range of concentrations of CD52 antibodies in the presence of 20% pooled human normal serum (NHS). CDC efficacy is shown as the area under the dose-response curve, normalized to the non-binding control antibodies IgG1-b12 (0%) and IgG1-Campath (100%). [Figure 14] Figure 1 shows the effect of substitution of amino acid K322 on the CDC efficacy of IgG isotype variants of Campath-E430G with enhanced Fc-Fc interactions. Wien 133 cells were incubated with a range of concentrations of CD52 antibodies in the presence of 20% pooled normal human serum (NHS). CDC efficacy is shown as the area under the dose-response curve, normalized to the non-binding control antibodies IgG1-b12 (0%) and IgG1-Campath (100%). [Figure 15]The effect of substitution of amino acid P329 (top) or K322 (bottom) on the CDC efficacy of IgG1-Campath variants with different Fc-Fc interaction-enhancing mutations is shown. Wien 133 cells were incubated with a range of concentrations of CD52 antibodies in the presence of 20% pooled human normal serum (NHS). CDC efficacy is shown as the area under the dose-response curve, normalized to the non-binding control antibodies IgG1-b12 (0%) and IgG1-Campath (100%). [Figure 16] Figure 1 shows the effect of substitution of amino acids K322 or P329 on the CDC efficacy of anti-CD20 antibodies with enhanced Fc-Fc interactions. Wien 133 cells were incubated with a range of concentrations of CD20 antibodies in the presence of 20% pooled normal human serum (NHS). CDC efficacy is shown as the percentage of lysis determined by the percentage of propidium iodide (PI)-positive cells. Antibody IgG1-b12 was used as a non-binding isotype control. [Figure 17A] Figure 1 shows the effect of substitution of amino acids K322 or P329 on FcγR binding of anti-CD38 IgG1-005 antibodies with E430G-enhanced Fc-Fc interactions, as measured by ELISA. A series of concentrations of the indicated antibodies were captured onto microtiter plate wells and incubated with fixed concentrations of FcγRIIA, FcγRIIB, or FcγRIII, or added to FcγRI-coated wells. Mutants P329D-E430G, P329K-E430G, and P329R-E430G reduced FcγRI binding to background levels; K322E-E430G retained similar binding to wild-type (WT) IgG1-005 to all FcγR mutants tested. The mutants L234A / L235A / P329G / E430G (AAGG) and L234F / L235E / P329D / E430G (FEDG) reduced binding of all FcγR mutants tested to background levels. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D]See legend to Figure 17A. [Figure 17E] See legend to Figure 17A. [Figure 17F] See legend to Figure 17A. [Figure 18] The effect of substitution of amino acid P329 on the CDC efficacy of IgG1-Campath or IgG1-11B8 variants with Fc-Fc interaction-enhancing mutations is shown. Wien 133 cells were incubated with a range of concentrations of a mixture of CD20 and CD52 antibodies in the presence of 20% pooled human normal serum (NHS). CDC efficacy is shown as (upper panel) the percentage of lysis, determined by the percentage of propidium iodide (PI)-positive cells, and (lower panel) the area under the dose-response curve, normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-Campath-E430G + IgG1-11B8-E430G (100%). [Figure 19] The effect of substitution of amino acid K322 on the CDC efficacy of IgG1-Campath or IgG1-11B8 variants with Fc-Fc interaction-enhancing mutations is shown. Wien 133 cells were incubated with a range of concentrations of a CD20 and CD52 antibody mixture in the presence of pooled 20% normal human serum (NHS). CDC efficacy is shown as (upper panel) the percentage of lysis, determined by the percentage of propidium iodide (PI)-positive cells, and (lower panel) the area under the dose-response curve, normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-Campath-E430G + IgG1-11B8-E430G (100%). [Figure 20-1]Effect of substitution of amino acids K322, K439, and S440 on CDC efficacy by IgG1-Campath or IgG1-11B8 mutants with Fc-Fc interaction-enhancing mutations. Daudi, Raji, Ramos, REH, U266B1, U-698-M, and Wien 133 cells were incubated with 30.0 μg / mL of CD20 and CD52 antibodies, either as single agents or as a mixture, in the presence of 20% pooled normal human serum (NHS). CDC efficacy is shown as percentage lysis, determined by the percentage of propidium iodide (PI)-positive cells, normalized to the non-binding control antibody IgG1-b12 (0%) and either IgG1-Campath-E430G (100%, for REH, U266B1, and Wien 133 cells) or IgG1-11B8-E430G (100%, for Daudi, Raji, Ramos, and U-698-M cells), depending on which antibody induced the highest degree of lysis. EGE = K322E / E430G / K439E; EGK = K322E / E430G / S440K. [Figure 20-2] See description of Figure 20-1. [Figure 20-3] See description of Figure 20-1. [Figure 20-4] See description of Figure 20-1. [Figure 20-5] See description of Figure 20-1. [Figure 20-6] See description of Figure 20-1. [Figure 20-7] See description of Figure 20-1. [Figure 21A]The effect of substitution of amino acids K322 or P329 on the relative OX40 response of IgG1-SF2 mutants with the Fc-Fc enhancing mutation E345R is shown. Thaw-and-Use GloResponse NFκB-luc2 / OX40 Jurkat cells were incubated for 5 hours with 2.5 μg / mL of antibody in the presence of 5% serum (final) from different sources. Responses in the OX40 assay were recorded by luminescence detected after stimulation of OX40 with anti-OX40 antibody or 1.5 μg / mL of OX40 ligand, which induces expression of a luciferase reporter gene. Luminescence signals were normalized to the response measured in control incubations without antibody (0%) and with OX40 ligand (100%). FBS: fetal bovine serum; NHS: human normal serum; WT: wild-type IgG1-SF2 reference antibody. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 21D] See legend to Figure 21A. [Figure 21E] See legend to Figure 21A. [Figure 22]Sequence alignment using Clustal 2.1 software 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, numbered according to the EU numbering system given in Kabat. The sequences shown are residues 130 to 330 of the human IgG1 heavy chain constant region (SEQ ID NO:1; UniProt Accession No. P01857) and 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:3; UniProt Accession No. P01861). NO:4; Uniprot accession number P01861); and residues 225-428 of the IgE constant region (Uniprot accession number P01854); and residues 133-353 of the IgA1 constant region (Uniprot accession number P01876); and residues 120-340 of the IgA2 constant region (Uniprot accession number P01877); and residues 230-452 of the IgM constant region (Uniprot accession number P01871); and residues 176-384 of the IgD constant region (Uniprot accession number P01880). DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description of the Invention In describing embodiments of the present invention, specific terminology will be used for the sake of clarity. However, it is understood that the present invention is not intended to be limited to the specific terminology so selected, and that each specific term encompasses all technical equivalents that function in a similar manner to accomplish a similar purpose.

[0031] definition The term "parent polypeptide" or "parent antibody" is understood to be a polypeptide or antibody that is identical to a polypeptide or antibody according to the invention, but which has a first mutation that is an Fc-Fc enhancing mutation, for example at position E345, E430, or S440, and as a result has increased Fc-Fc mediated oligomerization, increased Fc effector function, such as CDC, and may also have other enhanced effector functions.

[0032] The term "polypeptide comprising an immunoglobulin Fc region and a binding region" in the context of the present invention refers to a polypeptide comprising an immunoglobulin Fc region and a binding region capable of binding to any molecule, e.g., a polypeptide, present on, for example, a cell, bacterium, or virion. The immunoglobulin Fc region is typically defined as an antibody fragment that can be generated after digestion of an antibody with papain (known to those skilled in the art) and comprises the two CH2-CH3 regions of the immunoglobulin and a connecting region, e.g., a hinge region. The constant domain of the antibody heavy chain defines the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region, together with cell surface receptors called Fc receptors and proteins of the complement system, mediates the effector functions of the antibody. The binding region can be a polypeptide sequence, e.g., a protein, a protein ligand, a receptor, an antigen-binding region, or a ligand-binding region capable of binding to a cell, bacterium, or virion. When the binding region is, for example, a receptor, the "polypeptide comprising an immunoglobulin Fc region and a binding region" may be prepared as a fusion protein of the immunoglobulin Fc region and the binding region. When the binding region is an antigen-binding region, the "polypeptide comprising an immunoglobulin Fc domain and a binding region" may be an antibody such as a chimeric, humanized, or human antibody, or a heavy chain-only antibody, or an ScFv-Fc fusion. A polypeptide comprising an immunoglobulin Fc region and a binding region typically comprises a linking region, such as a hinge region and two CH2-CH3 regions of the immunoglobulin heavy chain. Therefore, a "polypeptide comprising an immunoglobulin Fc region and a binding region" may be a "polypeptide comprising at least an immunoglobulin Fc region and a binding region." The term "immunoglobulin Fc region" in the context of the present invention means the presence of joining regions, such as the hinge and CH2 and CH3 regions, depending on the subtype of immunoglobulin, e.g., human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE antibody. The polypeptide is not limited to human origin, but can be of any origin, such as mouse or cynomolgus monkey origin.

[0033] The terms "Fc-region," "Fc region," "Fc-domain," and "Fc domain," as used herein, are intended to refer to a fragment of the crystallizable region of an antibody. These different terms may be used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the present invention. The term "parent polypeptide" or "parent antibody" is understood to be a polypeptide or antibody that is identical to a polypeptide or antibody according to the present invention, but that lacks a second mutation and has a first mutation that is an Fc-Fc enhancing mutation, for example, at position E345, E430, or S440, such that the parent polypeptide or antibody has increased Fc-Fc mediated oligomerization, increased Fc effector functions, such as CDC, and may also have other enhanced effector functions. As noted above, unless otherwise indicated or clearly contradicted by context, the term "parent polypeptide" or "parent antibody" refers to a polypeptide or antibody that has an Fc-Fc-enhancing first mutation but does not have a second mutation that reduces one or more Fc effector functions. Thus, the polypeptide or antibody contains one or more mutations compared to the "parent polypeptide" or "parent antibody."

[0034] The term "hinge region," as used herein, is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0035] The term "CH2 region" or "CH2 domain," as used herein, is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any other subtype described herein.

[0036] The term "CH3 region" or "CH3 domain," as used herein, is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any other subtype described herein.

[0037] The term "immunoglobulin" refers to a type of structurally related glycoprotein consisting of two pairs of polypeptide chains: one pair of low-molecular-weight light (L) chains and one pair of heavy (H) chains, all four of which are tightly interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. Heavy chains are interconnected by disulfide bonds at the so-called "hinge region." Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in sequence and / or structure-defined loop configurations), also called complementarity-determining regions (CDRs), and interspersed, more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the 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 indicated or contradicted by context, references herein to amino acid positions within constant regions follow EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition - 1991 NIH Publication No.91-3242).

[0038] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either of these, capable of specifically binding to an antigen. The antibody of the present invention comprises an immunoglobulin Fc domain and an antigen-binding region. Antibodies generally comprise two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least an Fc domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule comprise binding domains that interact with antigens. The constant or "Fc" region of an antibody can mediate the binding of the immunoglobulin to host tissues or elements, such as various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., C1q, the first component of the classical pathway of complement activation. The antibody may also be a multispecific antibody, e.g., a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be present on the same target or on different targets. When epitopes are present on different targets, such targets may be present on the same cell or on different cells or cell types. As noted above, unless otherwise specified or clearly contradicted by the context, the term antibody as used herein encompasses antibody fragments that contain at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be obtained by any known method, such as enzymatic cleavage, peptide synthesis, and recombinant expression. It has been shown that the antigen-binding function of an antibody can also be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies consisting of only two heavy chains, such as those naturally occurring in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche,WO2011069104), asymmetric strand-exchange engineered domains (SEED or Seed-body) and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), dual variable domain immunoglobulins (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); dual domain double-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-Diabody (ImClone / Eli Lilly), Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), Electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), dual targeting domain antibodies (GSK / Domantis), two-in-one antibodies or dual action Fabs that recognize two targets (Genentech,NovImmune, Adimab), cross-linked Mab (Karmanos Cancer Center), covalent fusion mAb (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecific (ImClone / Eli Lilly, Shen, J., et al.J Immunol Methods, 2007.318(1-2):p.65-74), TIG-body, DIG-body and PIG-body (Pharmabcine), dual affinity retargeting molecule (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybody (Zyngenia), a generic light chain approach (Crucell / Merus, US7262028) or a generic heavy chain approach (κλBody by NovImmune, WO2012023053), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment comprising an Fc domain, such as scFv fusions, e.g., BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009.393(3):p.672-92), scFv fusions by Genetech / Roche, scFv fusions by Novartis, scFv fusions by Immunomedics, scFv fusions by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb by f-Star, 2(WO2008 / 003116), and double scFv fusions. It should also be understood that the term antibody, unless otherwise specified, encompasses polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies) obtained by techniques such as those used by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. Potentially, the antibodies produced can have any isotype.

[0039] The term "full-length antibody," as used herein, refers to an antibody that includes all of the heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that isotype.

[0040] 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 somatic mutations in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0041] The term "chimeric antibody," as used herein, refers to an antibody in which both chain types are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (whether of non-human species origin, or synthetic or engineered from any species, including humans) linked to a constant region of human origin. The variable domain of a chimeric chain, when analyzed as a whole, has a V-region amino acid sequence that is closer to that of the non-human species than to that of humans.

[0042] The term "humanized antibody," as used herein, refers to an antibody in which both chain types have been humanized as a result of antibody engineering. Humanized chains are typically chains in which the complementarity-determining regions (CDRs) of the variable domains are foreign (nonhuman or synthetic in origin), while the remainder of the chain is of human origin. Because the assessment of humanization is based on the resulting amino acid sequence, not the methodology itself, protocols other than grafting can be used. The variable domains of humanized chains, when analyzed as a whole, have V-region amino acid sequences that are more human than those of other species. The terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like, as used herein, refer to a preparation of Ab molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an Ab exhibiting a single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas, which comprise B cells obtained from transgenic or transchromosomic non-human animals, e.g., transgenic mice, whose genomes have been rearranged to produce functional human antibodies and contain human heavy chain and light chain transgene repertoires, and fused with immortalized cells.

[0043] The term "isotype," as used herein, refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotypes thereof, e.g., IgG1m(za) and IgG1m(f)) that is encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be associated with either a kappa (κ) or lambda (λ) light chain. The term "mixed isotype," as used herein, refers to an immunoglobulin Fc region obtained by combining structural forms of one isotype with analogous regions from another isotype, thereby generating a hybrid isotype. A mixed isotype may comprise an Fc region having a sequence composed of two or more isotypes selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, thereby producing combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0044] The terms "antigen-binding region," "region that binds to an antigen," "binding region," or antigen-binding domain, as used herein, refer to the region of an antibody capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody. The VH and VL domains can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in the form of sequence- and / or structure-defined loops), also called complementarity-determining regions (CDRs), and interspersed, more conserved regions called framework regions (FRs). An antigen can be any molecule, e.g., a polypeptide, present on, for example, a cell, bacterium, or virion.

[0045] The term "target," as used herein, refers to a molecule to which the antigen-binding region of an antibody binds. Targets include any antigen against which an antibody is generated. The terms "antigen" and "target" are used interchangeably in reference to antibodies and may constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0046] The term "epitope" refers to a protein determinant capable of specific binding to an antibody variable domain. Epitopes usually consist of surface groupings of molecules, such as amino acids, sugar side chains, or a combination thereof, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes may include amino acid residues directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues not directly involved in binding.

[0047] The term "antibody variant" or "variant of a parent antibody" of the present invention refers to an antibody molecule containing one or more mutations compared to a "parent antibody." These different terms are used interchangeably and may have the same meaning and purpose with respect to any aspect or embodiment of the present invention. Similarly, a "variant of a polypeptide comprising an immunoglobulin Fc region and a binding region" or a "variant of a polypeptide comprising an immunoglobulin Fc region and a binding region" of the present invention refers to a "polypeptide comprising an immunoglobulin Fc region and a binding region" that contains one or more mutations compared to a "polypeptide comprising an Fc region and a binding region of a parent immunoglobulin." These different terms are used interchangeably and may have 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 parent amino acid sequence. Amino acid substitutions may replace a natural amino acid with another naturally occurring amino acid or a non-naturally occurring amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within amino acid classes shown in one or more of the following three tables:

[0048] Classes of amino acid residues for conservative substitutions TIFF2025124873000001.tif52135

[0049] Classes of alternative conservative amino acid residue substitutions TIFF2025124873000002.tif39135

[0050] Alternative physical and functional classifications of amino acid residues TIFF2025124873000003.tif70139

[0051] In the context of the present invention, substitutions in the variants are: Original amino acid - position - substituted amino acid As shown; Amino acid residues are indicated using three-letter or one-letter codes, including the codes Xaa and X. Thus, the notation "E345R" or "Glu345Arg" means that the variant contains a substitution of arginine for glutamic acid at the amino acid position in the variant that corresponds to amino acid 345 of the parent antibody.

[0052] If the position itself is not present in the antibody, but the variant comprises an amino acid insertion, e.g. Use the notation position-substituted amino acid, e.g., "448E."

[0053] Such designations are particularly relevant in the context of one or more modifications in a series of homologous polypeptides or antibodies.

[0054] Similarly, if the identity of the substituted amino acid residue or residues is not important, The original amino acid position is represented as "E345".

[0055] Modifications in which one or more of the original amino acids and / or one or more substituted amino acids may include more than one but not all amino acids, such as when glutamic acid at position 345 becomes arginine, lysine, or tryptophan; "Glu345Arg,Lys,Trp" or "E345R,K,W" or "E345R / K / W" or "E345 to R, K or W" may be used interchangeably in the context of the present invention.

[0056] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids or with other amino acids, e.g., unnatural amino acids. For example, substitution of amino acid E at position 345 encompasses each of the substitutions 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345P, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is equivalent to the designation 345X, where X represents any amino acid. Such substitutions may also be designated as E345A, E345C, etc., or E345A, C, etc., as well as E345A / C / , etc. The same applies equally to any position described herein, and any one of such substitutions is specifically encompassed herein.

[0057] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, as opposed to the recognition or activation phase of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, e.g., cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, e.g., neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and exert specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing ADCC. For example, FcR-expressing monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells are involved in the specific killing of target cells and antigen presentation to other components of the immune system, or binding to cells that present antigens. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation delivers C3 fragments onto target cells. Such C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors, such as cytokines. For example, FcγRI expression has been shown to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose and dissolve target cells.In some embodiments, antibody-driven classical complement activation produces C3 fragments on target cells.These C3 cleavage products can directly promote the phagocytosis of effector cells or indirectly promote antibody-mediated phagocytosis.

[0058] The term "Fc effector function," as used herein, is intended to refer to a function that results from the binding of a polypeptide or antibody to its target, e.g., an antigen, on a cell membrane, where the Fc effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) binding of an opsonizing antibody to a complement receptor mediated by the antibody, (x) opsonization, and (xi) any combination of (i)-(x).

[0059] The term "reduced one or more Fc effector functions," as used herein, is intended to refer to a reduction in the Fc effector functions of a polypeptide or antibody when compared directly to the Fc effector function(s) of the parent polypeptide or antibody in the same assay.

[0060] The term "clustering-dependent function," as used herein, is intended to refer to a function that is the result of the formation of an antigen complex following oligomerization of a polypeptide or antibody bound to an antigen, optionally on a cell, cell membrane, virion, or another particle. Examples of clustering-dependent effector functions include (i) antibody oligomerization, (ii) antibody oligomer stability, (iii) antigen oligomerization, (iv) antigen oligomer stability, (v) induction of apoptosis, (vi) proliferation modulation, e.g., reducing, inhibiting, or stimulating proliferation, (vii) signal transduction modulation, e.g., reducing, inhibiting, or stimulating protein phosphorylation, and (viii) any combination of (i)-(vii).

[0061] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of directing transcription of a nucleic acid segment ligated into it. One type of vector is a "plasmid," which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment may be ligated into the viral genome. Some 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 mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0062] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because some modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphoid cells, as well as prokaryotic cells, such as Escherichia coli (E. coli), and other eukaryotic hosts, such as plant cells and fungi.

[0063] The term "transfectoma," as used herein, includes recombinant eukaryotic host cells, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungal, e.g., yeast cells, expressing an Ab or a target antigen.

[0064] The term "preparation" refers to a preparation of antibody variants and mixtures of different antibody variants that may have increased ability to oligomerize when interacting with an antigen associated with a cell, cell membrane, virion, or other structure (e.g., an antigen expressed on the cell surface), resulting in enhanced signaling and / or activation by the antigen.

[0065] As used herein, the term "affinity" refers to the strength of binding of one molecule, e.g., an antibody, to another, e.g., a target or antigen, at a single site, e.g., the strength of monovalent binding of an individual antigen-binding site of an antibody to an antigen.

[0066] As used herein, the term "avidity" refers to the total strength of binding between two structures, for example, between multiple antigen-binding sites of an antibody that simultaneously interact with a target, or between multiple binding sites, for example, an antibody and C1q. When there is more than one binding interaction, the two structures will only dissociate when all binding sites dissociate, and therefore the dissociation rate will be slower than for individual binding sites, resulting in a greater effective total binding strength (avidity) compared to the strength with which each individual binding site binds (affinity).

[0067] As used herein, the term "oligomer" refers to a molecule (e.g., an antibody) that is composed of more than one but a limited number of monomer units, as opposed to a polymer that is composed of, at least in principle, an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to denote the number of monomer units in an oligomer; for example, a tetramer is composed of four units, and a hexamer is composed of six units.

[0068] The term "oligomerization," as used herein, is intended to refer to the process of converting monomers to a finite degree of polymerization. It is observed herein that polypeptides, antibodies, and / or other dimeric proteins comprising a target-binding region according to the present invention can form oligomers, e.g., hexamers, through non-covalent association of Fc regions after target binding, e.g., on the cell surface. Antibody oligomerization can be assessed, for example, in a cell viability assay using an anti-DR5 antibody comprising an Fc-Fc-enhancing mutation, e.g., E430G or E345R (as described in Example 13). Fc-Fc-mediated oligomerization of polypeptides or antibodies occurs through intermolecular association of Fc regions between adjacent polypeptides or antibodies after target binding on the (cell) surface, and is enhanced by introducing a first mutation to the amino acid corresponding to E430, E345, or S440 (provided that the mutation at S440 is S440Y or S440W). Thus, the formation of Fc-Fc-mediated oligomerization upon target binding on the (cell) surface can be measured in an assay using the peptide DCAWHLGELVWCT, which blocks Fc-Fc interactions. Induction of oligomerization can be assessed by comparing the responses of the following groups in the assay: Group I) an antibody with a wild-type Fc region; Group II) an antibody identical to the antibody in Group I) except that it contains a first mutation according to the present invention, e.g., E430G; Group III) the DCAWHLGELVWCT peptide in combination with an antibody identical to the antibody in Group I) except that it contains a first mutation according to the present invention, e.g., E430G; and Group IV) an antibody identical to the antibody in Group I) except that it contains a first mutation according to the present invention, e.g., E430G, and a second mutation according to the present invention, e.g., P329D. By comparing the responses of Group I and Group II, it is possible to assess the response of enhanced oligomerization. By comparing the responses of Group II and Group III, it is possible to assess the response of enhanced oligomerization blocking. By comparing the responses of groups II and IV, it is possible to assess whether the enhanced oligomerization is maintained.The appropriate assay for evaluating oligomerization-dependent responses depends on the target antigen to which the antibody binds, as will be apparent to those skilled in the art. Thus, for antibodies that bind to target antigens that induce programmed cell death (PCD), such as TNFR-SF, that have intracellular death domains, e.g., DR5, FAS, DR4, and TNFR1, a suitable assay for examining oligomerization may be a viability assay, as described in Example 13. Viability assays may be performed on BxPC-3 cells in the presence of antibodies according to the above assay groups, i.e., Group I, Group II, Group III, and / or Group IV. BxPC-3 cells are incubated with 5 μg / mL or 10 μg / mL of antibodies according to the above assay groups at 37°C for 3 days. The percentage of viable cells may be determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat. No. G7571). For antibodies that bind to costimulatory immune receptors without death domains, such as TNFR-SF, e.g., OX40, CD40, CD30, CD27, 4-1BB, RANK, and GITR, a suitable assay for examining oligomerization can be an NFAT reporter bioassay. NFAT reporter bioassays can be performed using Jurkat NFAT reporter cells stably expressing a target antigen (which will be apparent to those skilled in the art), such as NFκB-luc2 / OX40 Jurkat cells expressing a luciferase reporter gene under the control of an NFAT response element and having membrane expression of OX40, in the presence of the above-mentioned assay groups, i.e., Group I, Group II, Group III, and / or Group IV. NFκB-luc2 / OX40 Jurkat cells are incubated with 1.5 or 5 μg / mL of an antibody according to the above-mentioned assay group at 37°C for one day. Luciferase expression induced by OX40 activation can be measured by measuring luminescence signals.

[0069] The term "clustering," as used herein, is intended to refer to the oligomerization of antibodies, polypeptides, antigens, or other proteins through non-covalent interactions.

[0070] The term "Fc-Fc enhancement," as used herein, is intended to refer to increasing the binding strength between Fc regions of polypeptides or stabilizing the interaction between Fc regions, such that two Fc region-containing antibodies or polypeptides form oligomers after target binding.

[0071] 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 an antigen. It is understood that antibody binding to an antigen, as described herein, occurs both in vivo and in vitro. C1q binding can be assessed, for example, by using an antibody immobilized on an artificial surface or by using an antibody bound to a predetermined antigen on a cell or virion surface (as described in Examples 3 and 11). C1q binding to an antibody oligomer is understood herein to be a multivalent interaction resulting in high-avidity binding. For example, reduced C1q binding due to the introduction of a second mutation into a polypeptide or antibody can be measured by comparing the C1q binding of the polypeptide or antibody to the C1q binding of its parent polypeptide or antibody without the second mutation in the same assay, as exemplified in Example 3. Briefly, cells of appropriate origin expressing the target antigen bound by the antigen-binding region of the antibody can be used in this assay, and such cell lines or cell types will be apparent to those skilled in the art. Thus, for antibodies that bind to a target antigen on cancer cells, such as DR5, cancer cells such as BxPC-3 human pancreatic cancer cells (ATCC CRL-1687) may be suitable for the assay. On the other hand, for antibodies that bind to OX-40 expressed on T cells, T cells such as Jurkat human T cells (ATCC TIB-152) may be suitable for the assay. The reduced C1q binding of antibodies according to the present invention is 1×10 6The binding activity of the antibody can be evaluated by incubating appropriate cells at a concentration of 1000 μg / mL in a 96-well round-bottom polystyrene plate with: i) a range of concentrations (0.0003-100 μg / mL) of an antibody comprising the first and second mutations according to the present invention in the presence of 20% C4-depleted serum; and ii) a range of concentrations (0.0003-100 μg / mL) of a parent antibody comprising the first mutation but not the second mutation in the presence of 20% C4-depleted serum, wherein the antibodies in i) and ii) are incubated with the appropriate cells for 30 minutes at 4°C, followed by incubation with a labeled anti-human C1q antibody, e.g., FITC-labeled rabbit anti-HuC1q, and C1q binding is measured by flow cytometry. Alternatively, the reduced C1q binding of the antibodies of the present invention can be measured in an enzyme-linked immunosorbent assay (ELISA) for C1q binding by coating (in 100 μL of PBS) 96-well plates with i) serial dilutions (from 0.001 to 20 μg / mL) of antibodies containing the first and second mutations of the present invention; and ii) serial dilutions (from 0.001 to 20 μg / mL) of antibodies containing the first mutation but not the second mutation, respectively, at 4° C. overnight, followed by subsequent incubations with 200 μL / well of 0.5×PBS supplemented with 0.025% Tween 20 and 0.1% gelatin for 1 hour at room temperature (blocking), with washing between incubations, and 100 μL of 3% NHS (Sanquin, The reaction is carried out at 37°C for 1 hour with 100 μL of rabbit anti-human C1q (DAKO, Cat. No. A0136, 1 / 4,000) for 1 hour at room temperature, and with 100 μL of swine anti-rabbit IgG horseradish peroxidase (HRP) (DAKO, Cat. No. P0399, 1 / 10,000) as detection antibody for 1 hour at room temperature; finally, with 100 μL of substrate containing 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Cat. No. 11112 597001) for approximately 15 minutes at room temperature; the reaction is stopped by adding 100 μL of 2% oxalic acid and can be evaluated by measuring the absorbance at 405 nm.

[0072] As used herein, the term "complement activation" refers to activation of the classical complement pathway, initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on a surface. C1 is a complex consisting of six recognition proteins, C1q, and the serine protease heterotetramer C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions beginning with the cleavage of C4 to C4a and C4b and C2 to C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves complement component C3 to C3b and C3a, forming C5 convertase. The C5 convertase cleaves C5 into C5a and C5b, the latter of which is deposited on the membrane, triggering the later events of complement activation, in which terminal complement components C5b, C6, C7, C8, and C9 are assembled into the membrane attack complex (MAC). The complement cascade results in the generation of pores that cause cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be assessed by using C1q availability, CDC kinetics, CDC assays (as described in WO2013 / 004842, WO2014 / 108198), or by cellular deposition of C3b and C4b, as described in Beurskens et al., April 1, 2012, vol. 188, no. 7, pp. 3532-3541.

[0073] The term "complement-dependent cytotoxicity" ("CDC"), as used herein, is intended to refer to the process of antibody-mediated complement activation, in which pores in the membrane created by the MAC construct result in lysis of antibodies bound to targets on cells or virions. CDC can be assessed by in vitro assays, for example, by CDC assays in which normal human serum is used as a complement source, as described in Examples 2, 3, 4, and 6, or over a range of C1q concentrations. For example, reduced CDC activity due to the introduction of a second mutation into a polypeptide or antibody can be measured by comparing the CDC activity of the polypeptide or antibody to the CDC activity of its parent polypeptide or antibody lacking the second mutation in the same assay, as exemplified in Examples 3 and 4.

[0074] The term "antibody-dependent cell-mediated cytotoxicity" ("ADCC"), as used herein, is intended to refer to the mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of the bound antibody. ADCC can be measured using methods such as the ADCC assay described in Example 10 or the Luminescent ADCC Reporter BioAssay described in Example 9. For example, reduced ADCC activity due to the introduction of a second mutation into a polypeptide or antibody can be measured by comparing the ADCC activity of the polypeptide or antibody to the ADCC activity of its parent polypeptide or antibody without the second mutation in the same assay, as exemplified in Examples 10 and 9.

[0075] The term "antibody-dependent cellular phagocytosis" ("ADCP"), as used herein, is intended to refer to the mechanism of removal of antibody-coated target cells or virions by internalization by phagocytes. The internalized antibody-coated target cells or virions are encapsulated in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes. ADCP can be assessed by using an in vitro cytotoxicity assay using macrophages as effector cells and video microscopy, as described by van Bij et al. in Journal of Hepatology, Volume 53, Issue 4, October 2010, Pages 677-685.

[0076] The term "complement-dependent cytotoxicity" ("CDCC"), as used herein, is intended to refer to the mechanism of killing of target cells or virions by cells expressing complement receptors that recognize cleavage products of the third component of complement (C3) that is covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC can be assessed in a manner similar to that described for ADCC.

[0077] The term "plasma half-life," as used herein, refers to the time it takes for the concentration of a polypeptide in plasma to decrease to half of its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase is typically 1-3 days, during which there is an approximately 50% decrease in plasma concentration due to redistribution between plasma and tissues. Plasma half-life can be measured by methods well known in the art.

[0078] The term "plasma clearance rate," as used herein, is a quantitative measure of the rate at which a polypeptide is removed from the blood when administered to a living organism. The plasma clearance rate can be calculated as Dose / AUC (mL / day / kg), where the AUC value (area under the curve) is determined from the concentration-time curve.

[0079] The term "antibody-drug conjugate," as used herein, refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that couples the drug to, for example, the antibody. The linker is cleavable or non-cleavable in the presence of malignant cells; in this case, the antibody-drug conjugate kills the malignant cells.

[0080] The term "antibody-drug conjugate uptake," as used herein, refers to the process by which an antibody-drug conjugate binds to a target on a cell and is then internalized / encapsulated by the cell membrane, thereby being drawn into the cell. Antibody-drug conjugate uptake can be assessed as described in WO 2011 / 157741, "Antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay."

[0081] The term "apoptosis," as used herein, refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events can lead to characteristic cellular changes (morphology) and death. These changes include cytoplasmic blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis can be induced by antibody binding to certain receptors.

[0082] As used herein, the term " programmed cell death " or " PCD " refers to any form of cell death mediated by intracellular program.Different forms of PCD exist, and the commonality of various types of PCD is that they are carried out by active cellular process, which can be disrupted by interfering with intracellular signal transduction.In a specific embodiment, the occurrence of any form of PCD in cell or tissue can be examined by staining cell or tissue with conjugated Annexin V, and correlating with phosphatidylserine exposure.

[0083] The term "annexin V," as used herein, refers to a protein of the annexin family that binds to phosphatidylserine (PS) on the cell surface.

[0084] Fc receptor binding can be measured indirectly as described in Example 9. Fc receptor binding can be measured directly as described in Example 21. For example, a reduction in Fc receptor binding due to the introduction of a second mutation into a test antibody or polypeptide can be measured by comparing the ADCC activity of the polypeptide or antibody to the ADCC activity of its parent polypeptide or antibody without the additional mutation in the same assay, as exemplified in Example 21.

[0085] The terms "Fc gamma receptor," "Fc gamma R," "Fc gamma receptor," and "FcγR" may be used interchangeably herein to describe the Fc gamma receptor class. This receptor class includes several family members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different molecular structures and therefore different antibody affinities. FcγRI binds IgG more strongly than FcγRII or FcγRIII.

[0086] The term "FcRn," as used herein, is intended to refer to the neonatal Fc receptor, an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk through the intestinal epithelium of neonatal rodents into the neonatal bloodstream. Further studies have revealed a similar receptor in humans. However, in humans, it is found in the placenta, where it helps facilitate the transport of maternal IgG to the developing fetus and has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at an acidic pH of 6.0–6.5 but not at neutral or higher pH. Thus, FcRn can bind IgG from the intestinal lumen (inside the intestine), which has a slightly acidic pH, ensuring efficient unidirectional transport to the basolateral side (inside the body), where the pH is neutral to basic (pH 7.0–7.5). This receptor also plays a role in IgG recycling in adults by being present in the endocytic pathway within endothelial cells. FcRn receptors in acidic endosomes bind internalized IgG by pinocytosis, recycling it to the cell surface and releasing it into the basic pH of the blood, thereby preventing lysosomal degradation, which may explain the longer half-life of IgG in the blood compared with other isotypes.

[0087] The term "Protein A," as used herein, is intended to refer to a 56-kDa MSCRAMM surface protein first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cell osmolality, and a two-component system called ArlS-ArlR. It has found utility in biochemical studies due to its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains folded into a three-helix bundle. Each domain has the ability to bind proteins from many mammalian species, most notably IgG. It binds to the Fc region of most immunoglobulin heavy chains (overlapping with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. This interaction in serum allows IgG molecules to bind to bacteria via their Fc region, rather than their Fab region alone, thereby disrupting opsonization, complement activation, and phagocytosis.

[0088] The term "protein G," as used herein, is intended to refer to an immunoglobulin-binding protein expressed by group C and group G streptococci, which is similar to, but has different specificity than, protein A. It is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) cell surface protein that has found use in antibody purification due to its binding to the Fc region.

[0089] Specific Aspects of the Invention The present invention is based on the discovery of a need for therapeutic polypeptide and antibody agents that have enhanced Fc-Fc interactions when bound to their corresponding antigen on the surface of a target cell, and thus form oligomers upon binding to the antigen, but lack the enhanced Fc effector functions, e.g., CDC and / or ADCC, typically seen in polypeptides and antibodies that form oligomers, e.g., hexamers, upon binding. Surprisingly, the inventors have found that by introducing a second mutation corresponding to amino acid position K322 or P329 into the Fc region of a polypeptide or antibody bearing a first mutation corresponding to one of amino acid positions E430, E345, or S440, enhanced Fc-Fc interactions can be maintained, but Fc effector functions, e.g., CDC and / or ADCC, can be reduced compared to the parent of the same polypeptide or antibody bearing only the first mutation but not the second mutation. In some embodiments, one or more effector functions can be reduced to levels below those seen in a wild-type polypeptide or antibody, i.e., an otherwise identical polypeptide or antibody lacking the first and second mutations.

[0090] In some embodiments, the introduction of the second mutation reduces Fc effector function to a level equal to or lower than that seen for the wild-type polypeptide or antibody, hi some embodiments, the introduction of the second mutation reduces Fc effector function to a level equal to or lower than that seen for the same antibody or polypeptide having only the first mutation, i.e., the parent polypeptide or antibody.

[0091] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region and an antigen-binding region of a human IgG, wherein the Fc region comprises a CH2 and a CH3 domain, and wherein the Fc region comprises (i) a first mutation and (ii) a second mutation corresponding to the following amino acid positions in human IgG1 according to EU numbering: i. a first mutation at E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; and ii. A second mutation at K322 or P329 The present invention provides a polypeptide or antibody comprising:

[0092] The first mutation according to the present invention, located at one of amino acid positions E430, E345, or S440, results in enhanced Fc-Fc interaction and oligomerization in the polypeptide or antibody. Furthermore, enhanced oligomerization occurs when the antigen-binding region of the polypeptide or antibody is bound to its corresponding target antigen. This enhanced oligomerization results in the formation of oligomers, such as hexamers. The formation of oligomeric structures, such as hexamers, increases the C1q binding avidity of the polypeptide or antibody, thereby enhancing Fc effector functions, such as CDC and / or ADCC. The second mutation according to the present invention, located at one of amino acid positions K322 or P329, reduces Fc effector functions in the polypeptide or antibody. Such reduced Fc effector functions may be, for example, reduced C1q binding or CDC activity. Thus, the second mutation can counteract the enhanced Fc effector function introduced by the first mutation, thereby generating a polypeptide or antibody with enhanced Fc-Fc interaction and oligomerization but without increased Fc effector function. That is, the Fc effector function is reduced compared to a polypeptide or antibody with the first mutation but not the second mutation. In some cases where the wild-type polypeptide or antibody has increased Fc effector function, e.g., CDC, introduction of the first and second mutations may increase the level of oligomerization but reduce the level of CDC to a level lower than that seen in the wild-type polypeptide or antibody. The polypeptides or antibodies of the present invention are particularly advantageous when Fc effector function is undesirable, for example, when activating effector cells.

[0093] In one aspect of the invention, the Fc region does not comprise mutations at amino acid positions corresponding to L234 and L235. That is, in one aspect of the invention, the Fc region comprises wild-type amino acids L and L at positions corresponding to L234 and L235 in human IgG1, where the positions are according to EU numbering.

[0094] In one embodiment of the invention, the Fc region comprises a first and a second mutation, provided that the Fc region comprises L and L at positions corresponding to L234 and L235.

[0095] In one embodiment of the invention, the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. In a preferred embodiment of the invention, the first mutation is selected from E430G or E345K. This provides an embodiment that allows for enhanced oligomerization of the polypeptide or antibody upon binding to an antigen on a cell surface.

[0096] In one embodiment of the invention, a polypeptide comprises at least one mutation that is an Fc-Fc enhancing mutation and at least one mutation that reduces Fc effector function, i.e., in one embodiment of the invention, the polypeptide comprises i) at least one first mutation at an amino acid position corresponding to E430, E345, or S440 (provided that the mutation at S440 is S440Y or S440W), and ii) at least one second mutation at an amino acid position corresponding to K322 or P329.

[0097] In one embodiment, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and one of the first mutations described above may be present in the first and / or second heavy chain. In one embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and the first mutation is present in both the first and second heavy chains. In a preferred embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and the first and second mutations are present in both the first and second heavy chains. In one embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and the first mutation is present in the first and second heavy chains, and the second mutation is present in both the first and second heavy chains.

[0098] In one embodiment of the present invention, the second mutation is selected from the group consisting of K322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y. In one embodiment of the present invention, the second mutation is K322E. This provides an embodiment that allows inhibition of one or more Fc effector functions. In one embodiment, the second mutation reduces the Fc effector function enhanced by the first mutation. In one embodiment, the second mutation partially reduces the Fc effector function enhanced by the first mutation. In one embodiment, the second mutation in the polypeptide or antibody is capable of reducing Fc effector function to a level lower than that seen in a polypeptide or antibody having the first mutation but not the second mutation, i.e., the parent polypeptide or antibody. In one embodiment, the second mutation in the polypeptide or antibody is capable of reducing Fc effector function to a level similar to or lower than that seen in a polypeptide or antibody lacking the first and second mutations, i.e., the wild-type polypeptide or antibody. In one embodiment, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and one of the above-described second mutations is present in the first and / or second heavy chain.

[0099] In one embodiment, the second mutation is selected from the group of K322E, K322D, and K322N and reduces CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is selected from the group of K322E, K322D, and K322N and reduces C1q binding. In one embodiment, the second mutation is K322E and reduces CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is K322E and reduces C1q binding.

[0100] In one embodiment, the second mutation is selected from the group of P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is selected from the group of P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one embodiment, the second mutation is selected from the group of P329R, P329K, P329D, P329E, and P329G and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is P329R and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is P329R and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is P329R and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one embodiment, the second mutation is P329K and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one embodiment, the second mutation is P329D and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one embodiment, the second mutation is P329E and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one embodiment, the second mutation is P329G and reduces ADCC, FcγR binding, CDC, and / or C1q binding.

[0101] In one aspect of the invention the second mutation is P329A. In one aspect the second mutation is P329A and reduces ADCC but not CDC.

[0102] In one aspect of the invention, the second mutation is at position P329, with the proviso that the second mutation is not P329A.

[0103] In one aspect of the invention, the second mutation is at amino acid position P329, with the proviso that the second mutation is not P329A or P329G.

[0104] In a preferred aspect of the invention, the polypeptide or antibody comprises a second mutation which is P329R, with the proviso that the polypeptide or antibody does not comprise mutations at positions corresponding to L234 and L235 in human IgG1.

[0105] In another aspect of the invention, the second mutation is selected from the group consisting of P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y.

[0106] In another aspect of the invention, the second mutation is selected from the group consisting of P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y.

[0107] In another aspect of the invention, the second mutation is selected from the group of P329R, P329K, and P329D.

[0108] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E430 and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0109] In one aspect of the invention, the Fc region comprises a first mutation at an amino acid position corresponding to E430 and a second mutation comprising: (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y and wherein the Fc region comprises L and L at positions corresponding to L234 and L235.

[0110] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E430 and the second mutation is i. K322E, K322D, and K322N, or ii. P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0111] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E430 and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0112] In one aspect of the invention, the first mutation is selected from the group consisting of E430G, E430S, E430F, and E430T, and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0113] In one aspect of the invention, the first mutation is E430G and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0114] In one embodiment of the invention, the Fc region comprises a first mutation that is E430G and a second mutation selected from the group consisting of K322E, P329R, P329K, and P329D, and the Fc region comprises amino acids L and L at positions corresponding to L234 and L235.

[0115] In one embodiment of the present invention, the first mutation is E430G and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E430G and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E430G and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E430G and the second mutation is P329A.

[0116] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E345 and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0117] In one aspect of the invention, the Fc region comprises a first mutation at an amino acid position corresponding to E345 and a second mutation comprising: (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y and wherein the Fc region comprises L and L at positions corresponding to L234 and L235.

[0118] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E345 and the second mutation is i. K322E, K322D, and K322N, or ii. P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0119] In one aspect of the invention, the first mutation is at an amino acid position corresponding to E345 and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0120] In one aspect of the invention, the first mutation is selected from the group consisting of E345K, E345R, and E345Y, and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0121] In one embodiment of the invention, the first mutation is E345K and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0122] In one embodiment of the present invention, the first mutation is E345K and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E345K and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E345K and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E345K and the second mutation is P329A.

[0123] In one aspect of the invention, the first mutation is E430S and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0124] In one embodiment of the present invention, the first mutation is E430S and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E430S and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E430S and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E430S and the second mutation is P329A.

[0125] In one aspect of the invention, the first mutation is E430F and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0126] In one embodiment of the present invention, the first mutation is E430F and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E430F and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E430F and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E430F and the second mutation is P329A.

[0127] In one embodiment of the invention, the first mutation is E430T and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0128] In one embodiment of the present invention, the first mutation is E430T and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E430T and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E430T and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E430T and the second mutation is P329A.

[0129] In one aspect of the invention, the first mutation is E345Q and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0130] In one embodiment of the present invention, the first mutation is E345Q and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E345Q and the second mutation is P329A.

[0131] In one aspect of the invention, the first mutation is E345R and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0132] In one embodiment of the present invention, the first mutation is E345R and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E345R and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E345R and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E345R and the second mutation is P329A.

[0133] In one aspect of the invention, the first mutation is E345Y and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0134] In one embodiment of the present invention, the first mutation is E345Y and the second mutation is K322E. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is K322D. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is K322N. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329H. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329K. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329R. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329D. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329E. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329M. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329F. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329G. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329I. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329L. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329N. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329S. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329T. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329V. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329W. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is E345Y and the second mutation is P329A.

[0135] In one aspect of the invention, the first mutation is selected from the group consisting of S440Y and S440W, and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0136] In one aspect of the invention, the first mutation is selected from the group consisting of S440Y and S440W, and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y and wherein the Fc region comprises L and L at positions corresponding to 234 and 235.

[0137] In one aspect of the invention, the first mutation is selected from the group consisting of S440Y and S440W, and the second mutation is i. K322E, K322D, and K322N, or ii. P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0138] In one aspect of the invention, the first mutation is selected from the group consisting of S440Y and S440W, and the second mutation is (i) K322E, K322D, and K322N, or (ii) P329H, P329K, P329R, P329D, P329E, P329F, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y is selected from one of the group consisting of:

[0139] In one embodiment of the invention, the first mutation is S440W and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0140] In one embodiment of the present invention, the first mutation is S440W and the second mutation is K322E. In one embodiment of the present invention, the first mutation is S440W and the second mutation is K322D. In one embodiment of the present invention, the first mutation is S440W and the second mutation is K322N. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329H. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329K. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329R. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329D. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329E. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329M. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329F. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329G. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329I. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329L. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329N. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329S. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329T. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329V. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329W. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is S440W and the second mutation is P329A.

[0141] In one aspect of the invention, the first mutation is S440Y and the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

[0142] In one embodiment of the present invention, the first mutation is S440Y and the second mutation is K322E. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is K322D. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is K322N. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329H. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329K. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329R. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329D. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329E. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329M. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329F. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329G. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329I. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329L. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329N. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329Q. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329S. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329T. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329V. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329W. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329Y. In one embodiment of the present invention, the first mutation is S440Y and the second mutation is P329A.

[0143] In one embodiment of the present invention, the Fc region comprises one or more additional mutations. The Fc region comprises a CH2 domain, a CH3 domain, and optionally a hinge region. In one embodiment of the present invention, the Fc region comprises one or more additional mutations in the CH2 or CH3 domain. In one embodiment, the one or more additional mutations are present in the CH2 domain. In another embodiment, the one or more additional mutations are present in the CH3 domain.

[0144] In one aspect of the invention, the Fc region comprises: (i) the first mutation, which is an Fc-Fc enhancing mutation; (ii) a second mutation that inhibits one or more Fc effector functions; (iii) a further mutation that suppresses oligomerization between Fc regions having the same further mutation; Includes.

[0145] In one embodiment of the present invention, the Fc region may comprise an additional mutation in the CH3 domain corresponding to K439, or if the first mutation is not present at S440, the additional mutation may be present at S440. In one embodiment of the present invention, the Fc region may comprise an additional mutation in the CH3 domain corresponding to one of positions S440 or K439, with the proviso that the first mutation is not present at S440. A polypeptide or antibody comprising the first and second mutations according to the present invention and an additional mutation at S440, e.g., S440K, does not oligomerize with a polypeptide or antibody comprising an additional mutation at S440, e.g., S440K. A polypeptide or antibody comprising the first and second mutations according to the present invention and an additional mutation at K439, e.g., K439E, does not oligomerize with a polypeptide or antibody comprising a mutation at K439, e.g., K439E. In one embodiment of the present invention, the additional mutation is selected from S440K or K439E. Polypeptides or antibodies containing an additional mutation, K439E or S440K, do not form oligomers with polypeptides containing the same mutation. Without being bound by theory, K439E and S440K can be viewed as complementary mutations, and therefore, an Fc region containing a K439E mutation does not form Fc-Fc interactions with another Fc region containing a K439E mutation. However, an Fc region containing a K439E mutation does form Fc-Fc interactions with another Fc region containing an S440K mutation. The same situation is observed with an Fc region containing an S440K mutation, which does not form Fc-Fc interactions with another Fc region containing an S440K mutation. Therefore, a polypeptide or antibody containing a K439E mutation forms oligomers with a polypeptide or antibody containing an S440K mutation in an alternating pattern.

[0146] In one embodiment of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation at K439 or S440, provided that if the additional mutation is present at S440, the first mutation is not present at S440.

[0147] In one embodiment of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional K439E or S440K mutation, provided that if the additional mutation is S440K, the first mutation is not present at S440; and wherein the Fc region comprises wild-type amino acids L and L at positions corresponding to L234 and L235.

[0148] In one embodiment of the invention, the Fc region comprises (i) a first mutation at an amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, wherein the second and further mutations are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K is selected from the group consisting of:

[0149] In one embodiment of the invention, the Fc region comprises (i) a first mutation at an amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, wherein the second and further mutations are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of: Here, the Fc region contains wild-type amino acids L and L at positions corresponding to L234 and L235.

[0150] In one embodiment of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (ii) a further mutation, wherein the mutations are: (i) E430G, E430S, E430F, and E430T; (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K is selected from the group consisting of:

[0151] In one embodiment of the invention, the Fc region comprises (i) a first mutation at an amino acid position corresponding to E345, and (ii) a second mutation and (iii) a further mutation, wherein the second and further mutations are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of: Here, the Fc region contains wild-type amino acids L and L at positions corresponding to L234 and L235.

[0152] In one embodiment of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, wherein the first mutation, the second mutation, and the further mutation are: (i) E345K, E345R, and E345Y; (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K is selected from the group consisting of:

[0153] In one embodiment of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, wherein the first mutation, the second mutation, and the further mutation are: (i) S440W and S440Y; (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii)K439E is selected from the group consisting of:

[0154] In one embodiment of the present invention, the Fc region comprises an additional mutation that is a hexamerization-inhibiting mutation corresponding to K439E or S440K (according to EU numbering) in human IgG1. That is, in one embodiment of the present invention, the Fc region comprises a hexamerization-enhancing mutation, for example, E430G, and a hexamerization-inhibiting mutation, for example, K439E. In one embodiment of the present invention, the Fc region comprises a hexamerization-enhancing mutation, for example, E345K, and a hexamerization-inhibiting mutation, for example, K439E. In another embodiment of the present invention, the Fc region comprises a hexamerization-enhancing mutation, for example, E430G, and a hexamerization-inhibiting mutation, for example, S440K. In one embodiment of the present invention, the Fc region comprises a hexamerization-enhancing mutation, for example, E345K, and a hexamerization-inhibiting mutation, for example, S440K. This provides an embodiment that enables exclusive hexamerization between the combination of an antibody comprising the K439E mutation and an antibody comprising the S440K mutation.

[0155] The polypeptide or antibody according to the present invention comprises at least the first and second mutations, but may also comprise further mutations to introduce further functionality into the polypeptide or antibody, as described above, hi one embodiment the Fc region comprises up to 10 mutations, such as 9 mutations, for example 8 mutations, for example 7 mutations, for example 6 mutations, for example 5 mutations, for example 4 mutations, for example 3 mutations, or for example 2 mutations.

[0156] This provides an embodiment that allows the polypeptides or antibodies of the present invention to have additional mutations that introduce additional characteristics into the polypeptides or antibodies. Furthermore, the additional mutations also allow for variation at positions within the Fc region that are not involved in Fc-Fc interactions as well as positions that are not involved in Fc effector function. Furthermore, the additional mutations may be due to allelic variation.

[0157] In one embodiment of the invention, the polypeptide or antibody has an Fc effector function that is reduced by at least 20% compared to a parent polypeptide or antibody that is identical to the antibody but lacking the second mutation. That is, in a polypeptide or antibody having a first and second mutation, the second mutation has the effect of reducing the effector function of the polypeptide or antibody by at least 20% compared to a parent polypeptide or antibody having only the first mutation. In another embodiment of the invention, the polypeptide or antibody has an Fc effector function that is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a parent polypeptide or antibody having only the first mutation.

[0158] In one embodiment of the invention, the polypeptide or antibody does not induce Fc effector function.

[0159] In one aspect of the present invention, it is understood that the reduced Fc effector function or activity of a polypeptide having a first and second mutation is when compared to a parent polypeptide having an identical antigen-binding region and an Fc region with the same first mutation, but without the second mutation in the Fc region.

[0160] In another aspect of the present invention, it is understood that the reduced Fc effector function or activity of a polypeptide having the first and second mutations is when the polypeptide is compared to a parent polypeptide, i.e., a wild-type antibody, which has the same antigen-binding region and Fc region but does not have the first and second mutations in the Fc region.

[0161] In one embodiment according to the present invention, the second mutation reduces at least one effector function. In one embodiment according to the present invention, the second mutation reduces more than one effector function. In one embodiment according to the present invention, the second mutation reduces CDC activity. In one embodiment according to the present invention, the second mutation reduces ADCC activity. In another embodiment, the second mutation reduces CDC and ADCC activity. In one embodiment according to the present invention, the second mutation reduces FcγRIIIa signaling. In a further embodiment according to the present invention, the second mutation reduces CDC activity but does not reduce ADCC activity or FcγRIIIa signaling. That is, in some embodiments according to the present invention, the second mutation reduces one or more effector functions while having no reducing effect on other effector functions. In one embodiment according to the present invention, the second mutation reduces CDC activity but still retains considerable ADCC activity.

[0162] In one embodiment of the present invention, the Fc effector function is selected from the group consisting of complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis, C1q binding, and FcγR binding. In one embodiment, the Fc effector function is FcγRIIIa signaling. That is, the second mutation according to the present invention can reduce at least one Fc effector function.

[0163] Some secondary mutations exhibit reduced effector functions. Certain mutations that reduce CDC activity also exhibit reduced ADCC activity and reduced FcγRIIIa binding. Such mutations include mutations selected from the group including P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y. However, other mutations that retain CDC activity but reduce FcγRIIIa binding and reduce ADCC activity have also been found. Such mutations include those in the group including P329A. Some secondary mutations, such as P329R and P329K, did not exhibit FcγRIa binding. Some secondary mutations, such as P329G and P329A, showed some reduction in binding to FcγRIa binding.

[0164] This provides novel polypeptide- or antibody-based therapeutic agents with reduced Fc effector function. The present invention also provides the ability of Fc-Fc-enhanced polypeptides or antibodies to exert Fc effector function more selectively.

[0165] In one aspect of the invention, the polypeptide is an antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody. In one aspect, the polypeptide is a monospecific polypeptide, a bispecific polypeptide, or a multispecific polypeptide.

[0166] The polypeptides of the present invention are not limited to antibodies with natural, e.g., human, Fc domains, but may also be antibodies with mutations other than those of the present invention, such as mutations affecting glycosylation or mutations that enable the antibody to become a bispecific antibody. The term "natural antibody" refers to any antibody that does not contain any genetically introduced mutations. It is therefore understood that antibodies containing naturally occurring modifications, e.g., different allotypes, are "natural antibodies" within the meaning of the present invention and can thereby be understood as parent antibodies. Such antibodies can serve as templates for one or more mutations according to the present invention, thereby providing mutant antibodies of the present invention. An example of a parent antibody containing mutations other than those of the present invention is the bispecific antibody described in WO2011 / 131746 (Genmab), which uses reducing conditions to promote half-molecule exchange between two antibodies containing IgG4-like CH3 regions, thus forming a bispecific antibody without the concomitant formation of aggregates. Other examples of parent antibodies include, but are not limited to, bispecific antibodies, such as heterodimeric bispecifics: Triomab (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); knob-into-hole (Genentech); electrostatic steering (Amgen, Chugai, Oncomed); SEEDbody (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof.

[0167] The polypeptide or antibody can be any human antibody of any isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA, optionally a full-length human antibody, e.g., a full-length human IgG1 antibody. In one embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment disclosed in Figure 22, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:1, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:2, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:3, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:4, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:5, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:6, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein.In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:7, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:8, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:9, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein. In one embodiment of the invention, a polypeptide or antibody comprises an Fc region comprising an Fc segment of SEQ ID NO:10, wherein the Fc segment further comprises a first mutation, a second mutation, and / or an additional or third mutation disclosed herein.

[0168] In one aspect of the invention, the polypeptide or antibody is a human IgG1 antibody, for example an IgG1m(za) or IgG1m(f) allotype.

[0169] In one embodiment of the present invention, a polypeptide or antibody has an Fc region that is a human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or a mixed isotype. That is, the Fc region of a polypeptide or antibody according to the present invention has at least first and second mutations introduced into the Fc region that correspond to human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or a mixed isotype. In one embodiment of the present invention, the Fc region is a mixed isotype selected from the group consisting of IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, and IgG3 / IgG4. In a mixed isotype, the Fc region is composed of amino acid sequences from more than one isotype.

[0170] In one embodiment of the invention, the polypeptide or antibody has an Fc region that is human IgG1, IgG2, IgG3, or IgG4.

[0171] In one preferred embodiment of the invention, the polypeptide or antibody has an Fc region that is of the human IgG1 isotype.

[0172] In one embodiment of the invention, the polypeptide or antibody has an Fc region that is of the IgG1m(f), IgG1m(a), IgG1m(z), IgG1m(x) allotype or mixed allotypes.

[0173] In one aspect of the invention, the polypeptide or antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0174] The tumor necrosis factor receptor superfamily (TNFRSF) is a group of cytokine receptors characterized by their ability to bind to ligands from the tumor necrosis factor superfamily (TNFSF) through an extracellular cysteine-rich domain. TNF receptors form trimeric complexes within the plasma membrane. The TNFRSFs included the following 29 proteins: TNFR1 (Uniprot P19438), FAS (Uniprot P25445), DR3 (Uniprot Q93038), DR4 (Uniprot O00220), DR5 (Uniprot O14763), DR6 (Uniprot O75509), NGFR (Uniprot P08138), EDAR (Uniprot Q9UNE0), DcR1 (Uniprot Q14798), DcR2 (Uniprot Q9UBN6), DcR3 (Uniprot O95407), OPG (Uniprot O00300), TROY (Uniprot Q92956), XEDAR (Uniprot Q9HAV5), LTbR (Uniprot P36941), and HVEM (Uniprot Q92956), TWEAKR (Uniprot Q9NP84), CD120b (Uniprot P20333), OX40 (Uniprot P43489), CD40 (Uniprot P25942), CD27 (Uniprot P26842), CD30 (Uniprot P28908), 4-1BB (Uniprot Q07011), RANK (Uniprot Q9Y6Q6), TACI (Uniprot O14836), BLySR (Uniprot Q96RJ3), BCMA (Uniprot Q02223), GITR (Uniprot Q9Y5U5), and RELT (Uniprot Q969Z4) are listed.

[0175] Some TNFRSFs are involved in apoptosis and contain intracellular death domains, such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR. Other TNFRSFs are involved in other signaling pathways, such as proliferation, survival, and differentiation, such as DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT. TNF receptors are expressed in a wide variety of mammalian tissues, especially in leukocytes.

[0176] In one embodiment, the antigen-binding region binds to a member of the TNFR-SF. In one embodiment, the antigen-binding region binds to a member of the TNFR-SF that does not contain an intracellular death domain. In one embodiment, the TNFR-SF is selected from the group of OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. In one embodiment, the TNFR-SF is selected from the group of FAS, DR4, DR4, TNFR1, DR6, DR3, EDAR, and NGFR.

[0177] The polypeptides or antibodies according to the invention may bind to any target, and examples of such targets or antigens according to the invention include, but are not limited to, TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, EDAR, DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, RELT.

[0178] multispecific antibodies In one aspect, the present invention provides a polypeptide or antibody comprising a first Fc region and a first antigen-binding region of human IgG, a second Fc region and a second antigen-binding region of human IgG, wherein the first and second Fc regions comprise (i) a first mutation and (ii) a second mutation and (iii) a third mutation corresponding to the following positions in human IgG1 according to EU numbering: (i) a first mutation at E430, E345, or S440; (ii) a second mutation at K322 or P329; (iii) a third mutation at F405 or K409 Includes; wherein the third mutation differs from the first Fc region and the second Fc region such that if the first Fc region has a mutation at position F405, then the second Fc region has a mutation at K409 (and vice versa); A polypeptide or antibody is provided.

[0179] This provides (iii) an embodiment in which the first Fc region and the second Fc region are not identical because the third mutation is not present in the same position in the first and second Fc regions.

[0180] It will be appreciated that any aspect of the invention described herein may be used in the multispecific antibody aspect described below.

[0181] Thus, in one aspect, the variant of the invention is an antibody selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0182] In one particular embodiment, the bispecific antibody has the format described in WO 2011 / 131746.

[0183] In another aspect, the invention provides a polypeptide or antibody that is a bispecific polypeptide or antibody comprising a first antigen-binding region, a second antigen-binding region, and an Fc region comprising a first CH2-CH3 heavy chain of an immunoglobulin and a second CH2-CH3 heavy chain of an immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and the first and / or second CH2-CH3 heavy chains are (i) 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; (ii) a second mutation selected from the group corresponding to E322E, P329R, P329K, and P329D; Including, the first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of human IgG1; the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of human IgG1, and the third mutation in the first polypeptide is different from the additional mutation in the second polypeptide; Concerning polypeptides or antibodies.

[0184] The bispecific antibodies of the present invention are not limited to a particular format and can be any of those described herein.

[0185] In one particular aspect of the invention, (i) the first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409, e.g., K409R; and (ii) the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405, e.g., F405L.

[0186] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (i) a first mutation corresponding to E430G; (ii) a second mutation selected from the group consisting of E322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329f, P329G, P329I, P329L, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y. Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0187] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (iii) a first mutation corresponding to E430G; (iv) a second mutation corresponding to E322E Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0188] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (i) a first mutation corresponding to E430G; (ii) a second mutation corresponding to P329R Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0189] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (i) a first mutation corresponding to E430G; (ii) a second mutation corresponding to P329K Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0190] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (i) a first mutation corresponding to E430G; (ii) a second mutation corresponding to P329D Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; the second Fc region comprises a third mutation at an amino acid residue corresponding to F405L.

[0191] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (i) the first mutation corresponding to E345K; (ii) a second mutation selected from the group consisting of E322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329f, P329G, P329I, P329L, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y. Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0192] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (iii) a first mutation corresponding to E345K; (iv) a second mutation corresponding to E322E Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0193] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (iii) a first mutation corresponding to E345K; (iv) a second mutation corresponding to P329R Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0194] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (iii) a first mutation selected from the group corresponding to E345K; (iv) a second mutation selected from the group corresponding to P329K; Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0195] In one embodiment of the invention, the first and / or second CH2-CH3 heavy chain comprises: (iii) a first mutation selected from the group corresponding to E345K; (iv) a second mutation selected from the group corresponding to P329D Including, The first CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to K409R; and the second CH2-CH3 heavy chain comprises a third mutation at an amino acid residue corresponding to F405L.

[0196] Methods for reducing Fc effector function of a polypeptide or antibody It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0197] In one aspect, the present invention relates to a method for reducing Fc effector function of a polypeptide or antibody comprising an Fc region and an antigen-binding region of a human immunoglobulin, the Fc region comprising CH2 and CH3 domains, the Fc region comprising a first mutation corresponding to (i) position E430, E345, or S440 in human IgG1 according to EU numbering, and a second mutation corresponding to (ii) position K322 or P329 in human IgG1 according to EU numbering. The first mutation according to the present invention, located at one of positions E430, E345, or S440, introduces the effect of enhancing Fc-Fc interaction of the polypeptide or antibody. The second mutation according to the present invention, located at one of positions K322 or P329, introduces the effect of reducing Fc effector function of the polypeptide or antibody.

[0198] In one embodiment of the present invention, the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, thereby providing an embodiment in which the first mutation enhances Fc-Fc interaction.

[0199] In one preferred embodiment of the invention, the first mutation is selected from E430G or E345K.

[0200] In one aspect, the present invention relates to a method for reducing Fc effector function or activity of a polypeptide or antibody having a first Fc-Fc-enhancing mutation by introducing a second mutation. It is understood that the method for reducing Fc effector function is measured when comparing the polypeptide or antibody to a parent polypeptide or antibody having the same antigen-binding region and Fc region with the same first mutation in the Fc region, but lacking the second mutation in the Fc region. In some aspects, the method for reducing Fc effector function or activity reduces effector function to a level lower than or equivalent to that of a parent polypeptide or antibody having the same antigen-binding region and Fc region, but lacking the first and second mutations in the Fc region.

[0201] In one aspect of the invention, the second mutation is selected from the group consisting of K322E, K322D, K322N, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y.

[0202] In one embodiment of the invention, the method comprises introducing a second mutation selected from the group of K322E, K322D, and K322N that reduces Fc effector function, e.g., CDC, CDCC, and / or C1q binding.

[0203] In one embodiment of the invention, the method comprises introducing a second mutation selected from the group of P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y for reducing Fc effector function, e.g., ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding.

[0204] In one preferred embodiment of the invention, the second mutation is selected from the group of K322E, P329R, P329K, and P329D.

[0205] In one aspect of the invention, the second mutation is at position P329, with the proviso that the second mutation is not P329A.

[0206] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E430G, the method comprising the step of introducing a second mutation corresponding to E322E.

[0207] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E430G, the method comprising introducing a second mutation corresponding to E322D. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E430G, the method comprising introducing a second mutation corresponding to E322N. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E430G, the method comprising introducing a second mutation corresponding to P329H.

[0208] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329K. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329R. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329D. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329E. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329M. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329F. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329G. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329I. In one aspect, the present invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329L.In one aspect, the present invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329N.In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329Q. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329S. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329T. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329V. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329W.In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E430G in its Fc region, the method comprising introducing a second mutation corresponding to P329Y.

[0209] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E430G, the method comprising the step of introducing a second mutation selected from the group consisting of K322E, P329R, P329K, and P329D.

[0210] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising the step of introducing a second mutation corresponding to E322E.

[0211] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E345K, the method comprising introducing a second mutation corresponding to E322D. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E345K, the method comprising introducing a second mutation corresponding to E322N. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E345K, the method comprising introducing a second mutation corresponding to P329H.

[0212] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329K. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329R. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329D. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329E. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329M. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329F. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329G. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329I. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329L.In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329N.In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329Q. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329S. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329T. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329V. In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329W.In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in its Fc region, the method comprising introducing a second mutation corresponding to P329Y.

[0213] In one aspect, the invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation in its Fc region corresponding to E345K, the method comprising the step of introducing a second mutation selected from the group consisting of K322E, P329R, P329K, and P329D.

[0214] In one aspect, the invention relates to a method, wherein the Fc region comprises one or more additional mutations in the CH3 domain.

[0215] In one aspect, the invention relates to a method in which the Fc region comprises an additional mutation in the CH3 domain corresponding to one of positions S440 or K439 in human IgG1 according to EU numbering. In one aspect of the invention, the Fc region comprises an additional mutation in the CH3 domain corresponding to one of positions S440 or K439, with the proviso that if the first mutation is present at S440, then the additional mutation is not present at position S440. A polypeptide or antibody comprising the first and second mutations according to the invention and an additional mutation at position S440, e.g., S440K, does not oligomerize with a polypeptide or antibody comprising a mutation at position S440, e.g., S440K. A polypeptide or antibody comprising the first and second mutations according to the invention and an additional mutation at position K439, e.g., K439E, does not oligomerize with a polypeptide or antibody comprising a mutation at position K439, e.g., K439E. This provides a method that allows the formation of oligomers between polypeptides or antibodies, where the first polypeptide or antibody comprises K439E mutation and the second polypeptide or antibody comprises S440K mutation.In this way, oligomers, such as hexamers, can be forced to form in some specific patterns between the first and second polypeptides.This can be important in methods where polypeptides bind to different targets or epitopes and oligomers should be formed in the combination of these different targets or epitopes.

[0216] In one aspect, the invention relates to a method, wherein said further mutation is selected from S440K or K439E.

[0217] In one aspect, the invention relates to a method of reducing Fc effector function, wherein the Fc effector function is reduced by at least 20% relative to the same parent polypeptide or antibody having the same first mutation but without the second mutation, hi another aspect of the invention, the polypeptide or antibody has an Fc effector function that is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% relative to the parent polypeptide or antibody having only the first mutation.

[0218] In one aspect, the invention relates to a method of reducing an Fc effector function, wherein the Fc effector function is selected from the group of complement dependent cytotoxicity (CDC), complement dependent cell-mediated cytotoxicity (CDCC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding.

[0219] In one aspect, the invention relates to a method of reducing ADCC, wherein the ADCC is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% relative to a comparison antibody that is identical to the antibody but does not contain the second mutation.

[0220] In one aspect, the invention relates to a method of reducing CDC, wherein CDC is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative to a comparison antibody that is identical to the antibody but does not contain the second mutation.

[0221] In one aspect, the invention relates to a method of reducing C1q binding, wherein C1q binding is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% relative to a comparison antibody that is identical to the antibody but does not contain the second mutation.

[0222] In one aspect, the invention relates to a method of reducing Fc gamma receptor binding, wherein Fc gamma receptor binding is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% relative to a reference antibody that is identical to the antibody but does not contain the second mutation.

[0223] In one aspect, the invention relates to a method of reducing Fc gamma receptor binding, wherein Fc gamma receptor binding is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% relative to a comparison antibody that is identical to the antibody but does not contain the first and second mutations.

[0224] In a preferred embodiment, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein Fc gamma receptor I binding is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference antibody that is identical to the antibody but does not contain the second mutation.

[0225] In one preferred aspect, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein Fc gamma receptor I binding is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a comparison antibody that is identical to the antibody but does not contain the first and second mutations.

[0226] In one preferred aspect, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein Fc gamma receptor I binding is reduced by at least 70%, preferably at least 80%, more preferably at least 90% or at least 100% compared to a comparison antibody that is identical to the antibody but does not contain the second mutation.

[0227] In one preferred aspect, the invention relates to a method for reducing Fc gamma receptor I binding, wherein the Fc gamma receptor I binding is reduced by at least 70%, preferably at least 80%, more preferably at least 90%, or at least 100% relative to a comparison antibody that is identical to the antibody but does not contain the first and second mutations. Thus, the method comprises reducing Fc gamma receptor I binding to a reduced level relative to a wild-type Fc region.

[0228] composition It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region having an immunoglobulin CH2-CH3 region and an antigen-binding region; the polypeptide or antibody may also be a multispecific polypeptide or antibody comprising a first antigen-binding region, a second antigen-binding region, and an Fc region comprising a first immunoglobulin CH2-CH3 heavy chain and a second immunoglobulin CH2-CH3 heavy chain.

[0229] The present invention also relates to compositions comprising the polypeptides or antibodies described herein and variant forms thereof. Specific aspects and embodiments are described below. Furthermore, such polypeptides or antibodies can be obtained according to any of the methods described herein.

[0230] In one aspect, the present invention relates to a composition comprising at least one polypeptide or antibody described herein.

[0231] In one embodiment of the invention, a composition comprises one or more polypeptides or antibodies according to any aspect or embodiment described herein.

[0232] In one embodiment of the invention, a composition comprises a first polypeptide or antibody and a second polypeptide or antibody according to any aspect or embodiment herein.

[0233] In one aspect of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies are (i) the first mutation, which is an Fc-Fc enhancing mutation; (ii) a second mutation that inhibits one or more Fc effector functions; (iii) an additional mutation that prevents oligomerization between Fc regions with the same additional mutation, wherein the first and second polypeptides or antibodies do not contain the same additional mutation. The Fc region comprises:

[0234] In one embodiment of the invention, a composition comprises a first polypeptide or antibody and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies comprise i) a first mutation, ii) a second mutation, and iii) an additional mutation, wherein the first and second polypeptides or antibodies do not comprise the same additional mutation. Thus, the composition comprises a first polypeptide or antibody comprising a first Fc region and a second polypeptide or antibody comprising a second Fc region.

[0235] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation at K439 or S440, provided that if the additional mutation is present at S440, the first mutation is not present at S440, and the first and second Fc regions do not contain additional mutations at the same amino acid position.

[0236] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation at K439 in the first Fc region and an additional mutation at S440 in the second Fc region, provided that if the additional mutation is present at S440, the first mutation is not present at S440.

[0237] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation at K439 in the second Fc region and an additional mutation at S440 in the first Fc region, provided that if the additional mutation is present at S440, the first mutation is not present at S440.

[0238] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation K439E in the first Fc region and an additional mutation S440K in the second Fc region.

[0239] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, which correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation S440K in the first Fc region and an additional mutation E439E in the second Fc region.

[0240] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (iii) a further mutation, and the first and / or second Fc region comprises (ii) a second mutation, which mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) a second mutation at E322 or P329; (iii) an additional mutation K439E in the first Fc region and an additional mutation S440K in the second Fc region.

[0241] This provides for embodiments in which either both the first and second polypeptides or antibodies have reduced Fc effector function, or in which only the first polypeptide or only the second polypeptide has reduced Fc effector function.

[0242] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation at an amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0243] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation at an amino acid position corresponding to E345, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0244] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0245] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0246] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G and (ii) a second mutation K322E, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0247] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G and (ii) a second mutation P329K, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0248] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G and (ii) a second mutation P329R, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0249] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E430G and (ii) a second mutation P329D, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0250] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0251] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0252] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K and (ii) a second mutation K322E, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0253] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K and (ii) a second mutation P329K, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0254] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K and (ii) a second mutation P329R, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0255] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345K and (ii) a second mutation P329D, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0256] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P3 29G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0257] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R, and (ii) a second mutation and (iii) a further mutation, wherein the second mutation and the further mutation are: (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0258] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R and (ii) a second mutation K322E, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0259] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R and (ii) a second mutation P329K, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0260] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R and (ii) a second mutation P329R, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0261] In one aspect of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation E345R and (ii) a second mutation P329D, and (iii) a further mutation, wherein the further mutation is (iii) K439E and S440K wherein the first and second Fc regions do not comprise the same additional mutations.

[0262] In another aspect of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies are (i) the first mutation, which is an Fc-Fc enhancing mutation; (ii) an additional mutation that prevents oligomerization between Fc regions with the same additional mutation, wherein the first and second polypeptides or antibodies do not contain the same additional mutation. an Fc region comprising (iii) either the first or second Fc region comprises the second mutation. Thus, in some embodiments, only the first polypeptide or antibody or the second polypeptide or antibody comprises the second mutation that reduces Fc effector function.

[0263] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation at an amino acid position selected from the group consisting of E430, E345, or S440, whereby the mutation at S440 is S440Y or S440W, (ii) a second mutation, and (iii) a further mutation E, which mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (iii) an additional K439E or S440K mutation, wherein the first and second Fc regions do not contain the same additional mutation, and if the first mutation is S440Y or S440W, then the additional mutation is not S440K; (ii) Either the first or second Fc region contains a second mutation at E322 or P329, but not both.

[0264] In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation at an amino acid position selected from the group consisting of E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W, and ii) a second mutation at an amino acid position selected from the group of E322 and P329, and iii) an additional mutation K439E; and the second Fc region comprises i) a first mutation at an amino acid position selected from the group consisting of E430 and E345, and an additional mutation S440K. This provides an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0265] In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first mutation at an amino acid position selected from the group consisting of E430 or E345, ii) a second mutation at an amino acid position selected from the group consisting of E322 and P329, and iii) an additional mutation S440K; and the second Fc region comprises (i) a first mutation at an amino acid position selected from the group consisting of E430, E345, or S440 (provided that the mutation at S440 is S440Y or S440W), and an additional mutation K439E. This provides an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0266] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second E322E mutation, and iii) a further K439E mutation; and the second Fc region comprises i) the first E430G mutation and a further S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0267] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second E322E mutation, and iii) a further S440K mutation; and the second Fc region comprises i) the first E430G mutation and a further K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0268] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329R mutation, and iii) a further K439E mutation; and the second Fc region comprises i) a first E430G mutation and a further S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0269] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329R mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E430G mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0270] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329K mutation, and iii) a further K439E mutation; and the second Fc region comprises i) a first E430G mutation and a further S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0271] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329K mutation, and iii) a further S440K mutation; and the second Fc region comprises i) the first E430G mutation and a further K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0272] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329D mutation, and iii) a further K439E mutation; and the second Fc region comprises i) a first E430G mutation and a further S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0273] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E430G mutation, ii) a second P329D mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E430G mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0274] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second E322E mutation, and iii) an additional K439E mutation; and the second Fc region comprises i) the first E345K mutation and an additional S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0275] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second E322E mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E345K mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0276] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329R mutation, and iii) an additional K439E mutation; and the second Fc region comprises i) a first E345K mutation and an additional S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0277] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329R mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E345K mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0278] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329K mutation, and iii) an additional K439E mutation; and the second Fc region comprises i) a first E345K mutation and an additional S440K mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0279] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329K mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E345K mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0280] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329D mutation, and iii) an additional K439E mutation; and the second Fc region comprises i) a first E345K mutation and an additional S440K mutation. This provides an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0281] In one embodiment of the invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) a first E345K mutation, ii) a second P329D mutation, and iii) an additional S440K mutation; and the second Fc region comprises i) the first E345K mutation and an additional K322E mutation, thereby providing an embodiment in which only the first polypeptide or antibody has reduced Fc effector function.

[0282] In one aspect of the invention, the composition comprises a polypeptide or antibody capable of binding to a member of the tumor necrosis factor receptor superfamily (TNFR-SF).

[0283] In one aspect of the invention, the composition comprises a polypeptide or antibody capable of binding to a member of the TNFR-SF having an intracellular death domain selected from the group consisting of TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, and EDAR.

[0284] In one embodiment of the present invention, the composition comprises a polypeptide or antibody capable of binding to a member of the TNFR-SF that does not have an intracellular death domain selected from the group consisting of DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0285] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a member of the TNFR-SF, which belongs to the group of immune activators consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0286] In one embodiment of the invention, a composition comprises polypeptides or antibodies, wherein a first polypeptide and a second polypeptide bind to different epitopes on one or more members of the TNFR-SF that do not have an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0287] In one embodiment of the invention, a composition comprises a polypeptide or antibody, wherein the binding of a first polypeptide to a member of the TNFR-SF that does not have an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT does not block the binding of a second antibody to a member of the TNFR-SF that does not have an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0288] In one embodiment of the present invention, a composition comprising a first polypeptide or antibody and a second polypeptide or antibody is prepared by dissolving the first polypeptide or antibody in a molar ratio of 1:49 to 49:1 in the composition, such as a 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:49 molar ratio, a 1:50 molar ratio, a 1:60 molar ratio, a 1:70 molar ratio, a 1:80 molar ratio, a 1:90 ... They are present in the following molar ratios: 1:35, 1:40, 1:45, 1:50, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1.

[0289] In one aspect of the invention, the composition comprising the first polypeptide and the second polypeptide and / or any further polypeptides are present in the composition in an equimolar ratio.

[0290] In one embodiment of the invention, the composition according to any aspect or embodiment is a pharmaceutical composition.

[0291] therapeutic use A polypeptide, antibody, bispecific antibody or composition according to any aspect or embodiment of the present invention may be used as a medicament, i.e. for therapeutic applications.

[0292] In one aspect, the present invention provides a polypeptide, antibody, or composition according to any aspect or embodiment disclosed herein for use as a medicament.

[0293] In another aspect, the present invention provides a polypeptide, antibody, or composition according to any aspect or embodiment disclosed herein for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0294] In another aspect, the invention relates to a method of treating an individual having a disease, comprising administering to the individual an effective amount of a polypeptide, antibody, or composition according to any aspect or embodiment disclosed herein.

[0295] In one aspect of the invention, the disease is selected from the group of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0296] In one embodiment of the invention, the method according to any aspect or embodiment disclosed herein relates to further administering an additional therapeutic agent.

[0297] In one aspect of the invention, the additional therapeutic agent is a chemotherapeutic agent (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), a kinase inhibitor (including but not limited to sorafenib, sunitinib, or everolimus), an apoptosis modulator (including but not limited to recombinant human TRAIL or birinapant), a RAS inhibitor, a proteasome inhibitor (including but not limited to and one or more anti-cancer agents selected from the group consisting of: anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR antibodies and antibody mimetics; and antibody-drug conjugates.

[0298] kit-of-parts It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0299] The present invention also relates to a kit of parts comprising the polypeptides or antibodies described herein for simultaneous, separate or sequential use in therapy. Furthermore, such variants may be obtained according to any of the methods described herein.

[0300] In one aspect, the present invention relates to a kit-of-parts comprising a polypeptide, antibody, or composition according to any aspect or embodiment described herein, wherein the polypeptide, antibody, or composition is present in one or more containers, e.g., vials.

[0301] In one aspect of the invention, the kit of parts comprises a polypeptide, an antibody or a composition according to any aspect or embodiment described herein for simultaneous, separate or sequential use in therapy.

[0302] In another aspect, the present invention relates to the use of a polypeptide, antibody, composition, or kit of parts according to any of the embodiments described herein for use in a diagnostic method.

[0303] In another aspect, the present invention relates to a diagnostic method comprising administering a polypeptide, antibody, composition or kit of parts according to any embodiment described herein to at least a part of the body of a human or other mammal.

[0304] In another aspect, the invention relates to the use of a polypeptide, antibody, composition, or kit of parts according to any of the embodiments described herein in imaging at least a portion of the body of a human or other mammal.

[0305] In another aspect, the present invention relates to a method for imaging at least a portion of the body of a human or other mammal, comprising administering a variant, composition, or kit-of-parts according to any embodiment described herein.

[0306] combination Further provided by the invention are preparations of any polypeptide or antibody according to any aspect or embodiment above, i.e., preparations comprising a plurality of polypeptides or antibodies. Also provided by the invention are compositions, e.g., pharmaceutical compositions, comprising a polypeptide or antibody according to any aspect or embodiment above. Also provided by the invention are the use of any such polypeptide or antibody, preparation, or composition as a medicament.

[0307] The present invention also provides combinations of polypeptides or antibodies in which one polypeptide or antibody comprises at least a first and a second mutation according to the invention, as well as preparations and pharmaceutical compositions of such combinations of variants and their use as medicines. Preferably, the two polypeptides or antibodies bind to the same antigen or different antigens that are typically expressed on the surface of the same cell, cell membrane, virion, and / or other particle.

[0308] Conjugates It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0309] In one aspect, the invention relates to a polypeptide or antibody in which the variant is conjugated to a drug, toxin, or radiolabel, e.g., a polypeptide or antibody in which the variant is conjugated to a toxin via a linker.

[0310] In one embodiment, the variant is part of a fusion protein.

[0311] In another aspect, the polypeptide or antibody of the present invention is not conjugated at the C-terminus to another molecule, such as a toxin or label. In one embodiment, the variant is conjugated to another molecule at a different site, typically at a site that does not interfere with oligomerization. For example, the antibody variant may be linked at another site to a compound selected from the group consisting of a toxin (e.g., a radioisotope), a prodrug, or a drug. Such a compound may more effectively kill target cells, for example, in cancer therapy. Thus, the resulting variant is an immunoconjugate.

[0312] Thus, in a further aspect, the present invention provides antibodies linked or conjugated to one or more therapeutic moieties, such as cytotoxins, chemotherapeutic agents, cytokines, immunosuppressants, and / or radioisotopes. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates." Immunoconjugates containing one or more cytotoxins are referred to as "immunotoxins."

[0313] Cytotoxins or cytotoxic agents include any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for forming the immunoconjugates of the invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, maytansine or an analog or derivative thereof, enediyne antitumor antibiotics such as neocarzinostatin, calicheamicin, esperamicin, dynemicin, lidamycin, kedarcidin or its analogs or derivatives, and the like. analogs or derivatives of, anthracyclines, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., 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; and duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin, or an analog or derivative of CC-1065), dolastatins, pyrrolo [2,1-c][1,4]benzodiazepines (PDB) or analogs thereof, antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), antimitotic agents (e.g., tubulin inhibitors), such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10;histone deacetylase inhibitors, such as the hydroxamic acids trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat, and the benzamides entinostat, CI994, mocetinostat, and aliphatic acid compounds, such as phenylbutyrate and valproic acid; proteasome inhibitors, such as danoprevir, bortezomib, amatoxins, such as α-amantin, diphtheria toxin and related molecules (e.g., diphtheria A chain and and active fragments and hybrid molecules thereof; ricin toxins (e.g., ricin A or deglycosylated ricin A chain toxins), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxins, C3 toxins, Shiga toxins, pertussis toxins, tetanus toxins, soybean Bowman-Birk protease inhibitors, Pseudomonas exotoxins, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, 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, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin. See, e.g., Pastan et al., Cell; 47 , 641(1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with the antibodies of the invention described elsewhere herein, such as anti-cancer cytokines or chemokines, are also potential therapeutic moieties useful for conjugation to the antibodies of the invention.

[0314] In one embodiment, the drug conjugate of the invention comprises an antibody disclosed herein conjugated to an auristatin or auristatin peptide analogs and derivatives (US Pat. No. 5,635,483; US Pat. No. 5,780,588). Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and to have anticancer (US Pat. No. 5,663,149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). The auristatin drug moiety can be attached to the antibody at the N-terminus or C-terminus of the peptide drug moiety via a linker.

[0315] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moieties DE and DF, as disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623, presented March 28, 2004, and described in US 2005 / 0238649.

[0316] An exemplary auristatin embodiment is MMAE (monomethylauristatin E). Another exemplary auristatin embodiment is MMAF (monomethylauristatin F).

[0317] In one embodiment, the 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., an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, the antibody of the present invention is conjugated to an aptamer or ribozyme.

[0318] In one embodiment, an antibody is provided that comprises one or more radiolabeled amino acids. Radiolabeled variants can be used for both diagnostic and therapeutic purposes (conjugation with radiolabeled molecules is another possible option). 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 (2 nd See, e.g., Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902. For example, radioisotopes can be conjugated by the chloramine-T method.

[0319] In one embodiment, the polypeptide or antibody of the present invention is conjugated to a radioisotope or a radioisotope-containing chelate. For example, the variant can be conjugated to a chelator linker, such as DOTA, DTPA, or tiuxetan, that allows the antibody to complex with a radioisotope. Alternatively, the variant can also include or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled variants can be used for both diagnostic and therapeutic purposes. In one embodiment, the variant of the present invention is conjugated to an alpha emitter. Non-limiting examples of radioisotopes include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 125 I, 111 In, 131 I, 186 Re, 213 Bs, 225 Ac and 227 Th is an example.

[0320] In one embodiment, a polypeptide or antibody of the invention may 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, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.

[0321] The polypeptides or antibodies of the present invention may also be chemically modified by covalent conjugation with a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching them to peptides are exemplified, for example, in US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000 to about 40,000, e.g., about 2,000 to about 20,000).

[0322] Any method known in the art for conjugating a polypeptide or antibody of the invention to one or more conjugated molecules, such as those described above, e.g., Hunter et al., Nature 144 , 945(1962), David et al., Biochemistry 13 , 1014(1974), Pain et al., J.Immunol.Meth. 40 , 219 (1981) and Nygren, J. Histochem. and Cytochem. 30 , 407 (1982) can be used. Such variants can be prepared by chemically conjugating other moieties to the N- or C-terminus of the variant or a fragment thereof (e.g., the heavy or light chain of an antibody) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, Chijin Shokan Publishing (1994)). Derivatives of such conjugated variants can also be prepared by conjugation at internal residues or sugar chains, as appropriate.

[0323] The above-mentioned agent can be coupled to the polypeptide or antibody of the present invention either directly or indirectly. An example of indirect coupling of a second agent is coupling to a cysteine ​​or lysine residue via a spacer or linker moiety in a bispecific antibody. In one embodiment, the polypeptide or antibody is conjugated via a spacer or linker to a prodrug molecule that can be activated into a therapeutic drug in vivo. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody into the intracellular environment. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). For example, the spacer or linker can be cleavable by tumor cell-binding enzymes or other tumor-specific conditions, thereby generating an active drug. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394, and WO201062171 (by Syntarga BV, et al.). Suitable antibody-prodrug technologies and duocarmycin analogs are also found in U.S. Patent No. 6,989,452 (Medarex), incorporated herein by reference. Alternatively or additionally, the linker may be a peptidyl linker that is cleaved, for example, by an intracellular peptidase or protease enzyme, such as, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).In a specific embodiment, the peptidyl linker cleavable by intracellular proteases is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, for example, US6214345, which describes the synthesis of doxorubicin with a Val-Cit linker and different examples of Phe-Lys linkers). Examples of Val-Cit and Phe-Lys linker structures include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB, or MC-Phe-Lys-GABA, where MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid. The advantage of using proteolytic intracellular release of therapeutic agents is that the agent is typically weakened when conjugated, and the serum stability of the conjugate is typically high.

[0324] In another embodiment, the linker unit is not cleavable, and the drug is released by degradation of the antibody (see US 2005 / 0238649). Typically, such linkers are substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment," in the context of a linker, means that when the mutant antibody drug conjugate compound is present in an extracellular environment (e.g., plasma), no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of the mutant antibody drug conjugate compound are cleaved. Whether a linker is substantially insensitive to the extracellular environment can be measured, for example, by incubating the mutant antibody drug conjugate compound with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures (Ab refers to antibody, and p represents drug loading (or the average number of cytostatic or cytotoxic agents per antibody molecule), which is 1 to about 8; e.g., p can be 4 to 6, e.g., 3 to 5, or p can be 1, 2, 3, 4, 5, 6, 7, or 8):

[0325] Examples of cleavable linkers in combination with auristatins include MC-vc-PAB-MMAF (also denoted as vcMMAF) and MC-vc-PAB-MMAF (also denoted as vcMMAE), where MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for Val-Cit (valine-citrulline)-based linker, and PAB is an abbreviation for p-aminobenzylcarbamate.

[0326] Other examples include auristatins in combination with non-cleavable linkers such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation for maleimidocaproyl).

[0327] In one embodiment, the drug linker moiety is vcMMAE. vcMMAE drug linker moieties and conjugation methods are disclosed in WO2004010957, US7659241, US7829531, US7851437, and US 11 / 833,028 (Seattle Genetics, Inc.), which are incorporated herein by reference, and the vcMMAE drug linker moiety is attached to the antibody at a cysteine ​​using methods similar to those disclosed therein.

[0328] In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation methods are disclosed in US7498298, US 11 / 833,954, and WO2005081711 (Seattle Genetics, Inc.), which are incorporated herein by reference, and the mcMMAF drug linker moiety is attached to the mutant at the cysteine ​​using methods similar to those disclosed therein.

[0329] In one embodiment, the polypeptide or antibody of the invention is linked to a chelator linker, such as tiuxetan, that allows the bispecific antibody to be conjugated to a radioisotope.

[0330] In one embodiment, each arm (or Fab arm) of the polypeptide or antibody is coupled directly or indirectly to the same therapeutic moiety or moieties.

[0331] In one embodiment, only one arm of the antibody is coupled directly or indirectly to one or more therapeutic moieties.

[0332] In one embodiment, each arm of the antibody is coupled, directly or indirectly, to a different therapeutic moiety. For example, in embodiments where the variant is a bispecific antibody and is prepared by controlled Fab arm exchange of two different monospecific antibodies described herein, e.g., a first and a second antibody, such a bispecific antibody can be obtained by using monospecific antibodies that are conjugated or associated with different therapeutic moieties.

[0333] Further Uses It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0334] In a further aspect, the present invention relates to a polypeptide, an antibody of the present invention as described above for use as a medicament, in particular for use as a medicament for the treatment of diseases or disorders, examples of such diseases and disorders including, but not limited to, cancer, and bacterial, viral, or fungal infections.

[0335] In another aspect, the present invention relates to the polypeptides, antibodies, bispecific antibodies, compositions, and kits of parts described herein for the treatment of diseases, such as cancer.

[0336] In another aspect, the present invention relates to a method for the treatment of a human disease comprising the administration of a variant, composition, or kit-of-parts described herein.

[0337] In another aspect, the invention relates to a method for the treatment of human cancer comprising the administration of the variant, composition, or kit of parts.

[0338] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of alleviating, ameliorating, arresting, or eradicating (curing) the symptoms or disease state.

[0339] An "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the dosage and for the period of time required. A therapeutically effective amount of an antibody may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0340] Dosage It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0341] The effective dosage and dosage regimen of the antibody depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range of a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg / kg, e.g., about 0.1 to 50 mg / kg, e.g., about 0.1 to 20 mg / kg, e.g., about 0.1 to 10 mg / kg, e.g., about 0.5, e.g., about 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0342] The polypeptide or antibody of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is intended for use in combination with one or more additional therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or angiogenesis inhibitors. Such combined administration may be simultaneous, separate, or sequential.

[0343] In a further aspect, the present invention provides a method for treating or preventing a disease, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a variant or pharmaceutical composition of the present invention in combination with radiation therapy and / or surgery.

[0344] Preparation method It will be understood that the embodiments described below with respect to polypeptides or antibodies refer to a polypeptide or antibody comprising an Fc region having a CH2-CH3 region of an immunoglobulin and an antigen-binding region, and that the polypeptide or antibody may also be a multispecific polypeptide or antibody having a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region and a second polypeptide or antibody having a second Fc region and a second antigen-binding region comprising a second CH2-CH3 region of an immunoglobulin.

[0345] The present invention also provides isolated nucleic acids and vectors encoding variants according to any one of the above aspects, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors, and expression systems for antibodies and variants thereof are known in the art and are described in the Examples. In embodiments in which the variant includes not only a heavy chain (or an Fc-containing fragment thereof) but also a light chain, the nucleotide sequences encoding the heavy and light chain portions can be present in the same nucleic acid or vector or in different nucleic acids or vectors.

[0346] The present invention also provides a method for producing a polypeptide or antibody according to any one of the above aspects in a host cell, wherein the polypeptide or antibody comprises at least an Fc region of a heavy chain, the method comprising the steps of: a) providing a nucleotide construct encoding a variant Fc region; b) expressing the nucleotide construct in a host cell; and c) recovering the antibody variant from the cell culture of host cells Also provided is a method comprising:

[0347] In some aspects, the antibody is a heavy chain antibody, however, in most aspects, the antibody also comprises a light chain, and therefore the host cell additionally expresses a light chain-encoding construct on either the same or a different vector.

[0348] Host cells suitable for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293T, PER-C6, NS / 0, and Sp2 / 0 cells. In one embodiment, the host cell is a cell in which Asn-linked glycosylation of proteins can be performed, e.g., a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In a further embodiment, the host cell is a non-human cell genetically modified to produce glycoproteins with human-like or human-type glycosylation. Examples of such cells are genetically modified methylotrophic yeast (Pichia pastoris) (Hamilton et al., Science 301(2003)1244-1246; Potgieter et al., J.Biotechnology 139(2009)318-325) and genetically modified duckweed (Lemna minor) (Cox et al., Nature Biotechnology 12(2006)1591-1597).

[0349] In one embodiment, the host cell is a host cell that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists several such antibody production systems, such as Sp2 / 0, NS / 0, or the mammary gland of transgenic animals (goats), in which only partial C-terminal lysine removal is obtained. In one embodiment, the host cell is a host cell with an altered glycosylation mechanism. Such cells have been reported in the art and can be used as host cells for expressing the variants of the present invention, thereby producing antibodies with altered glycosylation. See, e.g., Shields, R. Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP 1176195; WO 03 / 035835; and WO 99 / 54342. Additional methods for generating engineered glycoforms are known in the art and include, but are not limited to, Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation modification technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0350] The present invention also relates to antibodies obtained or obtainable by the methods of the invention described above.

[0351] In a further aspect, the present invention relates to a host cell capable of producing a polypeptide or antibody of the present invention. In one embodiment, the host cell is transformed or transfected with a nucleotide construct of the present invention.

[0352] This invention is further illustrated by the following examples which should not be construed as further limiting.

[0353] Array Table TIFF2025124873000004.tif30153TIFF2025124873000005.tif218153TIFF2025124873000006.tif141153 [Example]

[0354] Example 1: Antibody generation, production, and purification Antibody expression constructs For antibody expression, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into the pcDNA3.3 expression vector (ThermoFisher Scientific, US), which contains the IgG1 heavy and light chain constant regions. Desired mutations were introduced by either gene synthesis or site-directed mutagenesis. The antibodies described in this application have VH and VL sequences derived from the previously reported CD38 antibody HuMAB 005 (WO 2006 / 099875), DR5 antibodies hDR5-01 and hDR5-05 (WO 2014 / 009358), CD52 antibody IgG1-Campath (alemtuzumab; Crowe et al., Clin Exp Immunol. 1992, 87(1):105-110), and CD20 antibodies IgG1-7D8 and IgG1-11B8 (WO 2004 / 035607). In some examples, the human IgG1 antibody b12, a gp120-specific antibody, was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-23).

[0355] Transient expression Antibodies were expressed as IgG1,κ. Expi293T cells (Life / Thermo Scientific, USA) were transiently transfected with a mixture of plasmid DNAs encoding both the heavy and light chains of the antibodies using 293fectin (Invitrogen, US) essentially as described by Vink et al. (Vink et al., Methods, 65(1), 5-10 2014).

[0356] Protein purification and analysis Antibodies were purified by Protein A affinity chromatography. Culture supernatants were filtered through a 0.20 μm dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), washed, and eluted with 0.02 M sodium citrate-NaOH, pH 3. Immediately after purification, the eluate was loaded onto a HiPrep Desalting column (GE Healthcare), and the antibodies were buffer-exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B.Braun or Thermo Fisher). After buffer exchange, the samples were sterile-filtered through a 0.2 μm dead-end filter. Purified proteins were analyzed by multiple bioanalytical assays, including capillary electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (CE-SDS) and high-performance size-exclusion chromatography (HP-SEC). Concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2–8°C.

[0357] Example 2: Analysis of the effect of mutations previously shown to inhibit C1q binding and CDC in wild-type antibodies on the in vitro CDC efficacy of IgG-005 mutants with enhanced Fc-Fc interactions The C1q binding center in the CH2 domain of human IgG1 was mapped to residues D270, K322, P329, and P331 by alanine substitution (Idusogie et al., 2000 J. Immunol.). The mutations D270A, K322A, and P329A significantly reduced C1q binding and complement activation by rituximab in a complement concentration-dependent manner (Idusogie et al., 2000 J. Immunol.).

[0358] It has been shown that IgG hexamerization upon target binding on the cell surface supports efficient binding of C1q hexameric structures, resulting in strong C1q binding (Diebolder et al., Science 2014). IgG hexamerization on the cell surface is mediated by intermolecular noncovalent Fc-Fc interactions and can be enhanced by point mutations in the CH2 domain, such as E345R and E430G (Diebolder et al., Science 2014; De Jong et al., PloS Biology 2015). Fc-Fc-enhancing mutations increase C1q binding avidity in hexameric antibody structures on the cell surface, but C1q binding affinity is unaffected. Therefore, it is unpredictable whether mutations reported to reduce C1q binding affinity can block CDC by IgG1 antibody mutants with mutations for enhanced Fc-Fc interactions.

[0359] Here, the inventors analyzed the effect of introducing the D270A / K322A (AA) double mutation into IgG1-005 mutants IgG1-005-E430G and IgG1-005-E345R (WO2013 / 004842, WO2014 / 108198) and IgG1-005-E345R / E430G / S440Y (WO2014 / 006217), which have stabilized Fc-Fc interactions known to enhance complement activation.

[0360] 0.1 x 10 for CDC assay 6Daudi cells (ATCC No. CCL-213™) were preincubated in a polystyrene round-bottom 96-well plate (Greiner Bio-One Catalog No. 650101) with a concentration series of purified antibodies in a total volume of 80 μL for 15 minutes on a shaker at room temperature. Next, 20 μL of normal human serum (NHS; Catalog No. M0008, Sanquin, Amsterdam, The Netherlands) was added as a complement source and incubated for 45 minutes in a 37°C incubator (20% final NHS concentration; 3-fold dilutions yielding final antibody concentrations of 0.001 to 10.0 μg / mL). After placing the plate on ice, the cells were pelleted by centrifugation and the reaction was stopped by replacing the supernatant with 20 μL of 2 μg / mL propidium iodide solution (PI; Sigma Aldrich, Zwijnaarde, The Netherlands). The number of PI-positive cells was determined by FACS analysis on an Intellicyt iQue™ sorter (Westburg). Data were analyzed using a best-fit nonlinear dose-response fit and log-transformed concentration values ​​in GraphPad PRISM 5. Percent lysis was calculated as (number of PI-positive cells / total number of cells) × 100%.

[0361] Introduction of the D270A / K322A (AA) double mutation into wild-type (WT) IgG1-005 resulted in complete inhibition of CDC on Daudi cells (Figure 1). In contrast, in the presence of the Fc-Fc interaction-enhancing mutations E430G or E345R, introduction of D270A / K322A had only a minor effect on CDC efficacy (Figure 1): IgG1-005-E430G and IgG1-005-AA-E430G had the same maximal killing of 100% with EC50s of 0.01 ± 0.01 (μg / mL ± SD) and 0.06 ± 0.02 μg / mL, respectively; IgG1-005-E345R and IgG1-005-AA-E345R had maximal killing of 100% and 74.3%, with EC50s of 0.01 μg / mL and 0.14 μg / mL, respectively. In the presence of the triple mutation E345R / E430G / S440Y, which leads to hexamerization of the antibody in solution (Diebolder et al., 2014 Science; Wang et al., 2016 Mol. Cell), D270A / K322A had no effect on CDC (Figure 1).

[0362] These data indicate that mutations that inhibited the CDC activity of the WT IgG1 antibody were unable to block the CDC activity of antibody mutants with mutations for enhanced Fc-Fc interactions.

[0363] Example 3: Analysis of the effect of selected mutations at positions D270, K322, and P329 of the C1q-binding core on the in vitro CDC efficacy of IgG1-005 variants with enhanced Fc-Fc interactions Mutations at positions D270, K322, and P329 in the human IgG1 C1q binding site were designed to interfere with the protein-protein interactions established when C1q is bound to IgG1. Therefore, WT amino acids were replaced with new or oppositely charged amino acids: D270R, K322E, P329D, and P329R. These additional mutations were tested for their effect on the CDC efficacy of the IgG1-005 variant, which harbors the E430G mutation for enhanced Fc-Fc interactions. A concentration series of purified antibodies (3-fold dilutions to a final antibody concentration of 0.001 to 10.0 μg / mL) was tested in an in vitro CDC assay in Daudi cells using 20% ​​NHS as described in Example 2. TIFF2025124873000007.tif46128

[0364] Introduction of the K322E, P329D, or P329R mutations strongly inhibited CDC-mediated killing of Daudi cells by IgG1-005-E430G (Figure 2A). In contrast, introduction of D270R resulted in reduced potency and increased EC50 values ​​(0.005 μg / mL and 0.15 μg / mL for IgG1-005-E430G and IgG1-005-D270R / E430G, respectively), but did not reduce maximal killing of Daudi cells by IgG1-005-E430G. Data with D270A / K322A were included as a reference to demonstrate only a minor effect on CDC efficacy by IgG1-005-E430G, as described in Example 2.

[0365] For the K322E, P329D, and P329R mutations that inhibited the CDC efficacy of IgG1-005-E430G, their effect on C1q binding to antibody bound to Daudi cells was measured by FACS analysis. 6Daudi cells were incubated in 100 μL reactions in a polystyrene round-bottom 96-well plate with a concentration series of purified antibodies (3.33-fold dilutions, final antibody concentrations of 0.0003 to 100.0 μg / mL) and 20% C4-depleted serum as a C1q source for 30 minutes at 4°C. 100 μL of FACS buffer (PBS / 0.1% BSA / 0.01% Na azide) was added, and the cells were pelleted by centrifugation. Cells were washed with 150 μL of FACS buffer and incubated with 50 μL of FITC-labeled rabbit anti-HuC1q antibody (DAKO, catalog no. F0254; final concentration of 20 μg / mL) for 30 minutes at 4°C. Cells were washed twice with FACS buffer, resuspended in 30 μL of FACS buffer, and the mean fluorescence intensity was measured in an Intellicyt iQue™ sorter.

[0366] Introduction of the K322E, P329D, or P329R mutation inhibited C1q binding to IgG1-005-E430G bound to Daudi cells (Fig. 2B ).

[0367] Taken together, these data show that introducing K322E, P329D, or P329R mutations into IgG1-005-E430G resulted in inhibition of C1q binding and the concomitant CDC-mediated killing of Daudi cells.

[0368] Example 4: Effect of the K322X mutation on the in vitro CDC efficacy of IgG1-005 mutants with enhanced Fc-Fc interactions Antibodies were collected by harvesting the supernatants of transient transfections as described in Example 1. A range of antibody concentrations (0.001–30.0 μg / mL final concentration in 3-fold dilutions) was tested in an in vitro CDC assay in Daudi cells with 20% NHS, essentially as described in Example 2. Substitutions of lysine (K) at position 322 with alanine (A), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y), aspartic acid (D), glutamic acid (E), or asparagine (N) in combination with the E430G mutation were tested (Figure 3).

[0369] In this experiment, the specific mutations K322D, K322E, and K322N were able to block complement activation and CDC by IgG1-005-E430G, which has enhanced Fc-Fc interactions.

[0370] Example 5: Biophysical characterization of IgG1-005-E430G variants containing the mutations K322D, K322E, or K322N Purified antibody batches of IgG1-005-E430G mutants carrying the K322E, K322D, or K322N mutations were analyzed by CE-SDS and HP-SEC.

[0371] CE-SDS was performed under reducing and non-reducing conditions. Sample purity and fragmentation were analyzed using CE-SDS (Caliper Labchip GXII, PerkinElmer) on a Labchip GXII (High Sensitivity protocol) with little modification. Both non-reduced and reduced samples (with DTT) were prepared using the HT Protein Express Reagent Kit (CLS960008) and denatured by incubation at 70°C for 10 minutes. Samples were run on the HT Antibody Analysis 200 high sensitivity setting. Molecular weight and purity (% of total) data were analyzed using the Labchip GXII software. Figure 4A shows that IgG1-005-K322E / E430G behaved similarly to the wild-type IgG1 assay control with disulfide-linked heavy and light chains. A single molecular species with an apparent MW of approximately 150 kDa was visible under non-reducing conditions, whereas under reducing conditions, a heavy chain with an apparent MW of 50 kDa and a light chain of 26 kDa were visible. The antibody variants IgG1-005-K322D / E430G and IgG1-005-K322N / E430G contained high molecular weight aggregates under non-reducing conditions that appeared to disintegrate after reduction.

[0372] The HP-SEC fractions were separated using a TSK HP-SEC column (G3000SW xlThe analysis was performed using a Waters Alliance 2975 separation unit (Waters, Etten-Leur, The Netherlands) coupled to a Waters 2487 Dual λ absorbance detector (Waters; Toso Biosciences, via Omnilabo, Breda, The Netherlands). A 50 μL sample containing 1.25 μg / mL of protein was separated at 1 mL / min in 0.1 M NaSO / 0.1 M sodium phosphate buffer, pH 6.8. Results were processed using Empower software version 3 and presented for each peak as a percentage of the total peak area. Figure 4B shows that antibody IgG1-005-K322E / E430G eluted predominantly at the elution time expected for monomeric species (98% monomeric), whereas mutants IgG1-005-K322D / E430G (70% aggregated) and IgG1-005-K322N / E430G (43% aggregated) showed substantial amounts of higher molecular weight species. Thus, HP-SEC analysis suggested that the double mutant K322E / E430G was more homogeneous in solution than the double mutants K322D / E430G and K322N / E430G.

[0373] Example 6: Effect of the P329X mutation on the in vitro CDC efficacy of IgG1-005 mutants with enhanced Fc-Fc interactions Here, the effect of the P329X mutation on in vitro CDC efficacy was tested in the antibody IgG1-005-E430G, which has enhanced CDC. Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay in Daudi cells using 20% ​​NHS, essentially as described in Example 2.

[0374] The CDC efficacy of IgG1-005-E430G against Daudi cells was completely inhibited by substituting the proline at position P329 with aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), asparagine (N), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y) (Figure 5). In contrast, substitution of proline at position 329 with alanine (A) only partially reduced CDC efficacy, shifting the EC50 from 0.01 μg / mL for IgG1-005-E430G to 0.11 μg / mL for IgG1-005-P329A / E430G, but had no effect on maximal killing. These data indicate that substitution of proline at position 329 with another amino acid resulted in either inhibition (in the case of P329D / E / F / G / H / I / K / L / N / Q / R / S / T / V / W / Y) or no inhibition (in the case of P329A) of CDC efficacy by IgG1-005-E430G.

[0375] Example 7: Biophysical characterization of IgG1-005-E430G variants containing mutations at position P329 Purified antibody batches of IgG1-005-E430G mutant, in which the proline at position 329 was replaced with any other amino acid except cysteine, were analyzed by CE-SDS and HP-SEC.

[0376] CE-SDS was performed under reducing and non-reducing conditions as described in Example 5. All tested IgG1-005-E430G antibody variants, containing an additional mutation at amino acid P329, behaved similarly to the wild-type IgG1 assay control antibody with disulfide-linked heavy and light chains: a single molecular species with an apparent MW of approximately 150 kDa was visible under non-reducing conditions, whereas under reducing conditions, a heavy chain with an apparent MW of 50 kDa and a light chain of 26 kDa were visible (summarized in Table 1). These data suggest that under denaturing conditions, a monomeric molecule is formed that behaves typical of wild-type IgG1 antibodies.

[0377] HP-SEC fractionation was performed as described in Example 5. The tested IgG1-005-E430G antibody variants, in which amino acid P329 was further mutated, contained varying amounts of high molecular weight species (Table 1). The variants IgG1-005-P329R / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329T / E430G were essentially homogeneous in solution.

[0378] Table 1. Biophysical characterization of IgG1-005-P329X / E430G antibody variants (X represents any amino acid other than P or C) by CE-SDS and HP-SEC. TIFF2025124873000008.tif116143

[0379] Example 8: Analysis of the thermal stability of IgG1-005-E430G mutant containing a mutation at position P329 Purified antibody batches of the IgG1-005-E430G variant, in which the proline at position 329 has been replaced with any other amino acid except cysteine, were analyzed by differential scanning fluorimetry (DSF).

[0380] DSF was performed in an iQ5 96-well RT-PCR machine (Bio-Rad) capable of detecting changes in fluorescence intensity caused by binding of the exogenous dye Sypro-Orange (ThermoFisher-Scientific, S6651) to hydrophobic regions exposed by denaturation of IgG. Thermal melting curves were obtained by measuring the increase in fluorescence during controlled, stepwise thermal denaturation of the IgG under analysis. Therefore, duplicate samples were prepared: 5 μL of 0.6 mg / mL IgG protein mixed with 20 μL of 75 mM Sypro-Orange in either PBS (pH 7.4) (B. Braun, Netherlands) or 30 mM NaAc (pH 4). Fluorescence was recorded at temperatures ranging from 25°C to 95°C in 0.5°C increments, for a duration of 15 seconds, plus the time required to record the fluorescence of all wells.

[0381] For each antibody analyzed, the midpoint of the first thermal transition (Tm), observed as a sharp increase in fluorescence intensity upon temperature increase, averaged across both duplicates, is summarized in Table 2. Introduction of P329R or P329K in IgG1-005 and IgG1-005-E430G resulted in a gradual increase in the antibody Tm, whereas introduction of P329D reduced the Tm of both WT IgG1-005 and IgG1-005-E430G. These data suggest that introduction of P329R or P329KK increased the thermal stability of IgG1-005 and IgG1-005-E430G, whereas P329D reduced the thermal stability of these antibodies.

[0382] Table 2. DSF analysis of IgG1-005-P329X / E430G antibody mutant TIFF2025124873000009.tif108128 1 IgG1-005 antibody variants ranked according to Tm reduction 2 The midpoint of the first thermal transition observed during the temperature ramp. Each value represents the average of duplicate measurements.

[0383] Example 9: Effect of mutations at position P329 on FcγRIIIa activation by IgG1-005 mutants with enhanced Fc-Fc interactions The effect on ADCC induction was tested for the IgG1-005-E430G mutant, in which proline at position 329 was substituted with any amino acid other than cysteine, aspartic acid, methionine, or arginine. Activation of FcγRIIIa-mediated signaling by IgG1-005-E430G mutants containing mutations at position P329 (P329A / E / F / G / H / I / K / L / N / Q / S / T / V / W / Y) was quantified in Daudi cells using the Luminescent ADCC Reporter BioAssay (Promega, catalog number G7015) according to the manufacturer's recommendations (Promega, #TM383). The kit contains Jurkat human T cells engineered to stably express high-affinity FcγRIIIa (V158) and the nuclear factor of activated T cells (NFAT) response element, which drives firefly luciferase expression. Briefly, Daudi cells (5,000 cells / well) were seeded in ADCC assay buffer [RPMI-1640 medium (Lonza, catalog number BE12-115F) supplemented with 3.5% low IgG serum] in a 384-well white OptiPlate (Perkin Elmer catalog number 6007290) and incubated for 6 hours at 37°C / 5% CO2 in a total volume of 30 μL containing a series of antibodies (5-fold dilutions ranging from 0.128 to 2,000 ng / mL final concentrations) and thawed ADCC Bioassay Effector Cells. After incubating the plate for 15 minutes at room temperature (RT), 30 μL of Bio-Glo Assay Luciferase Reagent was added and incubated for 5 minutes at room temperature. Luciferase production was quantified by luminescence readout on an EnVision Multilabel Reader (Perkin Elmer). Luminescence signals were normalized by subtracting the background luminescence signal measured in a medium-only sample (no Daudi cells, no antibody, no effector cells).

[0384] Dose-responsive FcγRIIIa activation by IgG1-005-E430G was completely inhibited at all tested concentrations of all P329X mutants (not shown), as shown in Figure 6 for a range of antibody concentrations from 3.2 ng / nL - 16 ng / mL - 80 ng / mL. These data indicate that proline at position 329 is essential for FcγRIIIa binding and activation.

[0385] Example 10: Analysis of the effect of mutations at K322 and P329 on the ADCC efficacy of IgG1-005 variants with enhanced Fc-Fc interactions CDC-inhibitory variants of IgG1-005-E430G (described in Examples 4 and 6), which exhibit favorable biophysical characteristics (described in Examples 5, 7, and 8), were tested for their ADCC efficacy. IgG1-005-E430G variants containing the K322E, P329A, P329D, P329K, or P329R mutations were applied to Daudi cells in an in vitro ADCC assay, and freshly isolated peripheral blood mononuclear cells (PBMCs) from three different healthy donors were used as effector cells. PBMCs were isolated from buffy coats (Sanquin, Amsterdam, The Netherlands) using Lymphocyte Separation Medium (Lonza, catalog number 17-829E) for standard Ficoll density gradient centrifugation according to the manufacturer's instructions. After resuspension of the cells in RPMI-1640 medium (Lonza, catalog number BE12-115F) supplemented with 10% DBSI (Donor Bovine Serum with Iron, ThermoFischer, catalog number 10371029) and penicillin / streptomycin (Pen / Strep) (Lonza, catalog number DE17-603E), the cells were counted by trypan blue exclusion and collected at 1 × 10 7 The cells were concentrated to 100 cells / mL.

[0386] Daudi cells were harvested (5 × 10 6The cells were then washed (twice in PBS, 1200 rpm, 5 min) and collected in 1 mL of RPMI-1640 medium supplemented with 10% DBSI and Pen / Strep, to which 100 μCi of 51 Cr (chromium-51; PerkinElmer, catalog number NEZ030002MC) was added. The mixture was incubated in a shaking water bath at 37°C for 1 hour. After washing the cells (twice in 50 mL of PBS, 1200 rpm, 5 minutes), the cells were resuspended in RPMI-1640 medium supplemented with 10% DBSI and Pen / Strep, counted by trypan blue exclusion, and a cell count of 1 x 10 5 The cells were diluted to a concentration of 100 cells / mL.

[0387] For ADCC experiments, 50 μL 51 Cr-labeled Daudi cells (5,000 cells / well) were preincubated with a concentration series (0.3–1,000 ng / mL final concentration in 3-fold dilutions) of the IgG1-005-E430G antibody variant in a total volume of 100 μL of RPMI-1640 medium supplemented with 10% DBSI and Pen / Strep in a 96-well round-bottom microtiter plate (Greiner Bio-One; catalog no. 650101). After 20 min at room temperature, 50 μL of PBMCs (500,000 cells) were added to achieve an effector:target ratio of 100:1 and incubated for 4 h at 37°C / 5% CO2. To determine the maximum amount of cell lysis, 50 μL of PBMCs was added at 100:1 effector:target ratio. 51 Cr-labeled Daudi cells (5,000 cells) were incubated with 100 μL of 5% Triton-X100. 51 Cr-labeled Daudi cells were incubated in 150 μL of medium without any antibody or effector cells. The level of antibody-independent cytolysis was examined by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. 51To count the amount of Cr, the plate was centrifuged (1200 rpm, 10 min), and 25 μL of the supernatant was transferred to 100 μL of Microscint-40 solution (Packard, Cat. No. 6013641) in a 96-well plate. The plate was sealed and shaken at 800 rpm for 15 min to measure the amount of released Cr. 51 Cr 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 maximal lysis - cpm spontaneous lysis) × 100%.

[0388] Dose-responsive ADCC-mediated killing of Daudi cells by IgG1-005-E430G was completely inhibited by introducing the P329D, P329K, or P329R mutations, as shown for a range of antibody concentrations from 0.3 ng / mL to 30 ng / mL to 300 ng / mL in Figure 7. In contrast, IgG1-005-E430G mutants with the K322E or P329A mutation retained substantial ADCC efficacy against Daudi cells.

[0389] Summarizing the CDC data described in Examples 4 and 6, the ADCC reporter data described in Example 9, and the in vitro ADCC data described in this Example, introduction of the P329D, P329K, or P329R mutations resulted in inhibition of both the CDC and ADCC activities of IgG1-005-E430G, despite the enhancing effect of the E430G mutation on Fc-Fc interactions and hexamerization upon target binding on the cell surface. In contrast, the K322E and P329A mutations resulted in CDC inhibition, but retained ADCC efficacy by IgG1-005-E430G.

[0390] Example 11: The P329D mutation is generally applicable to inhibit complement activation and CDC by IgG1 antibodies with mutations for enhanced Fc-Fc interactions Examples 3 and 6 show that introducing the P329D mutation into an anti-CD38 mAb IgG1-005 mutant containing an E430G mutation for enhanced Fc-Fc interaction resulted in complete inhibition of CDC activity against Daudi cells. Next, we tested whether introducing the P329D mutation had the same effect on IgG1-005 mutants containing other Fc-Fc-enhancing mutations. Therefore, the P329D mutation was introduced into IgG1-005 mutants containing one or more of the E345R, E345K, or E345R / E430G / S440Y (RGY) mutations, and C1q binding was tested in an in vitro CDC assay in Daudi cells.

[0391] C1q binding to antibody bound to Daudi cells was measured by FACS analysis as described in Example 3. For the CDC assay, a range of antibody concentrations (0.0003 to 100.0 μg / mL final concentration at 3.33-fold dilutions) was tested on Daudi cells with 20% NHS as described in Example 2.

[0392] Introduction of the P329D mutation resulted in complete inhibition of C1q binding (Figure 8A) and CDC efficacy (Figure 8B) by IgG1-005 mutants harboring either the E345K, E345R, or E345R / E430G / S440Y mutations for enhanced Fc-Fc interactions in Daudi cells.

[0393] The data with the E345K, E345R, and RGY mutations presented in this example, together with the data with E430G described in Example 3, indicate that C1q binding and CDC efficacy by IgG1-005 antibodies with mutations for enhanced Fc-Fc interactions can generally be inhibited by the introduction of the P329D mutation.

[0394] Example 12: Biophysical characterization of hexameric IgG1-005-E345R / E430G / S440Y mutants containing K322E or P329D mutations To test the effect of K322E and P329D on IgG1 hexamerization, we utilized the triple mutant IgG1-005-E345R / E430G / S440Y, which combines three Fc-Fc interaction-enhancing mutations, E345R, E430G, and S440Y (RGY), and has been shown to form antibody hexamers in solution (Diebolder et al., Science 2014). K322E or P329D was introduced into IgG1-005-RGY to generate IgG1-005-K322E / E345R / E430G / S440Y (IgG1-005-ERGY) and IgG1-005-P329D / E345R / E430G / S440Y (IgG1-005-DRGY), and the effect on antibody hexamerization was analyzed by CE-SDS, HP-SEC, and native mass spectrometry. HP-SEC fractionation was performed as described in Example 5. Consistent with the behavior observed with IgG1-005-RGY (Diebolder et al., Science 2014), both IgG1-005-ERGY and IgG1-005-DRGY retained the ability to oligomerize in solution (Figure 9A). Two peaks corresponding to oligomers (elution time approximately 6.3 min) and monomers (elution time approximately 9–9.3 min) were observed, with intermediate intensities likely resulting from dynamic conversion between the oligomeric and monomeric states. The oligomeric fraction in IgG1-005-ERGY was measured to be 58.4%, while 31.4% was in the monomeric form; 10.2% eluted as an intermediate species. The oligomeric fraction in IgG1-005-DRGY was measured to be 78.8%, while the monomeric fraction was 12.5%; 8.7% of the intermediate species was observed.

[0395] CE-SDS was performed under reducing and non-reducing conditions. Consistent with the results observed with IgG1-005-RGY (Diebolder et al., Science 2014), both IgG1-005-ERGY and IgG1-005-DRGY exhibited a single molecular species with an apparent MW of approximately 150 kDa under non-reducing conditions, whereas under reducing conditions, a heavy chain with an apparent MW of 50 kDa and a light chain of 26 kDa were visible (Figure 9B). These data indicate that IgG1-005-ERGY and IgG1-005-DRGY behaved similarly to the WT monomeric IgG1 assay control antibody, indicating that hexamerization was disrupted under denaturing CE-SDS conditions, consistent with non-covalent Fc-Fc interactions.

[0396] Native mass spectrometry analysis of 2 μM IgG1-005-DRGY was performed in the absence or presence of excess C1q, buffered in 150 mM ammonium acetate, pH 7.5, using a modified LCT time-of-flight (Waters, UK) mass spectrometer tuned for optimal performance at high mass detection. Samples were sprayed through a borosilicate glass capillary mounted on a standard stationary nanospray source. Data analysis was performed using MassLynx (Waters, UK) and Origin Pro (Origin Lab, USA) software. IgG1-005-DRGY formed hexamers, similar to those observed with IgG1-005-RGY (Figure 9). Addition of C1q to IgG1-005-DRGY hexamers did not result in detectable C1q binding, whereas IgG1-005-RGY readily bound C1q under equivalent conditions (Figure 9C).

[0397] In summary, the biophysical analyses described in this example demonstrate that introduction of the C1q-binding inhibitor mutations P329D or K322E did not block hexamerization of IgG1-005-RGY in solution (HP-SEC, native MS), but completely abolished C1q binding (native MS). Furthermore, the oligomers formed in solution by the antibody mutants IgG1-005-ERGY and IgG1-005-DRGY were formed by non-covalent interactions (CE-SDS), consistent with the Fc-Fc interactions reported for IgG1-005-RGY (Diebolder et al., Science 2014).

[0398] Example 13: Analysis of the efficacy of agonistic DR5 antibodies with enhanced Fc-Fc interactions and the P329D mutation to induce killing Agonistic death receptor 5 (DR5) antibodies can induce the death of DR5-positive tumor cells by activating the extrinsic apoptotic pathway through DR5 hyperclustering, leading to recruitment of the adaptor protein, Fas-associated protein with death domain (FADD), to the intracellular DR5 death domain, which further leads to binding and activation of caspase-8 and formation of the DISC (death-inducing signaling complex) that initiates apoptosis. To demonstrate that Fc-Fc interactions are involved in killing by a combination of DR5 antibodies containing the E430G mutation for enhanced Fc-Fc interactions (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G), we utilized the 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science 2000 Feb 18;287(5456):1279-83), which binds to Fc in a region containing core amino acids within the hydrophobic patch involved in Fc-Fc interactions (Diebolder et al., Science. 2014 Mar 14;343(6176):1260-3). Viability assays were performed in BxPC-3 cells in the presence or absence of the DCAWHLGELVWCT peptide. Adherent BxPC-3 (ATCC, CRL-1687) cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation at 1,200 rpm for 5 minutes and resuspended in culture medium at a concentration of 0.5 × 10 5The cells were resuspended at a concentration of 5,000 cells / mL in RPMI 1640 containing 25 mM Hepes and L-glutamine (Lonza catalog no. BE12-115F) + 10% DBSI (Life Technologies catalog no. 10371-029) + Pen / Strep (Lonza catalog no. DE17-603E). 100 μL of the single-cell suspension (5,000 cells / well) was seeded into a 96-well polystyrene flat-bottom plate (Greiner Bio-One, catalog no. 655182) and incubated overnight at 37°C. The culture medium was removed and replaced with 100 μL of culture medium containing 100 μg / mL of the Fc-binding DCAWHLGELVWCT peptide, the nonspecific control peptide GWTVFQKRLDGSV, or no peptide. Next, 50 μL of the antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G (final concentration of 833 ng / mL) was added and incubated at 37°C for 3 days. To determine maximum killing, samples were incubated with 5 μM staurosporine (Sigma Aldrich, catalog number S6942). The percentage of viable cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, catalog number G7571), which quantifies the amount of ATP present, an indicator of metabolically active cells. From the kit, 20 μL of luciferin solution reagent was added per well and mixed by shaking the plate at 500 rpm for 2 minutes. The plate was then incubated at 37°C for 1.5 hours. 100 μL of the supernatant was transferred to a white OptiPlate-96 (PerkinElmer, Cat. No. 6005299), and luminescence was measured using an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software. The percentage of viable cells was calculated using the following formula: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of sample without antibody - luminescence of staurosporine sample)]. * Calculations were made using 100.

[0399] The ability of the antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G to induce killing of BxPC-3 cells was strongly inhibited by 100 μg / mL of the Fc-binding DCAWHLGELVWCT peptide (FIG. 10A). These data indicate that Fc-Fc interactions are required for the antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G harboring Fc-Fc-enhancing mutations to induce DR5 clustering on the cell surface of cancer cells and induce apoptosis.

[0400] Next, we performed a viability assay to examine the effect of introducing the P329D mutation on DR5 clustering and apoptosis induction by an agonistic DR5 antibody with the E430G mutation for enhanced Fc-Fc interaction. The viability assay was performed essentially as described above in BxPC-3 cells. Briefly, overnight-adhered BxPC-3 cells (5,000 cells / well) were incubated at 37°C for 3 days with a final antibody concentration of 5 μg / mL or 10 μg / mL in a total volume of 150 μL. The percentage of viable cells was determined using a CellTiter-Glo luminescent cell viability assay.

[0401] After introduction of the P329D (Figure 10B) or K322E (Figure 10C) mutation, the combination IgG1-hDR5-01-E430G + IgG1-hDR5-05-E430G, which harbors the E430G mutation for enhanced Fc-Fc interactions, was still able to induce killing of BxPC-3 cells at saturating antibody concentrations.

[0402] Taken together, these data indicate that the P329D and K322E mutations did not block Fc-Fc interactions required for clustering on target cells and induction of apoptosis upon DR5 binding by saturating concentrations of an agonistic DR5 antibody with the E430G mutation for enhanced Fc-Fc interactions.

[0403] Example 14: Glycosylation profiling of IgG1-005 variants with enhanced Fc-Fc interactions containing K322E, P329D, or P329R mutations The N-linked glycans of purified antibodies IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G were analyzed by mass spectrometry.

[0404] IgG samples were incubated with DTT for 1 hour at 37°C. The samples were then desalted on an Ultimate 3000 UPLC system (Dionex) using a 10-minute block gradient at 60°C on a Proswift RP-4H 1 × 250 mm column (Thermo Scientific) with MilliQ water (eluent A) and LC-MS-grade acetonitrile (eluent B), both containing 0.05% formic acid (Fluka). The UPLC system was coupled to a Q-Exactive Plus Orbitrap MS system (Thermo Scientific) equipped with a HESI source for electrospray ionization. Prior to analysis, the 800–3000 m / z scale was calibrated using LTQ Velos ESI positive calibration mix. Recorded mass spectra were deconvoluted using Protein Deconvolution software (Thermo Scientific) and used to quantify the relative abundance of individual N-linked glycans.

[0405] The antibody mutants IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G all exhibited glycosylation profiles similar to those generally observed for IgG1 antibodies expressed in EXPI293 cells, with low levels of mannose-5 or charged species, high levels of fucosylation, and 10% to 30% galactosylated species (Table 3). These data suggest that the mutations K322E, P329D, and P329R did not substantially affect the glycosylation profile of IgG1-005-E430G.

[0406] Table 3. Distribution of N-linked glycans in the IgG1-005-E430G mutant TIFF2025124873000010.tif75142

[0407] Example 15: Pharmacokinetic (PK) analysis of IgG-005 variants with enhanced Fc-Fc interactions containing K322E, P329D, or P329R mutations The effects of the K322E, P329D, and P329R mutations on the clearance rate of IgG1-005-E430G were examined in PK experiments in SCID mice. The clearance rates of IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G were compared with those of IgG1-005-E430G without the CDC-inhibitory mutation and WT IgG1-005 without the E430G mutation for enhanced Fc-Fc interactions.

[0408] The mice in this study were housed at the Central Laboratory Animal Facility (Utrecht, The Netherlands) and handled in an AAALAC and ISO 9001:2000-certified animal facility (GDL) in accordance with the experimental animal standards defined by FELASA. All experiments were conducted in accordance with the Dutch Animal Protection Act (WoD), which was translated from Directive (2010 / 63 / EU) and approved by the Animal Ethics Committee of Utrecht University. Female SCID (C.B-17 / IcrHan@Hsd-Prkdc<scid, Envigo) mice, aged 11 - 12 weeks (3 mice per group), were intravenously injected with 500 μg of antibody (25 mg / kg) at an injection volume of either 210 μL (for IgG1-005-K322E / E430G) or 200 μL (for other batches). Blood samples of 50 - 100 μL were collected from the retro-orbital vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days, and 21 days after antibody administration. The blood was collected into heparin-containing vials and centrifuged at 14,000 g for 10 minutes. Twenty microliters of plasma samples were diluted with 980 μL of PBST (PBS supplemented with 0.05% Tween 20) supplemented with 0.2% bovine serum albumin (BSA) and stored at -20°C until measurement of antibody concentration. Total human IgG concentration was measured using sandwich ELISA. Mouse anti-human IgG-kappa mAb clone MH16 (CLB Sanquin, catalog number M1268) was used as the capture antibody, and 96-well Microlon ELISA plates (Greiner, Germany) were coated at a concentration of 2 μg / mL (in PBS) with 100 μL at 4°C overnight. The plates were blocked by incubating with PBS supplemented with 0.2% BSA on a plate shaker at room temperature for 1 hour. After washing, 100 μL of diluted plasma samples were added and incubated on a plate shaker at room temperature for 1 hour.The plate was washed three times with 300 μL of PBST and then incubated with 100 μL of peroxidase-labeled goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA; 1:10,000 in PBST supplemented with 0.2% BSA) for 1 hour at room temperature on a plate shaker. The plate was washed again three times with 300 μL of PBST and then incubated with 100 μL of the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS; Roche, catalog number 11112 422001; one tablet in 50 mL of ABTS buffer (Roche, catalog number 11112 597001)] for 15 minutes at room temperature, protected from light. The reaction was stopped by adding 100 μL of 2% oxalic acid and incubating for 10 minutes at room temperature. Absorbance was measured at 405 nm on a microplate reader (Biotek, Winooski, VT). Concentrations were calculated using the injected material as a reference curve. Human myeloma protein containing IgG (The binding site, UK) was included as a plate control. Human IgG concentrations (units: μg / mL) were plotted using Graphpad Prism 6.0 (FIG. 11A), and the area under the curve (AUC) was calculated. Clearance by the last day of blood sampling (day 21) was calculated using the formula D. * 1.000 / AUC, where D is the injected dose (25 mg / kg) (FIG. 11B).

[0409] The CDC-inhibiting mutants IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G all exhibited clearance rates in the same range as IgG1-005-E430G and WT IgG1-005 (Figure 11). These data indicate that the clearance rate of the IgG1-005-E430G antibody, which has enhanced Fc-Fc interactions, was not affected by K322E (which inhibits CDC but retains ADCC efficacy) or P329D and P329R (which inhibit both CDC and ADCC efficacy).

[0410] Example 16: Effect of the P329X mutation on the in vitro CDC efficacy of IgG1-005 variants with enhanced Fc-Fc interactions Here, the effect of the P329X mutation on in vitro CDC efficacy was tested in antibody IgG1-005-E430G, which has enhanced CDC compared to IgG1-005. Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay in Daudi cells with 20% NHS, essentially as described in Example 2.

[0411] The CDC efficacy of IgG1-005-E430G against Daudi cells was completely inhibited by substituting proline at position P329 with methionine (M), aspartic acid (D), or arginine (R) (Figure 12). In contrast, substituting proline at position 329 with alanine (A) only partially reduced CDC efficacy, shifting the EC50 from 0.01 μg / mL for IgG1-005-E430G to 0.10 μg / mL for IgG1-005-P329A / E430G, but had no effect on maximal killing. These data indicate that substituting proline at position 329 with another amino acid resulted in either inhibition (in the case of P329M / D / R) ​​or no inhibition (in the case of P329A) of CDC efficacy by IgG1-005-E430G.

[0412] Example 17: Effect of P329R and P329D mutations on in vitro CDC efficacy of Campath IgG isotype variants with enhanced Fc-Fc interactions The effect of the P329R and P329D mutations on in vitro CDC efficacy was tested using different IgG isotype variants of the antibody IgG1-Campath-E430G, which has enhanced CDC compared to IgG1-Campath (Figure 13). Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay on Wien 133 cells using 20% ​​NHS essentially as described in Example 2. The area under the dose-response curve for triplicate experiments was calculated using GraphPad Prism 7.02 using log-transformed concentration axes and normalized to the cell lysis measured with the isotype control antibody IgG1-b12 (0%) and the cell lysis measured with IgG1-Campath (100%).

[0413] The area under the CDC dose-response curve for IgG1-Campath-E430G in Vienna 133 cells was increased approximately threefold compared to WT, but CDC activity was reduced to background levels by substituting arginine (R) or aspartic acid (D) for proline at position 329 (Figure 13). Similarly, CDC by IgG2-Campath-E430G was also reduced to background levels by introducing the P329R or P329D mutation. Furthermore, IgG3 and IgG4 isotype mutants containing both E430G and either the P329R or P329D mutation did not exhibit CDC lysis above background levels.

[0414] These data indicate that substitution of proline at position 329 with arginine or aspartic acid resulted in efficient inhibition of CDC efficacy by IgG1, IgG2, IgG3, and IgG4 isotype variants of IgG1-Campath-E430G.

[0415] Example 18: Effect of mutation K322E on in vitro CDC efficacy of Campath IgG isotype variants with enhanced Fc-Fc interactions The effect of mutation K322E on in vitro CDC efficacy was tested using different IgG isotype variants of antibody IgG1-Campath-E430G, which has enhanced CDC compared to IgG1-Campath (Figure 14). Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay on Wien 133 cells using 20% ​​NHS essentially as described in Example 2. The area under the dose-response curve for triplicate experiments was calculated using GraphPad Prism 7.02 using log-transformed concentration axes and normalized to the cell lysis measured with the isotype control antibody IgG1-b12 (0%) and the cell lysis measured with IgG1-Campath (100%).

[0416] The area under the CDC dose-response curve for IgG1-Campath-E430G in Vienna 133 cells was increased approximately threefold compared to WT, but was reduced to approximately 18% by substituting glutamic acid (E) for lysine at position 322 (Figure 14). Similarly, CDC by IgG2-Campath-E430G was reduced to background levels by introducing the K322E mutation. Furthermore, IgG3 and IgG4 isotype variants containing both the E430G and K322E mutations did not exhibit CDC lysis above background levels. These data indicate that substituting glutamic acid for lysine at position 322 resulted in efficient inhibition of the CDC efficacy of the IgG1, IgG2, IgG3, and IgG4 isotype variants of IgG1-Campath-E430G.

[0417] Example 19: Effect of mutations P329R and K322E on the in vitro CDC efficacy of Camphas variants with different mutations that induce enhanced Fc-Fc interactions The effects of the mutations P329R and K322E on in vitro CDC efficacy were tested using different Fc-Fc interaction-promoting mutants of the antibody IgG1-Campath. Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay on Wien 133 cells using 20% ​​NHS, essentially as described in Example 2. The area under the dose-response curve for triplicate experiments was calculated using GraphPad Prism 7.02 using log-transformed concentration axes and normalized to the cell lysis measured with the isotype control antibody IgG1-b12 (0%) and the cell lysis measured with IgG1-Campath (100%). The area under the CDC dose-response curve for IgG1-Campath-E345K, which contains the Fc-Fc interaction-promoting mutation E345K, in Wien 133 cells was increased approximately 2.4-fold compared to WT. Substitution of proline at position 329 with arginine (R) limited CDC to approximately 8%. Furthermore, introduction of P329R into two other mutants, E345R and E345R / E430G / S440Y (RGY), with increased Fc-Fc interactions limited CDC activity to levels lower than those observed with the parent IgG1-Campath antibody (Figure 15).

[0418] Substitution of lysine at position 322 with glutamic acid (E) in antibody IgG1-Campath-E345K reduced the area under the CDC dose-response curve from approximately 240% to 24% of that of the parent IgG1-Campath antibody. Introduction of K322E into mutant E345R, which has enhanced Fc-Fc interactions, limited the CDC activity of this mutant to 60% of that observed with the parent IgG1-Campath antibody (Figure 15). However, in contrast to mutation P329R, K322E was unable to limit the CDC of mutant RGY to levels below that of IgG1-Campath.

[0419] These data suggest that the inhibition of direct C1q binding by mutations P329R or K322E in the C1q-binding site can be partially compensated for by mutations that promote enhanced Fc-Fc interactions, such as E345R and RGY, which promote the formation of multivalent C1q-binding sites in IgG hexamers at the cell surface. Because IgG1-Campath-P329R-RGY exhibited lower CDC activity than IgG1-Campath-K322E-RGY, P329R appears to be a more potent inhibitor of direct C1q binding than K322E.

[0420] In summary, these data show that substituting proline at position 329 with arginine or lysine at position 322 with glutamic acid was able to inhibit the CDC efficacy of IgG1-Campath variants with different Fc-Fc interaction strengths.

[0421] Example 20: Effect of mutations P329R, P329D, and K322E on the in vitro CDC efficacy of anti-CD20 antibodies with enhanced Fc-Fc interactions The effects of mutations P329R, P329D, and K322E on in vitro CDC efficacy were tested using variants of the anti-CD20 antibodies IgG1-11B8 (type II) and IgG1-7D8 (type I) (WO 2004 / 035607). Different concentrations of purified antibody (ranging from 0.001 to 30.0 μg / mL final concentration) were tested in an in vitro CDC assay on Wien 133 cells using 20% ​​NHS, essentially as described in Example 2.

[0422] IgG1-11B8 did not exhibit detectable CDC, but introducing the mutation E430G, which induces enhanced Fc-Fc interactions, promoted efficient cell lysis (IgG1-11B8-E430G, Figure 16). Both the mutations P329R and K322E limited the CDC activity of IgG1-11B8-E430G to the background lysis level observed with the nonbinding isotype control antibody IgG1 b12.

[0423] IgG1-7D8 was capable of inducing CDC in Wien 133 cells, but CDC efficacy was stimulated by the introduction of the Fc-Fc interaction-enhancing mutation E430G. Introduction of the mutations P329R or P329D suppressed CDC activity to a level below that of the wild-type parent antibody IgG1-7D8. Without being limited by theory, Fc-independent ancillary CDC mediated by B cell receptor engagement may contribute to the residual CDC detected in IgG1-7D8-P329R-E...

Claims

[Claim 1] The invention described in the specification of this application.

Images