Polypeptide variants and their use

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 a polypeptide containing an Fc region, such as an antibody, in which the Fc effector function is reduced, for example, the binding to C1q is reduced, the complement-dependent cytotoxicity (CDC) is reduced, and due to one or more amino acid modifications in the Fc region, the activation of other effector functions may also be reduced.

Background Art

[0002] Background of the Invention The 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 the therapeutic window defined by efficacy and toxicity. CDC is initiated by the binding of C1q to the Fc region of the antibody. C1q is a multimeric protein consisting of six binding globular heads attached to a stalk. Each binding globular head has a low affinity for IgG, and C1q must acquire avidity by binding to many IgG1 molecules on the cell surface in order to trigger the classical complement pathway. ADCC and ADCP are initiated by the binding of the IgG Fc region to Fcγ receptors (FcγR) on effector cells.

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

[0004] WO2013 / 004842 (Patent Document 1) discloses an antibody or polypeptide comprising a mutant Fc region having one or more amino acid modifications that result in modification of effector functions, such as complement-dependent cytotoxicity (CDC).

[0005] WO2014 / 108198 (Patent Document 2) discloses a polypeptide, such as an antibody, comprising a mutant Fc region having one or more amino acid modifications that result in an increase in complement-dependent cytotoxicity (CDC).

[0006] WO2012 / 130831 (Patent Document 3) relates to an Fc region-containing polypeptide having an effector function modified as a result of one or more amino acid substitutions within the Fc region of a polypeptide. Such a polypeptide exhibits a reduced affinity for human FcyRIIIa and / or FcyRIIa and / or FcyRI as compared to a polypeptide containing a wild-type IgG Fc region, and shows a reduction of at least 20% in antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the polypeptide as compared to ADCC induced by a polypeptide containing a wild-type human IgG Fc region. WO2012 / 130831 (Patent Document 3) does not disclose an Fc region-containing polypeptide having enhanced Fc-Fc interaction and / or enhanced hexamer-forming ability.

[0007] As described above, previous efforts in enhancing Fc-Fc interaction between polypeptides and / or antibodies have the effect of enhancing effector functions, such as enhancing CDC and / or ADCC, thereby resulting in cell death of target cells to which the antibody or polypeptide binds.

[0008] Enhanced Fc-Fc interactions between antibodies can be used to amplify the effect of antibody binding to a target on the cell surface. However, when the target cell is an effector cell, such as a T cell, NK cell, or another effector cell whose mechanism of action involves binding to an effector cell (e.g., as in a bispecific antibody), the interaction with C1q or Fc gamma R and / or the activation of Fc effector functions, such as CDC and / or ADCC, may not be necessary. Thus, there is a need for antibodies that have enhanced Fc-Fc interactions but do not participate in C1q binding and / or do not have Fc gamma R interactions, and thereby do not activate Fc effector functions, such as CDC and / or ADCC.

[0009] Accordingly, an object of the present invention is to provide a mutant polypeptide or antibody comprising an Fc region and an antigen-binding region of human IgG, which has an increased Fc-Fc interaction and a decreased effector function, such as CDC and / or ADCC, compared to the parent polypeptide (wherein the parent polypeptide is human IgG of the same isotype and has the same antigen-binding region), and has a first mutation, which is an Fc-Fc enhancing mutation, at the amino acid position corresponding to E345, E430, or S440 in human IgG1 (provided 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 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 and ADCC. A further object of the present invention is to provide a polypeptide or antibody having enhanced Fc-Fc interaction and reduced Fc effector functions, such as reduced CDC and / or ADCC, compared to the parent polypeptide, and having only the 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 signal transduction without activating Fc effector functions, such as CDC and / or ADCC, when the antigen-binding region of the polypeptide or antibody is bound to the corresponding antigen, and optionally induces enhancement of signal transduction.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

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 functions, such as CDC and / or ADCC activity.

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

[0014] Without being bound by theory, when the polypeptide or antibody of the present invention binds to a target on the cell surface, a more stable binding interaction is possible between the Fc regions of two polypeptide or antibody molecules, which is thought to result in enhanced oligomerization, such as hexamer formation, without enhancing the Fc-mediated effector function. The polypeptide or antibody of the present invention further has reduced C1q binding and / or reduced Fc gamma R binding compared to its parent polypeptide or parent antibody that contains the first mutation but not the second mutation. The polypeptide or antibody of the present invention has reduced Fc effector function compared to its parent polypeptide or parent antibody that contains 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 the parent polypeptide or parent antibody. Some polypeptides or antibodies of the present invention have reduced Fc effector function, such as ADCC, compared to the parent polypeptide or parent antibody. Some polypeptides or antibodies of the present invention have reduced Fc effector function, such as CDC and ADCC, compared to the parent polypeptide or parent antibody. Some polypeptides or antibodies of the present invention further have a reduced Fc effector response compared to the same polypeptide or antibody that does not contain the first and second mutations, i.e., that contains the 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 of human IgG and an antigen-binding region, wherein the Fc region comprises CH2 and CH3 domains, and the Fc region has the following mutations at the following amino acid positions in human IgG1 according to EU numbering (Edelman et al., Proc Natl Acad Sci U S 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 and provides a polypeptide or antibody comprising the same.

[0016] That is, the inventors of the present invention have found that in a first aspect of the present invention, 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 interaction and thus oligomerization after target binding can reduce the Fc effector function. Mutations corresponding to amino acid positions K322 or P329 in the Fc region of a polypeptide or antibody have the effect of reducing one or more Fc effector functions to a reduced level compared to a parental polypeptide or antibody having the same first mutation but no second mutation. Thus, in one embodiment of the present invention, the polypeptide or antibody has at least one first mutation that can be selected from one of the 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 that can be selected from one of the positions K322 or P329.

[0017] In one aspect 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 aspect of the present invention, the first mutation is selected from E430G or E345K. In a preferred aspect, the first mutation is E430G.

[0018] In one aspect 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, P329A, and P329Y.

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

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

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

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

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

[0024] In another aspect, the present invention relates to a composition 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 present invention relates to a polypeptide, antibody, or composition described herein for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

[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] [Invention 1001] A polypeptide comprising an Fc region of human IgG and an antigen-binding region, wherein the Fc region comprises CH2 and CH3 domains, the Fc region has the following mutations at amino acid positions corresponding to those in human IgG1 according to EU numbering: (i) a first mutation and (ii) a second mutation: 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 comprising, the polypeptide. [Invention 1002] The polypeptide of Invention 1001, wherein the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [Invention 1003] The polypeptide of Invention 1001, wherein the first mutation is selected from E430G or E345K. [Invention 1004] The polypeptide of any of the above inventions, 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. [Invention 1005] The polypeptide of any of the above inventions, wherein the second mutation is K322E. [Invention 1006] A polypeptide of any one of the polypeptides of the present invention 1001 to 1004, wherein the second mutation is selected from the group consisting of P329R, P329K, and P329D. [The present invention 1007] Any polypeptide of the present invention, wherein the Fc region comprises one or more additional mutations. [The present invention 1008] Any polypeptide of the present 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 present invention 1007 or 1008, wherein the Fc region comprises an additional mutation corresponding to position K439 within the CH3 domain, or the additional mutation may be present at position S440 when the first mutation is not present at position S440. [The present invention 1010] The polypeptide of the present invention 1009, wherein the additional mutation is selected from S440K or K439E. [The present invention 1011] Any polypeptide of the present invention, wherein the Fc region comprises up to 10 mutations, such as 9 mutations, such as 8 mutations, such as 7 mutations, such as 6 mutations, such as 5 mutations, such as 4 mutations, such as 3 mutations, or such as 2 mutations. [The present invention 1012] Any polypeptide of the present invention having 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% reduced Fc effector function compared to the parental polypeptide that is identical to the polypeptide having the same first mutation but no second mutation. [The present invention 1013] Any polypeptide of the present invention that does not induce Fc effector function. [The present invention 1014] The polypeptide of the present invention 1012-1013, 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] The polypeptide of any one of the present invention, which is an antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody. [The present invention 1016] The polypeptide of any one of the present invention, wherein the Fc region is a human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or a mixed isotype. [The present invention 1017] The polypeptide of any one of the present invention, wherein the Fc region is a human IgG1 isotype. [The present invention 1018] The polypeptide of any one of the present invention, which is a human antibody, a humanized antibody, or a chimeric antibody. [The present invention 1019] The polypeptide of any one of the present invention, wherein the antigen-binding region binds to a member of TNFR-SF. [The present invention 1020] The polypeptide of the present invention 1019, wherein TNFR-SF does not contain an intracellular death domain. [The present invention 1021] The polypeptide of the present invention 1019, wherein the member of TNFR-SF is selected from the group consisting of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR. [The present invention 1022] The polypeptide of the present invention 1020, wherein 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 the Fc effector function of a polypeptide comprising an Fc region of a human immunoglobulin and an antigen-binding region, 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 comprises the step of introducing a second mutation corresponding to (ii) position K322 or P329 in human IgG1 according to EU numbering, the method. [Inventive concept 1024] The method of inventive concept 1023, wherein the first mutation is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [Inventive concept 1025] The method of inventive concept 1023 or 1024, wherein the first mutation is selected from E430G or E345K. [Inventive concept 1026] The method according to any one of inventive concepts 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. [Inventive concept 1027] The method according to any one of inventive concepts 1023 to 1026, wherein the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D. [Inventive concept 1028] The method according to any one of inventive concepts 1023 to 1027, wherein the Fc region comprises one or more additional mutations in the CH3 domain. [Inventive concept 1029] The method of inventive concept 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. [Inventive concept 1030] The method of inventive concept 1029, wherein the additional mutation is selected from S440K or K439E. [Invention 1031] The method according to any one 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 a parent polypeptide that is identical to a polypeptide having the same first mutation but no second mutation. [Invention 1032] The method according to any one of Inventions 1023 or 1031, wherein the Fc effector function is selected from the group consisting 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. [Invention 1033] The method according to Invention 1032, wherein 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% compared to a comparative antibody that is identical to the antibody except that it does not contain the second mutation. [Invention 1034] A composition comprising at least one polypeptide according to any one of Inventions 1001 to 1022. [Invention 1035] The composition according to Invention 1034, comprising one or more polypeptides according to any one of the above Inventions. [Invention 1036] The composition according to any one of Inventions 1034 to 1035, comprising a first polypeptide and a second polypeptide as defined in any one of Inventions 1001 to 1022. [Invention 1037] Comprising a first polypeptide comprising a first antigen-binding region and a first Fc region, a second polypeptide comprising a second antigen-binding region and a second Fc region, or an antibody, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, (iii) additional mutations, and these 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) a further mutation at K439 or S440, provided that if the further mutation is at S440, the first mutation is not at S440, and the first and second Fc regions do not contain the further mutation at the same amino acid position. The composition of the present invention 1036. [The present invention 1038] A composition according to any one of the present inventions 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. [The present invention 1039] A composition according to any one of the present inventions 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 no intracellular death domain, such as OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. [The present invention 1040] A composition according to any one of the present inventions 1036 to 1037, wherein a first polypeptide that binds to one member of the TNFR-SF having no intracellular death domain, such as OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR, does not block the binding of a second antibody that binds to one member of the TNFR-SF having no 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 molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 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, 2:1, and any composition of the present invention from 1034 to 1040. [Invention 1042] Any composition of the present invention from 1034 to 1040, wherein the first polypeptide, the second polypeptide, and / or any additional polypeptide are present in the composition in an equimolar ratio. [Invention 1043] Any composition of the present invention from 1034 to 1042, which is a pharmaceutical composition. [Invention 1044] Any polypeptide of the present invention from 1001 to 1022 or any composition of the present invention from 1034 to 1043 for use as a medicament. [Invention 1045] Any polypeptide of the present invention from 1001 to 1022 or any composition of the present invention from 1034 to 1044 for use in the treatment of cancer, autoimmune disease, inflammatory disease, or infectious disease. [Invention 1046] A method of treating an individual having a disease, comprising the step of administering to the individual an effective amount of any antibody or composition of the present invention. [Invention 1047] The method of the present invention 1046, wherein the disease is selected from the group consisting of cancer, autoimmune disease, inflammatory disease, and infectious disease. [Invention 1048] A method according to any one of the methods of the present invention 1046 - 1047, comprising the step of further administering a further therapeutic agent. [The present invention 1049] A method according to the present invention 1048, wherein the further therapeutic agent is one or more anti - cancer agents selected from the group consisting of chemotherapeutic drugs (including, but not limited to, paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5 - fluorouracil, pemetrexed), kinase inhibitors (including, but not limited to, sorafenib, sunitinib, or everolimus), apoptosis regulators (including, but not limited to, recombinant human TRAIL or birinapant), RAS inhibitors, proteasome inhibitors (including, but not limited to, bortezomib), histone deacetylase inhibitors (including, but not limited to, vorinostat), nutraceuticals, cytokines (including, but not limited to, IFN - γ), antibodies or antibody mimetics (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), antibody - drug conjugates. [The present invention 1050] A kit of parts comprising any polypeptide or composition of the present invention, wherein the polypeptide or composition is present in one or more containers, for example, vials. [The present invention 1051] The kit of parts according to the present invention 1050, wherein any polypeptide or composition of the present invention is for simultaneous, separate, or sequential use in therapy. [The present invention 1052] Use of any polypeptide or composition of the present invention 1001 - 1043 for the manufacture of a medicament for the treatment of a disease. [The present invention 1053] The use according to 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 present invention, in particular, various uses and therapeutic applications of the above - mentioned polypeptides or antibodies, will be further described in more detail below.

Brief Description of Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0030] DETAILED DESCRIPTION OF THE INVENTION In the description of the embodiments of the present invention, specific technical terms are used for clarity. However, it should be understood that the present invention is not intended to be limited to the specific terms so selected, and each such specific term includes all technical equivalents that function in a similar manner to achieve a similar purpose.

[0031] Definition The term "parent polypeptide" or "parent antibody" is identical to the polypeptide or antibody according to the present invention, but the parent polypeptide or parent antibody 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, and is understood to be a polypeptide or antibody.

[0032] The term "polypeptide comprising an Fc region and a binding region of an immunoglobulin" refers, in the context of the present invention, to a polypeptide comprising an Fc region of an immunoglobulin and a binding region having the ability to bind to any molecule present on, for example, a cell, bacterium, or virion, such as a polypeptide. The Fc region of an immunoglobulin is typically an antibody fragment that can be generated after digestion of an antibody with papain (which is known to those skilled in the art), and is defined as an antibody fragment comprising two CH2-CH3 regions of the immunoglobulin and a linking region, such as a hinge region. The antibody isotype, for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE, is defined by the constant domain of the antibody heavy chain. The Fc region mediates the effector functions of the antibody together with cell surface receptors called Fc receptors and proteins of the complement system. The binding region can be a polypeptide sequence, such as a protein, protein ligand, receptor, antigen-binding region, or ligand-binding region having the ability to bind to a cell, bacterium, or virion. When the binding region is, for example, a receptor, the "polypeptide comprising an Fc region and a binding region of an immunoglobulin" may be prepared as a fusion protein of the Fc region of the immunoglobulin and the binding region. When the binding region is an antigen-binding region, the "polypeptide comprising an Fc domain and a binding region of an immunoglobulin" can be an antibody such as a chimeric, humanized, or human antibody, or an antibody consisting of only the heavy chain, or a ScFv-Fc fusion. The polypeptide comprising an Fc region and a binding region of an immunoglobulin can typically comprise a linking region, such as a hinge region, and two CH2-CH3 regions of the heavy chain of the immunoglobulin, and thus the "polypeptide comprising an Fc region and a binding region of an immunoglobulin" can be a "polypeptide comprising at least an Fc region and a binding region of an immunoglobulin". The term "Fc region of an immunoglobulin" means, in the context of the present invention, that a linking region, such as a hinge, and CH2 and CH3 regions corresponding to the subtype of the antibody of the immunoglobulin, for example, human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE, are present. The polypeptide is not limited to human origin and 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 fragments of the crystallizable region of an antibody. These different terms are used interchangeably and can constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. The term "parent polypeptide" or "parent antibody" is identical to the polypeptide or antibody according to the present invention, but the parent polypeptide or parent antibody has no second mutation and has a first mutation that is an Fc-Fc enhancing mutation, for example, at position E345, E430, or S440. As a result, the parent polypeptide or parent antibody is understood to be a polypeptide or antibody that has increased Fc-Fc mediated oligomerization, increased Fc effector function, such as CDC, and may also have other enhanced effector functions. As described above, unless otherwise specified or clearly inconsistent with the context, the term "parent polypeptide" or "parent antibody" refers to a polypeptide or antibody that has a first mutation for Fc-Fc enhancement but no second mutation that reduces one or more Fc effector functions. Thus, a polypeptide or antibody contains one or more mutations compared to a "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 216-230 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] As used herein, the term "CH3 region" or "CH3 domain" 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 the EU numbering. However, the CH3 region may also be of any other subtype described herein.

[0037] The term "immunoglobulin" refers to a structurally related class of glycoproteins consisting of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four of which are strongly interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (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 are regions of hypervariability (or hypervariable regions, which can be hypervariable in the form of loops defined by sequence and / or structure) and are also referred to as complementarity determining regions (CDRs), and can be further subdivided into intervening, more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise specified or inconsistent with the context, references to amino acid positions within the constant regions herein follow EU numbering (Edelman et al., Proc Natl Acad Sci U S 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, having the ability to specifically bind to an antigen. The antibodies of the present invention comprise the Fc domain of an immunoglobulin and an antigen-binding region. Antibodies generally comprise two CH2-CH3 regions and a linking region, such as a hinge region, and at least the Fc domain. Thus, the antibodies of the present invention can be those comprising 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 an antigen. 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, such as C1q, the first component of the classical pathway of complement activation. Also, the antibody can be a multispecific antibody, such as a bispecific antibody or a similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes can be present on the same target or on different targets. When the epitopes are present on different targets, such targets can be present on the same cell or on different cells or cell types. As described above, unless otherwise specified or clearly inconsistent with the context, the term "antibody" in this specification includes antibody fragments that comprise 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 methods. It has been shown that the antigen-binding function of an antibody can also be exerted by fragments of a full-length antibody. Examples of binding fragments included within the term "Ab" or "antibody" include, without limitation, monovalent antibodies (described in WO2007059782 by Genmab); heavy chain antibodies consisting only of two heavy chains, such as those naturally occurring in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche,WO2011069104), an asymmetric chain exchange modified operation domain (SEED or Seed-body), and a bispecific antibody-like molecule (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 immunoglobulin (Abbott, DVD-Ig, US Patent No. 7,612,181); double domain double head antibody (Unilever; Sanofi Aventis, WO20100226923), di-diabody (ImClone / Eli Lilly), antibody format by Knobs-into-holes (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 antibody (GSK / Domantis), Two-in-one Antibodies or Dual action Fab that recognize two targets (Genentech,NovImmune, Adimab), bridged Mab (Karmanos Cancer Center), covalently linked fusion mAb (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecificity (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), bispecific affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybody (Zyngenia), approach using a common light chain (Crucell / Merus, US7262028) or approach using a common heavy chain (κλBody by NovImmune, WO2012023053), as well as fusion proteins containing polypeptide sequences fused to antibody fragments containing an Fc domain, such as scFv fusions, such as 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), as well as bispecific scFv fusions. The term "antibody" also includes, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies) (Oligoclonics) obtained by techniques such as those utilized by Symphogen and Merus, 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. The antibodies produced can potentially have any isotype.

[0039] The term "full-length antibody" as used herein refers to an antibody that includes all of the constant and variable domains corresponding to those normally found in the wild-type antibody of its 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 present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-directed mutagenesis in vitro or somatic mutagenesis 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, e.g., mouse, are 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 includes a foreign variable domain (derived from a non-human species or engineered from any species including synthetic or human) linked to a constant region of human origin. The variable domains of the chimeric chain, when analyzed as a whole, have a V-region amino acid sequence closer to that of a non-human species than to human.

[0042] As used herein, the term "humanized antibody" refers to an antibody in which both chain types have been humanized as a result of antibody modification procedures. A humanized chain typically has a variable domain in which the complementarity-determining regions (CDRs) are foreign (from a non-human species or synthetic in origin), while the remainder of the chain is of human origin. Since the evaluation of humanization is based on the resulting amino acid sequence rather than the methodology itself, it is possible to use protocols other than grafting. The variable domain of a humanized chain, when analyzed as a whole, has a V-region amino acid sequence that is closer to that of humans than to other species. As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", etc. refer to a preparation of Ab molecules of single-molecule 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 having a single binding specificity and having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas, which are transgenic or chromosomally introduced non-human animals, such as transgenic mice, having a genome rearranged to contain a human heavy-chain transgene repertoire and a light-chain transgene repertoire and producing functional human antibodies, and which contain B cells fused with immortalized cells.

[0043] As used herein, the term "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotype thereof, such as IgG1m(za) and IgG1m(f)). Further, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain. As used herein, the term "mixed isotype" refers to the Fc region of an immunoglobulin obtained by combining a structural entity of one isotype with a similar region derived from another isotype, thereby generating a hybrid isotype. The mixed isotype may include an Fc region having an array composed of two or more isotypes selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, whereby combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA can be produced.

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

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

[0046] The term "epitope" means a determinant of a protein that has the ability to specifically bind to an antibody variable domain. An epitope usually consists of a group of surface molecules, such as amino acids, sugar side chains, or a combination thereof, and usually has specific three-dimensional structural features as well as specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that the binding to the former is lost in the presence of a denaturing solvent while the binding to the latter is not lost. An epitope may include amino acid residues that are directly involved in the binding (also referred to as the immunodominant components of the epitope) and other amino acid residues that are not directly involved in the binding.

[0047] The term "antibody variant" or "variant of a parental antibody" in the present invention refers to an antibody molecule that contains one or more mutations as compared to the "parental antibody". These different terms are used interchangeably and may constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Similarly, the "variant of a polypeptide comprising an Fc region and a binding region of an immunoglobulin" or "variant of a polypeptide comprising an Fc region and a binding region of a parental immunoglobulin" in the present invention refers to a "polypeptide comprising an Fc region and a binding region of an immunoglobulin" that contains one or more mutations as compared to the "polypeptide comprising an Fc region and a binding region of a parental immunoglobulin". These different terms are used interchangeably and may constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary mutations include deletions, insertions, and substitutions of amino acids in the parental amino acid sequence. Amino acid substitutions can be such that a natural amino acid is replaced with another naturally occurring amino acid or an amino acid derivative that does not occur naturally. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions can be defined by substitutions within one or more of the amino acid classes shown in the following three tables.

[0048] Classes of Amino Acid Residues for Conservative Substitutions TIFF0007695293000001.tif52135

[0049] Classes of alternative conservative amino acid residue substitutions TIFF0007695293000002.tif39135

[0050] Alternative physical and functional classifications of amino acid residues TIFF0007695293000003.tif70139

[0051] In the context of the present invention, substitutions in mutants are shown as original amino acid - position - substituted amino acid and are denoted using the three - letter or one - letter code including code Xaa and X to indicate amino acid residues. Thus, the notation "E345R" or "Glu345Arg" means that the mutant contains a substitution of arginine for glutamic acid at the amino acid position corresponding to position 345 of the parental antibody in the mutant. The position itself does not exist in the antibody, but when the mutant contains an amino acid insertion, for example,

[0052] the notation "position - substituted amino acid", for example "448E", is used. Such notation is particularly relevant in the context of one or more modifications in a series of homologous polypeptides or antibodies.

[0053] Similarly, when the identity of one or more substituted amino acid residues is not important,

[0054] the original amino acid - position, i.e., is denoted as "E345". original amino acid - position, i.e., is denoted as "E345".

[0055] When one or more original amino acids and / or one or more substituted amino acids include modifications that include more than one but not all amino acids, and 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" can be used interchangeably in the context of the present invention.

[0056] Furthermore, the term "substitution" includes substitution with any one of the other 19 natural amino acids, or other amino acids such as non-natural amino acids. For example, substitution of the amino acid E at position 345 includes 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. Also, such substitutions can be denoted as E345A, E345C, etc., or E345A, C, etc., and E345A / C / , etc. The same applies to any position described herein, and any one of such substitutions is specifically included herein.

[0057] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as opposed to the recognition or activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, such as cytotoxic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and exhibit specific immune functions. In some embodiments, effector cells such as natural killer cells have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcRs are involved in the specific killing of target cells and antigen presentation to other components of the immune system, or binding to cells presenting antigens. In some embodiments, ADCC is further enhanced by antibody-driven classical complement activation, which can result in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs) expressed on myeloid cells, such as CR3. Recognition of complement fragments by CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. Such C3 cleavage products can directly promote complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. Expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFN γ) 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 lyse target cells. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. Such C3 cleavage products can directly or indirectly promote phagocytosis by effector cells, either directly or by enhancing antibody-mediated phagocytosis.

[0058] As used herein, the term "Fc effector function" 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 the cell membrane, where the Fc effector function is due 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 cell phagocytosis (ADCP), (vii) complement-dependent cell 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] As used herein, the term "reduced one or more Fc effector functions" is intended to refer to a reduction in the Fc effector function of a polypeptide or antibody when directly compared to the Fc effector function of a parental polypeptide or antibody in the same assay.

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

[0061] As used herein, the term "vector" is intended to refer to a nucleic acid molecule having the ability to induce transcription of a nucleic acid segment ligated into the vector. An example of a 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 case the nucleic acid segment can be ligated into the viral genome. Some specific vectors have the ability to self-replicate in the host cell into which the vector has been introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) may be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated together with the host genome. Furthermore, some specific vectors have the ability to direct the expression of a gene operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most common form of use of vectors, "plasmid" and "vector" may be used interchangeably herein. However, the present invention is intended to encompass such other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[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 a particular target cell but also to the progeny of such cell. Because such progeny may be modified by either mutation or environmental influences in subsequent generations, they may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Examples of 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 expressing an Ab or a target antigen, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, such as yeast cells.

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

[0065] As used herein, the term "affinity" is the strength with which one molecule, such as an antibody, binds to another, such as a target or antigen, at a single site, e.g., the strength of the 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 multiple binding sites between two structures, e.g., between multiple antigen-binding sites of an antibody that interact simultaneously with a target or between, for example, an antibody and C1q. When more than one binding interaction exists, the two structures dissociate only when all binding sites have dissociated, and thus the dissociation rate is slower than for individual binding sites, thereby resulting in a greater effective total binding strength (avidity) compared to the strength (affinity) with which individual binding sites bind.

[0067] As used herein, the term "oligomer" refers to a molecule (e.g., an antibody) consisting of more than one but a limited number of monomer units, as opposed to a polymer consisting of a potentially infinite number of monomers, at least in principle. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Often, Greek prefixes are used to denote the number of monomer units within an oligomer; for example, a tetramer consists of 4 units and a hexamer consists of 6 units.

[0068] The term "oligomerization" as used herein is intended to refer to the process of converting monomers to a finite degree of polymerization. In the present specification, it has been observed that polypeptides, antibodies, and / or other dimeric proteins containing the target binding region according to the present invention can form oligomers, such as hexamers, by non-covalent association of the Fc region, for example, after target binding on the cell surface. Oligomerization of antibodies can be evaluated, for example, in a cell viability assay using anti-DR5 antibodies containing Fc-Fc enhancing mutations such as E430G or E345R (as described in Example 13). Fc-Fc mediated oligomerization of polypeptides or antibodies occurs by intermolecular association of the Fc regions between adjacent polypeptides or antibodies after target binding on the (cell) surface, and is increased by the introduction of a first mutation 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 that blocks Fc-Fc interaction. Induction of oligomerization can be evaluated in an assay by comparing the responses of the following groups; Group I) an antibody having a wild-type Fc region, Group II) an antibody identical to the antibody of Group I) except that it contains a first mutation according to the present invention, such as E430G, Group III) the DCAWHLGELVWCT peptide in combination with an antibody identical to the antibody of Group I) except that it contains a first mutation according to the present invention, such as E430G, Group IV) an antibody identical to the antibody of Group I) except that it contains a first mutation according to the present invention, such as E430G and a second mutation according to the present invention, such as P329D. By comparing the responses of Group I and Group II, it is possible to evaluate the response of enhanced oligomerization. By comparing the responses of Group II and Group III, it is possible to evaluate the response that blocks enhanced oligomerization. By comparing the responses of Group II and Group IV, it is possible to evaluate whether enhanced oligomerization is maintained.Which assay is preferred for evaluating an oligomerization-dependent response depends on which target antigen the antibody binds to (which will be apparent to those skilled in the art). Thus, for antibodies that bind to target antigens that induce programmed cell death (PCD) having an intracellular death domain, such as DR5, FAS, DR4, and TNFR1, such as those that bind to TNFR-SF, an assay suitable for examining oligomerization may be a viability assay as described in Example 13. The viability assay can be performed in BxPC-3 cells in the presence of an antibody according to the above assay groups, i.e., Group I, Group II, Group III, and / or Group IV. BxPC-3 cells are incubated at 37°C for 3 days with an antibody according to the above assay groups at 5 μg / mL or 10 μg / mL. The percentage of viable cells can be determined in a CellTiter-Glo luminescent cell viability assay (Promega, catalog number G7571). For antibodies that bind to co-stimulatory immune receptors that do not have a death domain, such as TNFR-SF, such as OX40, CD40, CD30, CD27, 4-1BB, RANK, and GITR, an assay suitable for examining oligomerization may be an NFAT reporter bioassay. The NFAT reporter bioassay can be performed using Jurkat NFAT reporter cells that stably express the target antigen (which will be apparent to those skilled in the art), such as NFκB-luc2 / OX40 Jurkat cells that express a luciferase reporter gene under the control of an NFAT response element and have membrane expression of OX40, in the presence of the above assay groups, i.e., Group I, Group II, Group III, and / or Group IV. NFκB-luc2 / OX40 Jurkat cells are incubated at 37°C for 1 day with an antibody according to the above assay groups at 1.5 or 5 μg / mL. Luciferase expression induced by activation of OX40 can be measured by measuring the luminescence signal.

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

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

[0071] As used herein, the term "C1q binding" is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to an antigen. It is understood that the antibody bound to the antigen can occur both in vivo and in vitro in the situations described herein. C1q binding can be evaluated, for example, by using an antibody immobilized on an artificial surface or an antibody bound to a predetermined antigen on the cell surface or virion surface (as described in Examples 3 and 11). The binding of C1q to an antibody oligomer is understood herein to be a multivalent interaction that results in high avidity binding. For example, a reduction in 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 that of its parental polypeptide or antibody without the second mutation in the same assay, as exemplified in Example 3. Briefly, cells of a suitable origin expressing the target antigen to which the antigen-binding region of the antibody binds can be used in this assay, and such cell lines or cell types will be apparent to those skilled in the art. Thus, for an antibody that binds to a target antigen on a cancer cell, such as DR5, cancer cells, such as BxPC-3 human pancreatic cancer cells (ATCC CRL-1687), may be suitable for this assay. On the other hand, for an antibody that binds to OX-40 expressed on T cells, T cells, such as Jurkat human T cells (ATCC TIB-152), may be suitable for this assay. The reduced C1q binding of the antibody according to the present invention is 1×10 6Appropriate cells at a concentration of mL can be evaluated by incubating in a polystyrene round-bottom 96-well plate: i) in the presence of 20% C4-depleted serum with antibodies containing the first and second mutations according to the present invention at a series of concentrations (0.0003 to 100 μg / mL); and ii) in the presence of 20% C4-depleted serum with parental antibodies containing the first mutation but not the second mutation at a series of concentrations (0.0003 to 100 μg / mL). Here, the antibodies in i) and ii) are incubated with the appropriate cells at 4°C for 30 minutes, and then incubated with a labeled anti-human C1q antibody, such as FITC-labeled rabbit anti-HuC1q, and C1q binding is measured by flow cytometry. Alternatively, the reduced C1q binding of the antibodies according to the present invention can be determined in an enzyme-linked immunosorbent assay (ELISA) for C1q binding. A 96-well Microlon ELISA plate (Greiner, catalog number 655092) is coated with: i) a dilution series of antibodies containing the first and second mutations according to the present invention (from 0.001 to 20 μg / mL); and ii) a dilution series of antibodies containing the first mutation but not the second mutation (from 0.001 to 20 μg / mL) (in 100 μL of PBS), incubated overnight at 4°C. Subsequent incubations are carried out with washing between incubations, with 200 μL / well of 0.5× PBS supplemented with 0.025% Tween 20 and 0.1% gelatin at room temperature for 1 hour (blocking), 100 μL of 3% NHS (Sanquin, Ref. M0008AC) at 37°C for 1 hour, 100 μL of rabbit anti-human C1q (DAKO, catalog number A0136, 1 / 4,000) at room temperature for 1 hour, and 100 μL of porcine anti-rabbit IgG horseradish peroxidase (HRP) (DAKO, catalog number P0399, 1 / 10,000) as the detection antibody at room temperature for 1 hour; finally, it is carried out with 100 μL of substrate containing 1 mg / mL of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, catalog number 11112 597001) at room temperature for about 15 minutes; the reaction is stopped by the addition of 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 the activation of the classical complement pathway initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on the surface. C1 is a complex consisting of six recognition proteins C1q and a heterotetramer of serine proteases C1r2C1s2. C1 is the first protein complex in the initial events of the classical complement cascade, which involves a series of cleavage reactions starting with the cleavage of C4 into C4a and C4b and C2 into C2a and C2b. C4b deposits and together with C2a forms an enzymatic active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, and C5 convertase is formed. This C5 convertase cleaves C5 into C5a and C5b, and the final components deposit on the membrane, thereby inducing the later events of complement activation in which the terminal complement components C5b, C6, C7, C8, and C9 are assembled into a 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 evaluated by using C1q efficacy, CDC kinetics CDC assays (such as those described in WO2013 / 004842, WO2014 / 108198), or by the cellular deposition of C3b and C4b, which is the method described in Beurskens et al April 1, 2012 vol.188 no.7 3532-3541.

[0073] The term "complement-dependent cytotoxicity" ("CDC"), as used herein, is intended to refer to the process of antibody-mediated complement activation that results in lysis of an antibody bound to a target on a cell or virion as a result of pores in the membrane created by the MAC complex. CDC can be assayed in vitro, for example, by the CDC assay in which normal human serum is used as a source of complement, as described in Examples 2, 3, 4, and 6, or can be evaluated at a series of C1q concentrations. For example, a reduction in 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 parental polypeptide or antibody that does not have the second mutation in the same assay, as illustrated 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 death 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, a reduction in 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 parental polypeptide or antibody that does not have the second mutation in the same assay, as illustrated in Examples 10 and 9.

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

[0076] The term "complement-dependent cytotoxicity" ("CDCC"), as used herein, is intended to refer to the mechanism of death of target cells or virions by cells expressing complement receptors that recognize cleavage products of complement component 3 (C3) covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC can be evaluated 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 decline to half of its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase is typically 1 to 3 days, during which a reduction of approximately 50% in plasma concentration is seen due to redistribution between plasma and tissues. The 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] As used herein, the term "antibody-drug conjugate" refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that couples the drug, for example, to 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] As used herein, the term "antibody-drug conjugate uptake" refers to the process by which, after an antibody-drug conjugate binds to a target on a cell, it is taken up / encapsulated by the cell membrane and thereby drawn into the cell. Antibody-drug conjugate uptake can be evaluated as described in "antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay" as described in WO 2011 / 157741.

[0081] As used herein, the term "apoptosis" refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events can result in characteristic changes (morphological structure) and death of the cells. Such changes include cytoplasmic blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and fragmentation of chromosomal DNA. Apoptosis can be induced by the binding of an antibody to a specific receptor.

[0082] As used herein, the term "programmed cell death" or "PCD" refers to any form of cell death mediated by an intracellular program. Different forms of PCD exist, and it is common for various types of PCD to be executed by active cell processes that can be disrupted by interfering with intracellular signaling. In one particular aspect, the occurrence of any form of PCD in a cell or tissue can be examined by staining the cell or tissue with conjugated annexin V and correlating it with phosphatidylserine exposure.

[0083] As used herein, the term "annexin V" refers to a group of annexin proteins that bind 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 with that of its parental polypeptide or antibody that does not have the additional mutation in the same assay, as exemplified in Example 21.

[0085] The terms "Fc gamma receptor", "Fc gamma R", "Fcγ receptor", "FcγR" can 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), FcγRIIIB (CD16b), which have different antibody affinities due to their different molecular structures. FcγRI binds IgG more strongly than FcγRII or FcγRIII.

[0086] As used herein, the term "FcRn" is intended to refer to the neonatal Fc receptor, which is an Fc receptor. This was first discovered in rodents as a unique receptor with the ability to transport IgG from mother's milk through the intestinal epithelium of neonatal rodents into the bloodstream of the neonates. Further studies have revealed a similar receptor in humans. However, in humans, this has been found in the placenta, which aids in facilitating the transport of maternal IgG to the developing fetus, and has also been shown to play a role in monitoring IgG catabolism. FcRn binds to IgG at an acidic pH of 6.0 - 6.5 but not at neutral or higher pH. Thus, FcRn can bind to IgG from the slightly acidic intestinal lumen (inside the intestine) and ensure 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 adult IgG recycling by being present within the endocytosis pathway in endothelial cells. The FcRn receptor within the acidic endosome binds to IgG that has undergone internalization by endocytosis, allows it to be recycled to the cell surface, and releases it into the bloodstream at a basic pH, thereby suppressing its degradation by lysosomes. This mechanism may explain why the half-life of IgG in the blood is longer compared to other isotypes.

[0087] The term "Protein A", as used herein, is intended to refer to the 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, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found utility in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a 3-helix bundle. Each domain has the ability to bind proteins from many mammalian species, most notably IgG. It binds to the Fc region of the heavy chain of most immunoglobulins (overlapping the conserved binding site for the FcRn receptor) and also interacts with the Fab region of the human VH3 family. Due to such interactions in serum, IgG molecules bind to bacteria by their Fc regions rather than by their Fab regions alone, thereby disturbing opsonization, complement activation, and phagocytosis by this bacterium.

[0088] The term "Protein G", as used herein, is intended to refer to immunoglobulin-binding proteins expressed by group C and G streptococci, which are similar to Protein A but have different specificities. 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 embodiments of the present invention The present invention is based on the discovery of a need for therapeutic agents of polypeptides and antibodies that have enhanced Fc-Fc interactions when bound to corresponding antigens on the surface of target cells and thus form oligomers upon binding to the antigen, but do not have enhanced Fc effector functions, such as CDC and / or ADCC, which are commonly found in oligomer-forming polypeptides and antibodies, such as hexamers. 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 having a first mutation corresponding to one of amino acid positions E430, E345, or S440, the enhanced Fc-Fc interaction can be maintained, but the Fc effector functions, such as CDC and / or ADCC, are reduced compared to the parent of the same polypeptide or antibody having only the first mutation and no second mutation. In some embodiments, one or more effector functions can be reduced to levels below those seen in the wild-type polypeptide or antibody, i.e., the same one except having no first and second mutations.

[0090] In some embodiments, introduction of the second mutation results in a reduction of the Fc effector function to a level equal to or lower than that seen in the wild-type polypeptide or antibody. In some embodiments, introduction of the second mutation results in a reduction of the Fc effector function to a level equal to or lower than that seen in 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 human IgG, wherein the Fc region comprises CH2 and CH3 domains, and the Fc region has the following mutations 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 in K322 or P329 is provided in a polypeptide or an antibody.

[0092] The first mutation according to the present invention is at one of amino acid positions E430, E345, or S440, and introduces the effects of enhanced Fc-Fc interaction and oligomerization in the polypeptide or antibody. Furthermore, the enhanced oligomerization occurs when the antigen-binding region of the polypeptide or antibody is bound to the corresponding target antigen. This enhanced oligomerization results in the formation of oligomers such as hexamers, for example. The formation of an oligomer structure, such as a hexamer, has the effect of increasing Fc effector functions such as CDC and / or ADCC by increasing the C1q-binding avidity of the polypeptide or antibody. The second mutation according to the present invention, which is present at one of amino acid positions K322 or P329, introduces the effect of reducing the Fc effector function in the polypeptide or antibody. Such reduced Fc effector function can be, for example, reduced C1q binding or CDC activity. Thus, the second mutation counteracts the enhanced Fc effector function introduced by the first mutation, whereby a polypeptide or antibody having enhanced Fc-Fc interaction and oligomerization but not having an increased Fc effector function can be produced. That is, the Fc effector function is reduced compared to a polypeptide or antibody having the first mutation but not having the second mutation. In some cases where the wild-type polypeptide or antibody has an increased Fc effector function, such as CDC, the introduction of the first and second mutations can increase the oligomerization level, but the CDC level can be reduced to a level lower than that observed in the wild-type polypeptide or antibody. The polypeptide or antibody according to the present invention is particularly advantageous when an Fc effector function is not desirable, for example, when activating effector cells.

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

[0094] In one aspect of the present invention, the Fc region contains the first and second mutations, provided that the Fc region contains L and L at the positions corresponding to L234 and L235.

[0095] In one aspect 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 a preferred aspect of the present invention, the first mutation is selected from E430G or E345K. Thereby, an aspect is provided that enables enhanced oligomerization of the polypeptide or antibody upon binding to an antigen on the cell surface.

[0096] In one aspect of the present invention, the polypeptide contains at least one mutation that is an Fc-Fc enhancing mutation and at least one mutation that reduces the Fc effector function. That is, in one aspect of the present invention, the polypeptide contains i) at least one first mutation at the 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 the amino acid position corresponding to K322 or P329.

[0097] In one aspect, 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 within the first and / or second heavy chains. In one aspect 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 within both the first and second heavy chains. In a preferred aspect 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 and the second mutation are present within both the first and second heavy chains. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, the first mutation is present within the first and second heavy chains, and the second mutation is present within both the first and second heavy chains.

[0098] 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. In one aspect of the invention, the second mutation is K322E. This provides an aspect that enables inhibition of one or more Fc effector functions. In one aspect, the second mutation reduces the Fc effector function enhanced by the first mutation. In one aspect, the second mutation partially reduces the Fc effector function enhanced by the first mutation. In one aspect, the second mutation in the polypeptide or antibody can reduce the 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 parental polypeptide or parental antibody. In one aspect, the second mutation in the polypeptide or antibody can reduce the Fc effector function to a level equal to or lower than that seen in a polypeptide or antibody having no first and second mutations, i.e., the wild-type polypeptide or antibody. In one aspect, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, and one of the second mutations described above is present within the first and / or second heavy chains.

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

[0100] In one aspect, 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 and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one aspect, 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 and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one aspect, the second mutation is selected from the group consisting of P329R, P329K, P329D, P329E, and P329G and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one aspect, the second mutation is P329R and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one aspect, the second mutation is P329R and reduces ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding. In one aspect, the second mutation is P329R and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one aspect, the second mutation is P329K and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one aspect, the second mutation is P329D and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one aspect, the second mutation is P329E and reduces ADCC, FcγR binding, CDC, and / or C1q binding. In one aspect, 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 does not reduce CDC.

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

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

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

[0105] In another aspect of the present 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 present 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 present invention, the second mutation is selected from the group of P329R, P329K, and P329D.

[0108] In one aspect of the present invention, the first mutation is present at the 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 selected from one of the group consisting of.

[0109] In one embodiment of the present invention, the Fc region contains a first mutation at the 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 selected from one of the group consisting of, provided that the Fc region contains L and L at the positions corresponding to L234 and L235.

[0110] In one embodiment of the present invention, the first mutation is present at the 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 selected from one of the group consisting of.

[0111] In one embodiment of the present invention, the first mutation is present at the 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 selected from one of the group consisting of.

[0112] In one embodiment of the present 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 groups consisting of.

[0113] In one aspect of the present 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 aspect of the present invention, the Fc region includes 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 includes amino acids L and L at positions corresponding to L234 and L235.

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

[0116] In one aspect of the present invention, the first mutation is present at the 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 groups consisting of.

[0117] In one aspect of the present invention, the Fc region contains a first mutation at the 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 groups consisting of, provided that the Fc region contains L and L at the positions corresponding to L234 and L235.

[0118] In one aspect of the present invention, the first mutation is present at the 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 groups consisting of.

[0119] In one aspect of the present invention, the first mutation is present at the 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 selected from one of the group consisting of

[0120] In one aspect of the present 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 selected from one of the group consisting of

[0121] In one aspect of the present 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 aspect of the present invention, the first mutation is E345K and the second mutation is K322E. In one aspect of the present invention, the first mutation is E345K and the second mutation is K322D. In one aspect of the present invention, the first mutation is E345K and the second mutation is K322N. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329H. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329K. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329R. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329D. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329E. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329M. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329F. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329G. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329I. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329L. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329N. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329S. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329T. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329V. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329W. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E345K and the second mutation is P329A.

[0123] In one aspect of the present 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 aspect of the present invention, the first mutation is E430S and the second mutation is K322E. In one aspect of the present invention, the first mutation is E430S and the second mutation is K322D. In one aspect of the present invention, the first mutation is E430S and the second mutation is K322N. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329H. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329K. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329R. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329D. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329E. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329M. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329F. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329G. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329I. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329L. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329N. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329S. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329T. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329V. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329W. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E430S and the second mutation is P329A.

[0125] In one aspect of the present 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 aspect of the present invention, the first mutation is E430F and the second mutation is K322E. In one aspect of the present invention, the first mutation is E430F and the second mutation is K322D. In one aspect of the present invention, the first mutation is E430F and the second mutation is K322N. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329H. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329K. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329R. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329D. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329E. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329M. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329F. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329G. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329I. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329L. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329N. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329S. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329T. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329V. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329W. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E430F and the second mutation is P329A.

[0127] In one aspect of the present 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 aspect of the present invention, the first mutation is E430T and the second mutation is K322E. In one aspect of the present invention, the first mutation is E430T and the second mutation is K322D. In one aspect of the present invention, the first mutation is E430T and the second mutation is K322N. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329H. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329K. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329R. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329D. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329E. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329M. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329F. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329G. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329I. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329L. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329N. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329S. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329T. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329V. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329W. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E430T and the second mutation is P329A.

[0129] In one aspect of the present 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 aspect of the present invention, the first mutation is E345Q and the second mutation is K322E. In one aspect of the present invention, the first mutation is E345Q and the second mutation is K322D. In one aspect of the present invention, the first mutation is E345Q and the second mutation is K322N. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329H. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329K. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329R. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329D. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329E. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329M. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329F. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329G. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329I. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329L. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329N. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329S. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329T. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329V. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329W. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E345Q and the second mutation is P329A.

[0131] In one aspect of the present 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 aspect of the present invention, the first mutation is E345R and the second mutation is K322E. In one aspect of the present invention, the first mutation is E345R and the second mutation is K322D. In one aspect of the present invention, the first mutation is E345R and the second mutation is K322N. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329H. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329K. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329R. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329D. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329E. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329M. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329F. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329G. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329I. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329L. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329N. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329S. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329T. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329V. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329W. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E345R and the second mutation is P329A.

[0133] In one aspect of the present 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 aspect of the present invention, the first mutation is E345Y and the second mutation is K322E. In one aspect of the present invention, the first mutation is E345Y and the second mutation is K322D. In one aspect of the present invention, the first mutation is E345Y and the second mutation is K322N. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329H. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329K. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329R. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329D. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329E. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329M. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329F. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329G. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329I. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329L. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329N. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329Q. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329S. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329T. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329V. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329W. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329Y. In one aspect of the present invention, the first mutation is E345Y and the second mutation is P329A.

[0135] In one aspect of the present 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 groups consisting of

[0136] In one aspect of the present 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 groups consisting of, provided that the Fc region contains L and L at positions corresponding to 234 and 235.

[0137] In one aspect of the present 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 groups consisting of

[0138] In one aspect of the present 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 selected from one of the group consisting of

[0139] In one aspect of the present 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 aspect of the present invention, the first mutation is S440W and the second mutation is K322E. In one aspect of the present invention, the first mutation is S440W and the second mutation is K322D. In one aspect of the present invention, the first mutation is S440W and the second mutation is K322N. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329H. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329K. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329R. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329D. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329E. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329M. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329F. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329G. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329I. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329L. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329N. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329Q. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329S. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329T. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329V. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329W. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329Y. In one aspect of the present invention, the first mutation is S440W and the second mutation is P329A.

[0141] In one aspect of the present 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 aspect of the present invention, the first mutation is S440Y and the second mutation is K322E. In one aspect of the present invention, the first mutation is S440Y and the second mutation is K322D. In one aspect of the present invention, the first mutation is S440Y and the second mutation is K322N. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329H. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329K. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329R. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329D. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329E. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329M. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329F. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329G. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329I. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329L. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329N. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329Q. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329S. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329T. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329V. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329W. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329Y. In one aspect of the present invention, the first mutation is S440Y and the second mutation is P329A.

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

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

[0145] In one aspect of the invention, the Fc region comprises further mutations within the CH3 domain corresponding to K439 or, if the first mutation is not at position S440, may be at position S440. In one aspect of the invention, the Fc region comprises further mutations within the CH3 domain corresponding to one of positions S440 or K439, provided that the first mutation is not at S440. Polypeptides or antibodies according to the invention comprising the first and second mutations and further mutations at position S440, such as S440K, do not form oligomers with polypeptides or antibodies comprising further mutations at position S440, such as S440K. Polypeptides or antibodies according to the invention comprising the first and second mutations and further mutations at position K439, such as K439E, do not form oligomers with polypeptides or antibodies having a mutation at position K439, such as K439E. In one aspect of the invention, the further mutation is selected from S440K or K439E. Polypeptides or antibodies comprising a further mutation that is K439E or S440K do not form oligomers with polypeptides having the same mutation. Without being bound by theory, K439E and S440K can be viewed as complementary mutations, and thus an Fc region comprising the K439E mutation does not result in Fc-Fc interactions with another Fc region comprising the K439E mutation. However, an Fc region comprising the K439E mutation does result in Fc-Fc interactions with another Fc region comprising the S440K mutation. The same situation is seen with Fc regions comprising the S440K mutation, which do not result in Fc-Fc interactions with another Fc region comprising the S440K mutation. Thus, polypeptides or antibodies comprising the K439E mutation form oligomers with polypeptides or antibodies comprising the S440K mutation in an alternating pattern.

[0146] In one aspect of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, (iii) a further mutation, which mutations correspond to the following amino acid positions within human IgG1 according to EU numbering: (i) the 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) A further mutation at K439 or S440, provided that if the further mutation is at S440, the first mutation is not at S440.

[0147] In one aspect of the invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, and these mutations correspond to the following amino acid positions within 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; (ii) A second mutation at E322 or P329; (iii) A further K439E or S440K mutation, provided that if the further mutation is S440K, the first mutation is not at S440; wherein the Fc region contains the wild-type amino acids L and L at positions corresponding to L234 and L235.

[0148] In one aspect of the invention, the Fc region comprises (i) a first mutation at the amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, and the second and the further mutations are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of.

[0149] In one aspect of the invention, the Fc region comprises (i) a first mutation at the amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, and the second and the further mutations are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of; wherein the Fc region contains the wild-type amino acids L and L at positions corresponding to L234 and L235.

[0150] In one aspect of the invention, the Fc region contains (i) a first mutation, (ii) a second mutation, and (ii) a further mutation, and these mutations are (i) E430G, E430S, E430F, and E430T; (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of.

[0151] In one aspect of the invention, the Fc region contains (i) a first mutation at the amino acid position corresponding to E345, and (ii) a second mutation and (iii) a further mutation, and the second and the further mutations are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of; wherein the Fc region contains the wild-type amino acids L and L at positions corresponding to L234 and L235.

[0152] In one aspect of the present invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, and 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, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of.

[0153] In one aspect of the present invention, the Fc region comprises (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, and 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, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E selected from the group consisting of.

[0154] In one aspect of the present invention, the Fc region comprises additional mutations that are hexamerization-inhibiting mutations corresponding to K439E or S440K (according to EU numbering) in human IgG1. That is, in one aspect of the present invention, the Fc region comprises a hexamerization-enhancing mutation, such as E430G, and a hexamerization-inhibiting mutation, such as K439E. In one aspect of the present invention, the Fc region comprises a hexamerization-enhancing mutation, such as E345K, and a hexamerization-inhibiting mutation, such as K439E. In another aspect of the present invention, the Fc region comprises a hexamerization-enhancing mutation, such as E430G, and a hexamerization-inhibiting mutation, such as S440K. In one aspect of the present invention, the Fc region comprises a hexamerization-enhancing mutation, such as E345K, and a hexamerization-inhibiting mutation, such as S440K. Thereby, an aspect is provided that enables exclusive hexamerization between a combination of an antibody containing the K439E mutation and an antibody containing the S440K mutation.

[0155] The polypeptide or antibody according to the present invention has at least the first and second mutations, but as described above, may also have additional mutations for introducing additional functions into the polypeptide or antibody. In one aspect, the Fc region comprises up to 10 mutations, such as 9 mutations, such as 8 mutations, such as 7 mutations, such as 6 mutations, such as 5 mutations, such as 4 mutations, such as 3 mutations, or such as 2 mutations.

[0156] Thereby, an aspect is provided that enables the polypeptide or antibody of the present invention to have additional mutations for introducing additional properties into the polypeptide or antibody. Further, the additional mutations also allow for variations at positions that do not participate in Fc-Fc interactions within the Fc region and at positions that do not participate in Fc effector functions. Further, the additional mutations may be due to allelic mutations.

[0157] In one aspect of the present invention, the polypeptide or antibody has an Fc effector function that is at least 20% reduced compared to the parental polypeptide or antibody that is identical to the antibody except for not containing the second mutation. That is, in the polypeptide or antibody having the first and second mutations, the second mutation has the effect of reducing the effector function of the polypeptide or antibody by at least 20% compared to the parental polypeptide or antibody having only the first mutation. In another aspect of the present 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%, at least 95% compared to the parental polypeptide or antibody having only the first mutation.

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

[0159] It is understood that in one aspect according to the present invention, the reduction in the Fc effector function or activity of the polypeptide having the first and second mutations is in the case of comparing the polypeptide with a parental polypeptide having the same first mutation in the same antigen-binding region and an Fc region but no second mutation in the Fc region.

[0160] It is understood that in another aspect according to the present invention, the reduction in the Fc effector function or activity of the polypeptide having the first and second mutations is in the case of comparing the polypeptide with a parental polypeptide having the same antigen-binding region and Fc region and no first and second mutations in the Fc region, i.e., a wild-type antibody.

[0161] In one aspect according to the present invention, the second mutation reduces at least one effector function. In one aspect according to the present invention, the second mutation reduces more than one effector function. In one aspect according to the present invention, the second mutation reduces CDC activity. In one aspect according to the present invention, the second mutation reduces ADCC activity. In another aspect, the second mutation reduces both CDC and ADCC activities. In one aspect according to the present invention, the second mutation reduces FcγRIIIa signaling. In a further aspect according to the present invention, the second mutation reduces CDC activity but does not reduce ADCC activity or FcγRIIIa signaling. That is, in some aspects 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 aspect according to the present invention, the second mutation reduces CDC activity but still retains a substantial ADCC activity.

[0162] In one aspect 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 aspect, 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 of the second mutations show a reduction of more than one effector function. Specific mutations that reduce CDC activity were also characterized by reduced ADCC activity and reduced FcγRIIIa binding. Such mutations include mutations selected from the group consisting of 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 were also found. Such mutations include those of the group containing P329A. Some of the second mutations, such as P329R and P329K, did not show FcγRIa binding. Some of the second mutations, such as P329G and P329A, showed some reduction in binding to FcγRIa binding.

[0164] Thereby, a novel polypeptide or antibody-based therapeutic agent having reduced Fc effector function is provided. Further, the present invention provides a more selective Fc effector function exertion ability of an Fc-Fc enhancing polypeptide or antibody.

[0165] In one aspect of the present 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 polypeptide of the present invention is not limited to antibodies having a natural, e.g., human, Fc domain, and may be an antibody having mutations other than those of the present invention, such as mutations that affect glycosylation or mutations that enable the antibody to become a bispecific antibody. The term "natural antibody" is intended to mean any antibody that does not contain any genetically introduced mutations. Thus, it will be understood that antibodies containing naturally occurring modifications, e.g., different allotypes, are "natural antibodies" in the meaning of the present invention and can thereby be understood as parental antibodies. Such antibodies can serve as templates for one or more mutations according to the present invention, whereby mutant antibodies of the present invention can be provided. An example of a parental antibody containing mutations other than those of the present invention is the bispecific antibody described in WO2011 / 131746 (Genmab), which uses reducing conditions to promote half-molecule exchange of two antibodies containing an IgG4-like CH3 region and thus forms a bispecific antibody without accompanying formation of aggregates. Other examples of parental antibodies include, but are not limited to, bispecific antibodies, e.g., heterodimeric bispecific ones: 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 parental 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 isotype, e.g., any human antibody of 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 aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment disclosed in Figure 22, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:1, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:2, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:3, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:4, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:5, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein. In one aspect of the invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:6, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or third mutation disclosed herein.In one aspect of the present invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:7, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or the third mutation disclosed herein. In one aspect of the present invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:8, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or the third mutation disclosed herein. In one aspect of the present invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:9, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or the third mutation disclosed herein. In one aspect of the present invention, the polypeptide or antibody comprises an Fc region comprising the Fc segment of SEQ ID NO:10, and this Fc segment further comprises the first mutation, the second mutation, and / or a further mutation or the third mutation disclosed herein.

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

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

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

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

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

[0173] In one aspect of the present 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 the ability to bind ligands of the tumor necrosis factor superfamily (TNFSF) via an extracellular cysteine-rich domain. TNF receptors form trimeric complexes within the plasma membrane. The following 29 proteins are listed as TNFRSF: 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), 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), RELT (Uniprot Q969Z4).

[0175] Some TNFRSFs are involved in apoptosis and include 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, RELT. TNF receptors are expressed in a wide variety of mammalian tissues, particularly leukocytes.

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

[0177] The polypeptide or antibody according to the present invention can bind to any target, and such target or antigen according to the present invention non-limitingly includes, for example, 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] Bispecific antibody In one aspect, provided by the present invention is a polypeptide or antibody comprising a first Fc region and a first antigen-binding region of human IgG, and a second Fc region and a second antigen-binding region of human IgG, wherein the first and second Fc regions correspond to the following positions in human IgG1 according to EU numbering: (i) a first mutation and (ii) a second mutation and (iii) a third mutation: (i) A first mutation in E430, E345, or S440; (ii) A second mutation in K322 or P329; (iii) A third mutation in F405 or K409 comprising; wherein, if the first Fc region has a mutation at position F405, the second Fc region has a mutation at K409 (and vice versa), different from the first Fc region and the second Fc region, provides a polypeptide or an antibody.

[0179] Thereby, (iii) since the third mutation does not exist at the same position in the first and second Fc regions, an aspect is provided in which the first Fc region and the second Fc region are not identical.

[0180] It will be understood that any aspect of the invention described herein can be used in the context of the multispecific antibodies 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 a particular aspect, the bispecific antibody has the format described in WO 2011 / 131746.

[0183] In another aspect, the invention is a polypeptide or an antibody which 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 the human IgG1 heavy chain, (ii) A second mutation selected from the group corresponding to E322E, P329R, P329K, P329D comprising 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 further mutation in the second polypeptide, relating to a polypeptide or an antibody.

[0184] The bispecific antibody of the present invention is not limited to a specific format and can be any of those described herein.

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

[0186] In one aspect of the present invention, the first and / or second CH2-CH3 heavy chain (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, P329Y comprising the first CH2-CH3 heavy chain comprises a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain comprises a third mutation at the amino acid residue corresponding to F405L.

[0187] In one embodiment of the present invention, the first and / or second CH2-CH3 heavy chains are (iii) a first mutation corresponding to E430G, (iv) a second mutation corresponding to E322E and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

[0188] In one embodiment of the present invention, the first and / or second CH2-CH3 heavy chains are (i) a first mutation corresponding to E430G, (ii) a second mutation corresponding to P329R and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

[0189] In one embodiment of the present invention, the first and / or second CH2-CH3 heavy chains are (i) a first mutation corresponding to E430G, (ii) a second mutation corresponding to P329K and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

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

[0191] In one aspect of the present invention, the first and / or second CH2-CH3 heavy chains are (i) a 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, P329Y and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

[0192] In one aspect of the present invention, the first and / or second CH2-CH3 heavy chains are (iii) a first mutation corresponding to E345K, (iv) a second mutation corresponding to E322E and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

[0193] In one aspect of the present invention, the first and / or second CH2-CH3 heavy chains are (iii) a first mutation corresponding to E345K, (iv) a second mutation corresponding to P329R and include the first CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain includes a third mutation at the amino acid residue corresponding to F405L.

[0194] In one aspect of the present invention, the first and / or second CH2-CH3 heavy chains are (iii) a first mutation selected from the group corresponding to E345K, (iv) A second mutation selected from the group corresponding to P329K comprising the first CH2-CH3 heavy chain comprises a third mutation at the amino acid residue corresponding to K409R; the second CH2-CH3 heavy chain comprises a third mutation at the amino acid residue corresponding to F405L.

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

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

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

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

[0200] In one aspect, the present invention relates to a method for reducing the Fc effector function or activity of a polypeptide or antibody having a first Fc-Fc enhancing mutation by introducing a second mutation. It should be understood that the method for reducing the Fc effector function is determined when comparing the polypeptide or antibody with a parental polypeptide or antibody having the same antigen-binding region and an Fc region having the same first mutation within the Fc region but no second mutation within the Fc region. In some embodiments, by the method for reducing the Fc effector function or activity, the effector function is lower than or reduced to a comparable level compared to the level of a parental polypeptide or antibody having the same antigen-binding region and Fc region but no first and second mutations within the Fc region.

[0201] In one aspect 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.

[0202] In one aspect of the present invention, the method relates to reducing Fc effector functions such as CDC, CDCC, and / or C1q binding, and includes the step of introducing a second mutation selected from the group consisting of K322E, K322D, and K322N.

[0203] In one aspect of the present invention, the method relates to reducing Fc effector functions such as ADCC, ADCP, FcγR binding, CDC, CDCC, and / or C1q binding, and includes the step of introducing a second mutation selected from the group consisting of P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y.

[0204] In a preferred aspect of the present invention, the second mutation is selected from the group consisting of K322E, P329R, P329K, and P329D.

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

[0206] 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to E322E.

[0207] 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to E322D. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to E322N. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329H.

[0208] 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329K. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329R. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329D. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329E. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329M. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329F. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329G. 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 the Fc region, the method comprising the step of 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 the Fc region, the method comprising the step of 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329N.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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329Q. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329S. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329T. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329V. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329W. 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 the Fc region, the method comprising the step of introducing a second mutation corresponding to P329Y.

[0209] 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 the Fc region, 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 present invention relates to a method for reducing the effector function of a polypeptide or antibody comprising a first mutation corresponding to E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to E322E.

[0211] 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to E322D. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to E322N. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329H.

[0212] 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329K. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329R. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329D. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329E. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329M. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329F. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329G. 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 E345K in the Fc region, the method comprising the step of 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 E345K in the Fc region, the method comprising the step of 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329N.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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329Q. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329S. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329T. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329V. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329W. 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 E345K in the Fc region, the method comprising the step of introducing a second mutation corresponding to P329Y.

[0213] 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 E345K in the Fc region, 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 present invention relates to a method in which the Fc region comprises one or more additional mutations within the CH3 domain.

[0215] In one aspect, the present invention relates to a method in which the Fc region contains a further mutation within the CH3 domain corresponding to one of positions S440 or K439 in human IgG1 according to EU numbering. In one aspect of the present invention, the Fc region contains a further mutation within the CH3 domain corresponding to one of positions S440 or K439, provided that if the first mutation is present at S440, the further mutation is not present at position S440. Polypeptides or antibodies comprising the first and second mutations according to the present invention and further mutations at position S440, such as S440K, do not form oligomers with polypeptides or antibodies containing a mutation at position S440, such as S440K. Polypeptides or antibodies comprising the first and second mutations according to the present invention and further mutations at position K439, such as K439E, do not form oligomers with polypeptides or antibodies containing a mutation at position K439, such as K439E. Thereby, a method is provided that enables the formation of oligomers between a first polypeptide or antibody containing the K439E mutation and a second polypeptide or antibody containing the S440K mutation. In this way, it can be forced that oligomers, such as hexamers, etc., are formed in some specific patterns of the first and second polypeptides. This can be important in a method in which the polypeptides bind to different targets or epitopes and oligomers should be formed in such combinations of different targets or epitopes.

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

[0217] In one aspect, the present invention relates to a method for reducing Fc effector function, wherein the Fc effector function is at least 20% lower compared to a parental polypeptide or parental antibody that is identical to a polypeptide having the same first mutation but no second mutation. In another aspect of the present 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%, at least 95% compared to a parental polypeptide or antibody having only the first mutation.

[0218] In one aspect, the present invention relates to a method for reducing Fc effector function, wherein the Fc effector function is selected from the group consisting 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 present invention relates to a method for 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% compared to a comparative antibody that is identical to the antibody except for not containing the second mutation.

[0220] In one aspect, the present invention relates to a method for reducing CDC, wherein the CDC is reduced by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% compared to a comparative antibody that is identical to the antibody except for not containing the second mutation.

[0221] In one aspect, the present invention relates to a method for reducing C1q binding, wherein the 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% compared to a comparative antibody that is identical to the antibody except for not containing the second mutation.

[0222] In one aspect, the present invention relates to a method for reducing Fc gamma receptor binding, wherein the 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% compared to a comparative antibody that is identical to the antibody except for not containing a second mutation.

[0223] In one aspect, the present invention relates to a method for reducing Fc gamma receptor binding, wherein the 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% compared to a comparative antibody that is identical to the antibody except for not containing the first and second mutations.

[0224] In a preferred aspect, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein the 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%, at least 100% compared to a comparative antibody that is identical to the antibody except for not containing a second mutation.

[0225] In a preferred aspect, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein the 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%, at least 100% compared to a comparative antibody that is identical to the antibody except for not containing the first and second mutations.

[0226] In a preferred aspect, the present 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% compared to a comparative antibody that is identical to the antibody except for not containing a second mutation.

[0227] In a preferred embodiment, the present invention relates to a method for reducing Fc gamma receptor I binding, wherein the Fc gamma receptor I binding is at least 70%, preferably at least 80%, more preferably at least 90%, or at least 100% reduced compared to a comparative antibody that is identical to the antibody except that it does not contain the first and second mutations. Thus, the method includes reducing Fc gamma receptor I binding to a level that is reduced compared to the wild-type Fc region.

[0228] Composition The embodiments described below with respect to a polypeptide or antibody are understood to refer to a polypeptide or antibody comprising an Fc region having the CH2-CH3 region of an immunoglobulin and an antigen-binding region, and 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 CH2-CH3 heavy chain of an immunoglobulin and a second CH2-CH3 heavy chain of an immunoglobulin.

[0229] The present invention also relates to a composition comprising the polypeptide or antibody described herein and its isoforms. Specific aspects and embodiments are described below. Further, such a polypeptide or antibody can be obtained according to any method 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 present invention, the composition comprises one or more polypeptides or antibodies according to any aspect or embodiment described herein.

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

[0233] In one aspect of the present invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies are (i) a first mutation that 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, wherein the first and second polypeptides or antibodies do not contain the same further mutation and comprise an Fc region.

[0234] In one aspect of the present invention, the 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) a further mutation, wherein the first and second polypeptides or antibodies do not contain the same further 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 aspect of the present invention, the 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, (iii) a further mutation, and these mutations correspond to the following amino acid positions within human IgG1 according to EU numbering: (i) the first mutation E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) the second mutation at E322 or P329; (iii) a further mutation at K439 or S440, provided that if the further mutation is at S440, the first mutation is not at S440, and the first and second Fc regions do not contain the further mutation at the same amino acid position.

[0236] In one aspect of the invention, the 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, (iii) additional mutations, and these mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) The first mutation is E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) The 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 at S440, the first mutation is not at S440.

[0237] In one aspect of the invention, the 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, (iii) additional mutations, and these mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) The first mutation is E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W; (ii) The 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 at S440, the first mutation is not 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) a further mutation K439E in the first Fc region and a further mutation S440K in the second Fc region.

[0241] Thereby, there is provided an aspect in which both the first and second polypeptides or antibodies have reduced Fc effector function, or either only the first polypeptide or only the second polypeptide has reduced Fc effector function.

[0242] In one aspect of the invention, the 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 the amino acid position corresponding to E430, and (ii) a second mutation and (iii) a further mutation, the second mutation and the further mutation being (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0243] In one aspect of the invention, the 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 the amino acid position corresponding to E345, and (ii) a second mutation and (iii) a further mutation, the second mutation and the further mutation being (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same additional mutations.

[0244] In one aspect of the invention, the 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) additional mutations, and the second mutation and additional mutations are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same additional mutations.

[0245] In one aspect of the invention, the 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) additional mutations, and the second mutation and additional mutations are (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same additional mutations.

[0246] In one aspect of the invention, the 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, which further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0247] In one aspect of the invention, the 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, which further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0248] In one aspect of the invention, the 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, which further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0249] In one aspect of the present invention, the 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, and this further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0250] In one aspect of the present invention, the 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, and the second mutation and the further mutation are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0251] In one aspect of the present invention, the 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, and the second mutation and the further mutation are (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0252] In one aspect of the invention, the 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) further mutations, which further mutations are (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0253] In one aspect of the invention, the 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) further mutations, which further mutations are (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0254] In one aspect of the invention, the 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) further mutations, which further mutations are (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0255] In one aspect of the present invention, the 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, and this further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0256] In one aspect of the present invention, the 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, and the second mutation and the further mutation are (ii) K322E, K322D, K322N, P329A, P329H, P329K, P329R, P329D, P329E, P329F, P329G, P329I, P329L, P329M, P329N, P329Q, P329S, P329T, P329V, P329W, and P329Y; (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[0257] In one aspect of the present invention, the 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, and the second mutation and the further mutation are (ii) K322E, P329K, P329R, P329D; (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0258] In one aspect of the invention, the 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, the further mutation being (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0259] In one aspect of the invention, the 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, the further mutation being (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0260] In one aspect of the invention, the 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, the further mutation being (iii) K439E and S440K Selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutations.

[0261] In one aspect of the invention, the 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, which further mutation is (iii) K439E and S440K selected from the group consisting of, wherein the first and second Fc regions do not contain the same further mutation.

[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 (i) a first mutation that is an Fc-Fc enhancing mutation; (ii) a further mutation that suppresses oligomerization between Fc regions having the same further mutation, wherein the first and second polypeptides or antibodies do not contain the same further mutation and comprise an Fc region comprising (iii) either the first or the second Fc region contains a second mutation. Thus, in some aspects, only the first polypeptide or antibody or the second polypeptide or antibody contains a second mutation that reduces Fc effector function.

[0263] In one aspect of the invention, the 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, provided that the mutation at S440 is S440Y or S440W, (ii) a second mutation, (iii) a further mutation E, and these mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (iii) Further K439E or S440K mutations, wherein the first and second Fc regions do not contain the same further mutation, and if the first mutation is S440Y or S440W, the further mutation is not S440K; (ii) Either the first or the second Fc region contains a second mutation at E322 or P329, but not both.

[0264] In one aspect of the invention, the 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 consisting of E322 and P329, and (iii) a further 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 a further mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0265] In one aspect of the invention, the 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) a further 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 a further mutation K439E. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0266] In one aspect of the present invention, the 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, (ii) a second mutation E322E, and (iii) an additional mutation K439E; and the second Fc region comprises (i) a first mutation E430G, and an additional mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0267] In one aspect of the present invention, the 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, (ii) a second mutation E322E, and (iii) an additional mutation S440K; and the second Fc region comprises (i) a first mutation E430G, and an additional mutation K322E. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0268] In one aspect of the present invention, the 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, (ii) a second mutation P329R, and (iii) an additional mutation K439E; and the second Fc region comprises (i) a first mutation E430G, and an additional mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0269] In one aspect of the present invention, the 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, (ii) a second mutation P329R, and (iii) a further mutation S440K; and the second Fc region comprises (i) a first mutation E430G and a further mutation K322E. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0270] In one aspect of the present invention, the 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, (ii) a second mutation P329K, and (iii) a further mutation K439E; and the second Fc region comprises (i) a first mutation E430G and a further mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0271] In one aspect of the present invention, the 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, (ii) a second mutation P329K, and (iii) a further mutation S440K; and the second Fc region comprises (i) a first mutation E430G and a further mutation K322E. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0272] In one aspect of the present invention, the 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, (ii) a second mutation P329D, and (iii) a further mutation K439E; and the second Fc region comprises (i) a first mutation E430G and a further mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0273] In one aspect of the present invention, the 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, (ii) a second mutation P329D, and (iii) a further mutation S440K; and the second Fc region comprises (i) a first mutation E430G and a further mutation K322E. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0274] In one aspect of the present invention, the 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, (ii) a second mutation E322E, and (iii) a further mutation K439E; and the second Fc region comprises (i) a first mutation E345K and a further mutation S440K. Thereby, an embodiment is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0275] In one aspect of the present invention, the 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, (ii) a second mutation E322E, and (iii) an additional mutation S440K; and the second Fc region comprises (i) a first mutation E345K, and an additional mutation K322E. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0276] In one aspect of the present invention, the 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, (ii) a second mutation P329R, and (iii) an additional mutation K439E; and the second Fc region comprises (i) a first mutation E345K, and an additional mutation S440K. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0277] In one aspect of the present invention, the 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, (ii) a second mutation P329R, and (iii) an additional mutation S440K; and the second Fc region comprises (i) a first mutation E345K, and an additional mutation K322E. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0278] In one aspect of the present invention, the 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, (ii) a second mutation P329K, and (iii) a further mutation K439E; and the second Fc region comprises (i) a first mutation E345K and a further mutation S440K. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0279] In one aspect of the present invention, the 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, (ii) a second mutation P329K, and (iii) a further mutation S440K; and the second Fc region comprises (i) a first mutation E345K and a further mutation K322E. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0280] In one aspect of the present invention, the 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, (ii) a second mutation P329D, and (iii) a further mutation K439E; and the second Fc region comprises (i) a first mutation E345K and a further mutation S440K. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

[0281] In one aspect of the present invention, the 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, (ii) a second mutation P329D, and (iii) a further mutation S440K; and the second Fc region comprises (i) a first mutation E345K and a further mutation K322E. Thereby, an aspect is provided in which only the first polypeptide or antibody has reduced Fc effector function.

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

[0283] In one aspect of the present invention, the composition comprises a polypeptide or antibody having binding ability to a member of 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 aspect of the present invention, the composition comprises a polypeptide or antibody having binding ability to a member of 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 aspect of the present invention, the composition comprises a polypeptide or antibody having binding ability to a member of TNFR-SF belonging to the group of immune activators consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0286] In one aspect of the present invention, the composition comprises a polypeptide or an antibody, and the first polypeptide and the second polypeptide bind to different epitopes on one or more members of 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 aspect of the present invention, the composition comprises a polypeptide or an antibody, and the binding of the first polypeptide to one member of 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 which is the binding of the second antibody to one member of 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 aspect of the present invention, a composition comprising a first polypeptide or antibody and a second polypeptide or antibody is present in a molar ratio of 1:49 to 49:1 in the composition, for example, a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 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, 2:1.

[0289] In one aspect of the present invention, a composition comprising a first polypeptide and a second polypeptide and / or any additional polypeptide(s) is present in an equimolar ratio in the composition.

[0290] In one aspect of the present invention, a 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 can be used as a medicament, i.e., for therapeutic use.

[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, autoimmune disease, inflammatory disease, or infectious disease.

[0294] 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 according to any aspect or embodiment disclosed herein.

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

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

[0297] In one aspect of the present 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 regulator (including, but not limited to, recombinant human TRAIL or birinapant), a RAS inhibitor, a proteasome inhibitor (including, but not limited to, bortezomib), 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), or one or more anti-cancer agents selected from the group consisting of antibody-drug conjugates.

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

[0299] The present invention also relates to a parts kit for simultaneous, separate, or sequential use in therapy, comprising the polypeptide or antibody described herein. Further, such variants can be obtained according to any method described herein.

[0300] In one aspect, the invention relates to a parts kit comprising a polypeptide, an antibody, or a 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 embodiment of the invention, the parts kit 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 invention relates to the use of a polypeptide, an antibody, a composition, or a parts kit according to any embodiment described herein for use in a diagnostic method.

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

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

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

[0306] Combination Furthermore, the present invention provides a preparation of any polypeptide or antibody according to any of the above aspects or embodiments, i.e., a preparation comprising a plurality of polypeptides or antibodies. The present invention also provides a composition comprising a polypeptide or antibody according to any of the above aspects or embodiments, such as a pharmaceutical composition. The present invention also provides the use of any such polypeptide or antibody, preparation, or composition as a medicament.

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

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

[0309] In one aspect, the present invention relates to a polypeptide or antibody in which the variant is conjugated to a drug, toxin, or radiolabel, for example, 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 invention is not conjugated at the C-terminus to another molecule, such as a toxin or a label. In one embodiment, the variant is conjugated to another molecule at another site, typically a site that does not interfere with oligomer formation. 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 compounds may, for example, be able to more effectively kill target cells in cancer therapy. Thus, the resulting variant is an immunoconjugate.

[0312] Accordingly, in a further aspect, the invention provides an antibody linked or conjugated to one or more therapeutic moieties, such as a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, and / or a radioisotope. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates". An immunoconjugate containing one or more cytotoxins is referred to as an "immunotoxin".

[0313] Cytotoxins or cytopathic agents include any agent that is harmful to cells (e.g., kills them). Suitable therapeutic agents for forming the immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitansine or its analogs or derivatives, enediyne antitumor antibiotics, e.g., neocarzinostatin, calicheamicin, esperamicin, dynemicin, lidamycin, kedarcidin or its analogs or derivatives, anthracyclines, mitozantrone, mitramycin, 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, e.g., carboplatin; and duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin), or analogs or derivatives of CC-1065), dolastatin, pyrrolo[2,1-c][1,4]benzodiazepine (PDB) or its analogs, antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitozantrone, plicamycin, anthramycin (AMC)), antimitotic agents (e.g., tubulin inhibitors), e.g., monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10;Histone deacetylase inhibitors such as hydroxamic acid-based trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat, and benzamide-based entinostat, CI994, mocetinostat, and aliphatic acid compounds such as phenylbutyrate and valproic acid, proteasome inhibitors such as danoprevir, bortezomib, amatoxins such as α-amanitin, diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules); ricin toxins (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk type protease inhibitor, Pseudomonas exotoxin, alloricin, saporin, modicine, gelanin, abrin A chain, modicine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crocin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugate-type molecules include antibacterial / soluble 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. For example, Pastan et al., Cell; 47 , 641(1986) and Goldenberg, Calif.A Cancer Journal for Clinicians 44, see 43(1994). Therapeutic agents that can be administered in combination with the antibodies of the present invention described elsewhere in this specification, for example, anti-cancer cytokines or chemokines, etc., are also candidates for therapeutic moieties useful for conjugation with the antibodies of the present invention.

[0314] In one aspect, the drug conjugate of the present invention comprises an antibody disclosed herein conjugated to an auristatin or an auristatin peptide analog and derivative (US5635483; US5780588). Auristatin interferes with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580 - 3584) and has been shown to have anti-cancer (US5663149) and anti-fungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961 - 2965). The auristatin drug moiety can be conjugated to the antibody at the N (amino) - terminus or C (terminus) of the peptide drug moiety via a linker.

[0315] Exemplary auristatin embodiments include the N - terminal - conjugated monomethyl auristatin drug moieties DE and DF 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 (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).

[0317] In one aspect, the antibody of the present invention comprises a conjugated nucleic acid or nucleic acid binding molecule. In such an aspect, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., siRNA molecule), or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In another aspect, the antibody of the present invention is conjugated to an aptamer or ribozyme.

[0318] In one aspect, an antibody comprising one or more radiolabeled amino acids is provided. The radiolabeled variant can be used for both diagnostic and therapeutic purposes (conjugation with a radiolabeled molecule is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2 nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) as well as U.S. 4,681,581, U.S. 4,735,210, U.S. 5,101,827, U.S. 5,102,990 (US RE35,500), U.S. 5,648,471 and U.S. 5,697,902. For example, the radioisotope can be conjugated by the chloramine-T method.

[0319] In one aspect, 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 thiouretane, that enables the antibody to complex with a radioisotope. Alternatively, one or more radiolabeled amino acids or other radiolabeled molecules can be included in or conjugated to the variant. The radiolabeled variant can be used for both diagnostic and therapeutic purposes. In one aspect, 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.

[0320] In one aspect, the polypeptide or antibody of the present invention can be conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansetimibe, and TNFα.

[0321] Alternatively, the polypeptide or antibody of the present invention may be chemically modified by covalent conjugation with a polymer, for example, to increase its circulating half-life. Exemplary polymers and methods for conjugating them to peptides are illustrated, for example, in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Further polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (for example, PEG having a molecular weight of about 1,000 to about 40,000, for example about 2,000 to about 20,000).

[0322] Any method known in the art for conjugating the polypeptide or antibody of the present invention to one or more conjugate-type molecules, for example, those described above, for example, 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) may be used. Such mutants can be prepared by chemically conjugating other moieties to the N-terminal or C-terminal side of the mutant or a fragment thereof (for example, the H chain or L chain of an antibody) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Also, derivatives of such conjugate-type mutants can be prepared, where appropriate, by conjugation at internal residues or sugar chains.

[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 portion in a bispecific antibody. In one aspect, the polypeptide or antibody is conjugated via a spacer or linker to a prodrug molecule that can be activated in vivo into a therapeutic drug. In some aspects, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug moiety from the antibody into the intracellular environment. In some aspects, the linker is cleavable by a cleaving factor present within the intracellular environment (e.g., within a lysosome or endosome or caveola). For example, the spacer or linker can be cleavable by a tumor cell-binding enzyme or other tumor-specific condition, thereby generating the active drug. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394, and WO201062171 (by Syntarga BV, et al.). Also, suitable antibody-prodrug technologies and duocarmycin analogs are found in U.S. Patent No. 6,989,452 (Medarex), which is incorporated herein by reference. Alternatively, the linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, such as, but not limited to, a lysosomal or endosomal protease. In some aspects, the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length. Cleaving agents include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives and result in the release of the active drug inside the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).In a specific embodiment, the cleavable peptidyl linker by intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, for example, US6214345, which describes the synthesis of doxorubicin having a Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of Val-Cit and Phe-Lys linkers include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB, or MC-Phe-Lys-GABA described below, where MC is the abbreviation of maleimidocaproyl, vc is the abbreviation of Val-Cit, PAB is the abbreviation of p-aminobenzylcarbamate, and GABA is the abbreviation of γ-aminobutyric acid. The advantage of using proteolytic intracellular release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stability of the conjugate is typically increased.

[0324] In yet another aspect, the linker unit is non-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" means that, in the context of the linker, when the mutant antibody-drug conjugate compound is present in the extracellular environment (e.g., plasma), in a sample of the mutant antibody-drug conjugate compound, 20% or less, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, about 3% or less, or about 1% or less of the linker is 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 time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma. Exemplary embodiments containing MMAE or MMAF and various linker components have the following structures (Ab means antibody, p represents the drug load (or the average number of cytostatic or cytotoxic drugs per molecule of antibody), which is from 1 to about 8, for example, p can be from 4 to 6, for example from 3 to 5, or p can be 1, 2, 3, 4, 5, 6, 7, or 8).

[0325] Examples of cleavable linkers combined with auristatin include MC-vc-PAB-MMAF (also designated vcMMAF) and MC-vc-PAB-MMAE (also designated vcMMAE), where MC is the abbreviation for maleimidocaproyl, vc is the abbreviation for a Val-Cit (valine-citrulline)-based linker, and PAB is the abbreviation for p-aminobenzylcarbamate.

[0326] Other examples include auristatin combined with a non-cleavable linker, such as mcMMAF (mc (MC is the same as mc in this context) is the abbreviation for maleimidocaproyl).

[0327] In one aspect, the drug linker moiety is vcMMAE. The vcMMAE drug linker moiety 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 conjugated to the antibody at cysteine using methods similar to those disclosed therein.

[0328] In one aspect, 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 conjugated to the variant at cysteine using methods similar to those disclosed therein.

[0329] In one aspect, the polypeptide or antibody of the invention is conjugated to a chelating agent linker, such as thiuxetan, that enables the bispecific antibody to be conjugated to a radioisotope.

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

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

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

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

[0334] In a further aspect, the invention relates to the use of the polypeptides, antibodies of the invention as described above for use as a medicament, in particular for use as a medicament for the treatment of a disease or disorder. Examples of such diseases and disorders include, but are not limited to, cancer, and infections by bacteria, viruses, or fungi.

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

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

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

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

[0339] "Effective amount" or "therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result at the required dosage and for the required period. The therapeutically effective amount of an antibody can vary depending on factors such as the state of the disease, the age, sex, and weight of the individual, and the ability of the antibody to induce a desired response in the individual. Also, a therapeutically effective amount is an amount in which the therapeutic beneficial effect outweighs any toxic or detrimental effects of the antibody or a portion of the antibody.

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

[0341] The effective dosage and dosage regimen of an antibody depend on the disease or medical condition to be treated and can be determined by one of ordinary skill in the art. Exemplary and non-limiting ranges of the therapeutically effective amount of the antibodies of the invention are from about 0.1 to 100 mg / kg, such as from about 0.1 to 50 mg / kg, such as from about 0.1 to 20 mg / kg, such as from about 0.1 to 10 mg / kg, such as about 0.5, such as 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., may be combined with other therapeutic agents related to the disease or condition to be treated. Thus, in one aspect, the antibody-containing medicament is for combination with one or more additional therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or angiogenesis inhibitors. Such combination 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, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a variant or pharmaceutical composition of the present invention in combination with radiotherapy and / or surgery.

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

[0345] The present invention also provides an isolated nucleic acid and vector encoding a variant according to any one of the above aspects, as well as a vector and expression system encoding the variant. Nucleic acid constructs, vectors, and expression systems suitable for antibodies and their variants are known in the art and are described in this example. In aspects where the variant includes not only the heavy chain (or its Fc-containing fragment) but also the light chain, the nucleotide sequences encoding the heavy chain portion and the light chain portion may 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 an antibody according to any one of the above aspects in a host cell, wherein the polypeptide or antibody comprises at least the Fc region of the heavy chain, and the method comprises the following steps: a) preparing a nucleotide construct encoding a mutant Fc region; b) expressing the nucleotide construct in a host cell; and c) recovering the antibody mutant from the cell culture of the host cell. The method also includes the steps.

[0347] In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody also includes a light chain, and thus the host cell further expresses a light chain encoding construct on 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 occur, such as a eukaryotic cell, such as a mammalian cell, such as a human cell. In a further embodiment, the host cell is a non-human cell genetically modified to produce a glycoprotein having human-like glycosylation 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 aspect, 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 aspect, the host cell is a host cell having a modified 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 and thereby producing antibodies having modified glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03 / 035835; and WO99 / 54342. Further methods for generating modified glycoforms are known in the art and include, without limitation, those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation 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 obtainable or obtained by the methods of the present invention as described above.

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

[0352] The present invention is further illustrated by the following examples, which should not be construed as further limitations.

[0353] Table of sequences TIFF0007695293000004.tif30153TIFF0007695293000005.tif218153TIFF0007695293000006.tif141153

Example

[0354] Example 1: Generation, production, and purification of antibodies Expression construct of antibody For antibody expression, the sequences of the variable heavy (VH) and variable light (VL) chains were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into the pcDNA3.3 expression vector (ThermoFisher Scientific, US) containing the constant regions of the heavy chain (HC) and light chain (LC) of IgG1. Desired mutations were introduced either by 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 (WO2006 / 099875), DR5 antibodies hDR5-01, hDR5-05 (WO2014 / 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 (WO2004 / 035607). In some examples, the human IgG1 antibody b12, which is 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 essentially as described by Vink et al. (Vink et al., Methods, 65(1), 5-10 2014) using a plasmid DNA mixture encoding both the heavy and light chains of the antibody and 293fectin (Invitrogen, US).

[0356] Protein purification and analysis The antibody was purified by protein A affinity chromatography. The culture supernatant was 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 antibody was buffer-exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B. Braun or Thermo Fisher). After buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. The purified protein was analyzed by multiple bioanalytical assays including capillary electrophoresis-SDS (CE-SDS) and high-performance size exclusion chromatography (HP-SEC) on sodium dodecyl sulfate-polyacrylamide gels. The concentration was measured by absorbance at 280 nm. The purified antibody was 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 variants 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 mutant D270A, K322A, and P329A were able to significantly reduce 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 assists the efficient binding of the C1q hexameric structure, resulting in strong C1q binding (Diebolder et al., Science 2014). IgG hexamerization on the cell surface is mediated by intermolecular non-covalent Fc-Fc interactions and can be enhanced by point mutations in the CH2 domain, such as E345R and E430G (Diebolder et al., 2014 Science; De Jong et al., 2015 PloS Biology). Fc-Fc enhancing mutations increase the C1q binding avidity in the hexameric antibody structure on the cell surface but do not affect the C1q binding affinity. Therefore, it is unpredictable whether mutations reported to reduce C1q binding affinity can block CDC by IgG1 antibody variants having mutations for enhanced Fc-Fc interactions.

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

[0360] For the CDC assay, 0.1×10 6Individual Daudi cells (ATCC number CCL-213 (trademark)) were pre-incubated for 15 minutes at room temperature on a shaker in a polystyrene round-bottom 96-well plate (Greiner bio-one catalog number 650101) containing a series of concentrations of purified antibody in a total volume of 80 μL. Next, 20 μL of human normal serum (NHS; catalog number M0008 Sanquin, Amsterdam, The Netherlands) was added as a complement source and incubated for 45 minutes in an incubator at 37 °C (final NHS concentration of 20%; final antibody concentration of 0.001 - 10.0 μg / mL at a 3-fold dilution). 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 a 2 μg / mL propidium iodide solution (PI; Sigma Aldrich, Zwijnaarde, The Netherlands). The number of PI-positive cells was examined by FACS analysis using an Intellicyt iQue (trademark) sorter (Westburg). The data were analyzed using the best-fit values of a non-linear dose-response fit and logarithmic transformed concentration values in GraphPad PRISM 5. The lysis rate 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 against Daudi cells (Figure 1). In contrast, in the presence of the Fc-Fc interaction-enhancing mutations E430G or E345R, the introduction of D270A / K322A had only a minor effect on CDC efficacy (Figure 1): for IgG1-005-E430G and IgG1-005-AA-E430G, the same maximum killing of 100% was achieved at EC50 0.01 ± 0.01 (μg / mL ± SD) and 0.06 ± 0.02 μg / mL, respectively; for IgG1-005-E345R and IgG1-005-AA-E345R, the maximum killing was 100% and 74.3% at EC50 0.01 μg / mL and 0.14 μg / mL, respectively. In the presence of the triple mutation E345R / E430G / S440Y (Diebolder et al., 2014 Science; Wang et al., 2016 Mol. Cell) that results in hexamerization of the antibody in solution, 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 variants with mutations for enhanced Fc-Fc interaction.

[0363] Example 3: Analysis of the effect of the selection of 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 interaction Mutations at positions D270, K322, and P329 of the human IgG1 C1q-binding site were designed to interfere with the protein-protein interactions established when C1q is bound to IgG1. Thus, the WT amino acids were replaced with charged amino acids having a new or opposite charge: D270R, K322E, P329D, and P329R. The effects of these additional variants on the CDC efficacy of the IgG1-005 variant having the E430G mutation for enhanced Fc-Fc interaction were tested. A series of concentrations of purified antibodies (final antibody concentrations of 0.001 - 10.0 μg / mL in 3-fold dilutions) were tested in Daudi cells in an in vitro CDC assay as described in Example 2 using 20% NHS. TIFF0007695293000007.tif46128

[0364] Introduction of the K322E, P329D, or P329R mutation strongly inhibited CDC-mediated killing of Daudi cells by IgG1-005-E430G (Figure 2A). In contrast, introduction of D270R resulted in a reduction in potency and an increase in the EC50 value (0.005 μg / mL and 0.15 μg / mL for IgG1-005-E430G and IgG1-005-D270R / E430G, respectively), but the maximum killing of Daudi cells by IgG1-005-E430G was not reduced. Data for D270A / K322A were included as a reference to show that it had only a minor effect on the CDC efficacy of 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, the effect on C1q binding to the antibody bound to Daudi cells was measured by FACS analysis. 0.1×10 6Individual Daudi cells were incubated for 30 minutes at 4°C in 100 μL of reaction solution in a polystyrene round-bottom 96-well plate containing a series of concentrations of purified antibody (final antibody concentrations of 0.0003 to 100.0 μg / mL at 3.33-fold dilution) and 20% C4-depleted serum as a C1q source. 100 μL of FACS buffer (PBS / 0.1% BSA / 0.01% sodium azide) was added and the cells were pelleted by centrifugation. The cells were washed with 150 μL of FACS buffer and incubated for 30 minutes at 4°C with 50 μL of FITC-labeled rabbit anti-HuC1q antibody (DAKO, catalog number F0254; final concentration of 20 μg / mL). The cells were washed twice with FACS buffer, resuspended in 30 μL of FACS buffer, and the mean fluorescence intensity was examined on an Intellicyt iQue™ sorter.

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

[0367] Collectively, these data indicate that introduction of the K322E, P329D, or P329R mutations into IgG1-005-E430G resulted in inhibition of C1q binding and the associated CDC-mediated killing of Daudi cells.

[0368] Example 4: Effect of K322X mutations on the in vitro CDC efficacy of IgG1-005 variants with enhanced Fc-Fc interactions Antibodies were collected by harvesting the supernatant of the transient transfection described in Example 1. A series of antibody concentrations (final concentrations of 0.001 - 30.0 μg / mL at 3-fold dilutions) were tested in Daudi cells in an in vitro CDC assay using 20% NHS essentially as described in Example 2. Combinations of substitution 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) and 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 with enhanced Fc-Fc interaction.

[0370] Example 5: Biophysical Characterization of IgG1-005-E430G Variants Containing the Mutations K322D, K322E, or K322N Purified antibody batches of IgG1-005-E430G variants with 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. The purity and fragmentation of the samples were analyzed with little modification using CE-SDS (Caliper Labchip GXII, PerkinElmer) with the Labchip GXII (High Sensitivity protocol). Both non-reducing and reducing samples (with DTT added) were prepared using the HT Protein Express Reagent Kit (CLS960008) and denatured by incubation at 70 °C for 10 minutes. The samples were electrophoresed with the HT antibody assay 200 high-sensitivity setting. Data on molecular weight and purity (percentage relative to the total) were analyzed using Labchip GXII software. Figure 4A shows that IgG1-005-K322E / E430G exhibited behavior equivalent to that of 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, while under reducing conditions, a heavy chain with an apparent MW of 50 kDa and a light chain with an apparent MW 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, which appeared to be degraded after reduction.

[0372] HP-SEC fractions were analyzed using a TSK HP-SEC column (G3000SW xl;Performed using a Waters Alliance 2975 separation unit (Waters, Etten-Leur, The Netherlands) coupled to a Toso Biosciences, via Omnilabo, Breda, The Netherlands) and a Waters 2487 dual λ absorbance detector (Waters). A 50 μL sample containing 1.25 μg / mL of protein was separated at 1 mL / min in 0.1 M Na2SO4 / 0.1 M sodium phosphate buffer, pH 6.8. Results were processed using Empower software version 3 and shown as the percentage of the total peak area for each peak. Figure 4B shows that the antibody IgG1-005-K322E / E430G eluted predominantly at the elution time predicted for the monomeric species (98% monomeric), while the variants IgG1-005-K322D / E430G (70% aggregated) and IgG1-005-K322N / E430G (43% aggregated) showed significant amounts of high molecular weight species. Thus, HP-SEC analysis suggested that the double mutant K322E / E430G is 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 variants 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. Purified antibodies at different concentrations (final concentration range of 0.001 - 30.0 μg / mL) were tested in Daudi cells in an in vitro CDC assay 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 the proline at position 329 with alanine (A) only partially reduced the CDC efficacy, with a shift in the EC50 from 0.01 μg / mL for IgG1-005-E430G to 0.11 μg / mL for IgG1-005-P329A / E430G, but no effect on maximum killing was observed. These data indicate that substitution of the 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 the CDC efficacy by IgG1-005-E430G.

[0375] Example 7: Evaluation of Biophysical Properties of IgG1-005-E430G Variants Containing a Mutation at Position P329 Purified antibody batches of IgG1-005-E430G variants in which the proline at position 329 was substituted with any other amino acid other than cysteine were analyzed by CE-SDS and HP-SEC.

[0376] CE-SDS was performed as described in Example 5 under reducing and non-reducing conditions. All of the tested IgG1-005-E430G antibody variants containing further mutations at amino acid P329 showed behavior similar to that of 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, while under reducing conditions, a heavy chain with an apparent MW of 50 kDa and a light chain with an apparent MW of 26 kDa were visible (summarized in Table 1). These data suggest that under denaturing conditions, monomeric molecules are formed that exhibit behavior typical of wild-type IgG1 antibodies.

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

[0378] (Table 1) Biophysical property evaluation of IgG1-005-P329X / E430G antibody variants (X represents any amino acid other than P or C) by CE-SDS and HP-SEC TIFF0007695293000008.tif116143

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

[0380] DSF was performed on an iQ5 96-well RT-PCR machine (Bio-Rad) that can detect changes in fluorescence intensity caused by the binding of the extrinsic dye Sypro-Orange (ThermoFisher-Scientific, S6651) to hydrophobic regions exposed by the denaturation of IgG. The thermal melting curve can be obtained by measuring the increase in fluorescence during the controlled stepwise thermal denaturation of the IgG to be analyzed. Therefore, duplicate samples of 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) were prepared. Fluorescence was recorded at temperatures in the range of 25°C to 95°C, increasing stepwise by 0.5°C and adding the time required to record the fluorescence of all wells for a duration of 15 seconds.

[0381] For each antibody analyzed, the midpoint of the first temperature transition (Tm), observed as a sharp increase in fluorescence intensity upon increasing temperature, is averaged for both replicates and summarized in Table 2. Introduction of P329R or P329K in IgG1-005 and IgG1-005-E430G resulted in a moderate increase in the Tm temperature of the antibody, whereas introduction of P329D reduced the Tm temperature of both WT IgG1-005 and IgG1-005-E430G. These data suggest that introduction of P329R or P329GK enhanced 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 variants TIFF0007695293000009.tif108128 1 IgG1-005 antibody variants were ranked according to the reduction in Tm 2 Midpoint of the first heat transition observed upon increasing temperature. Each value represents the average of duplicate measurements.

[0383] Example 9: Effect of mutations at position P329 on FcγRIIIa activation by IgG1-005 variants with enhanced Fc-Fc interactions The effect on ADCC induction was tested for the IgG1-005-E430G variant in which the proline at position 329 was substituted with any amino acid other than cysteine, aspartic acid, methionine, or arginine. The activation of FcγRIIIa-mediated signal transduction by IgG1-005-E430G variants containing a mutation 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). As effector cells, the kit contains Jurkat human T cells that have been engineered to stably express high-affinity FcγRIIIa (V158) and the nuclear factor of activated T cells (NFAT) response element that drives firefly luciferase expression. Briefly, Daudi cells (5,000 cells / well) were seeded in ADCC assay buffer [RPMI-1640 medium supplemented with 3.5% low IgG serum (Lonza, catalog number BE12-115F)] in a 384-well white OptiPlate (Perkin Elmer catalog number 6007290) and incubated at 37°C / 5% CO2 for 6 hours in a total volume of 30 μL containing a series of concentrations (final concentrations of 0.128 to 2.000 ng / mL in 5-fold dilutions) of antibody and thawed ADCC Bioassay Effector Cells. After incubating the plate at room temperature (RT) for 15 minutes, 30 μL of Bio Glo Assay Luciferase Reagent was added and incubated at RT for 5 minutes. The production of luciferase was quantified by luminescence readout information using an EnVision Multilabel Reader (Perkin Elmer). The luminescence signal was normalized by subtracting the background luminescence signal measured in a sample of medium only (no Daudi cells, no antibody, no effector cells).

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

[0385] Example 10: Analysis of the effects of mutations at positions K322 and P329 on the ADCC efficacy of IgG1 - 005 variants with enhanced Fc - Fc interactions The CDC - inhibitory variants of IgG1 - 005 - E430G (described in Examples 4 and 6) that exhibit good 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 against 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 resuspending 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 concentrated to 1×10 7 cells / mL.

[0386] Daudi cells were harvested (5×10 6cells / mL), washed (twice in PBS, 1200 rpm, 5 minutes), 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 at 37 °C for 1 hour in a shaking water bath. 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 diluted to a concentration of 1×10 5 cells / mL.

[0387] For the ADCC experiment, 50 μL of 51 Cr-labeled Daudi cells (5,000 cells / well) were pre-incubated with a series of concentrations (final concentrations of 0.3 - 1,000 ng / mL 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 number 650101). After 20 minutes at room temperature, 50 μL of PBMC (500,000 cells) was added to give an effector:target ratio of 100:1, and incubated at 37 °C / 5% CO2 for 4 hours. To examine the maximum amount of cell lysis, 50 μL of 51 Cr-labeled Daudi cells (5,000 cells) were incubated with 100 μL of 5% Triton-X100. To examine the amount of spontaneous lysis, 5,000 51 Cr-labeled Daudi cells were incubated in 150 μL of medium without any antibody or effector cells. The antibody-independent cell lysis level was examined by incubating 5,000 Daudi cells with 500,000 PBMC without antibody. The released 51To count the amount of Cr, the plates were centrifuged (1200 rpm, 10 minutes) and 25 μL of the supernatant was transferred to 100 μL of Microscint-40 solution (Packard, catalog number 6013641) in a 96-well plate. The plate was sealed and shaken at 800 rpm for 15 minutes, and the released 51 Cr was counted using a gamma counter. The rate of antibody-mediated lysis was calculated as follows using the measured counts per minute (cpm): (cpm sample - cpm Ab-independent lysis) / (cpm maximum lysis - cpm spontaneous lysis) × 100%.

[0388] The dose-responsive ADCC-mediated killing of Daudi cells by IgG1-005-E430G was completely inhibited by introducing the P329D, P329K, or P329R mutation, as shown in Figure 7 for a series of antibody concentrations 0.3 ng / mL - 3 ng / mL - 30 ng / mL - 300 ng / mL. In contrast, IgG1-005-E430G variants with the K322E or P329A mutation retained significant ADCC efficacy against Daudi cells.

[0389] Summarizing the CDC data described in Example 4 and Example 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 mutation resulted in inhibition of both the CDC activity and the ADCC activity of IgG1-005-E430G, despite the enhancing effect of the E430G mutation on Fc-Fc interaction and hexamerization upon target binding on the cell surface. In contrast, the K322E and P329A mutations resulted in CDC inhibition, but the ADCC efficacy by IgG1-005-E430G was retained.

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

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

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

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

[0394] Example 12: Biophysical Property Evaluation of Hexameric IgG1-005-E345R / E430G / S440Y Variants Containing the K322E or P329D Mutation To test the effects of K322E and P329D on the hexamerization of IgG1, the inventors utilized the triple mutant IgG1-005-E345R / E430G / S440Y (RGY), which has been shown to form antibody hexamers in solution and combines three Fc-Fc interaction enhancing mutations, E345R, E430G, and S440Y (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 effects on antibody hexamerization were 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 for 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 the oligomer (elution time ~6.3 minutes) and monomer (elution time ~9 - 9.3 minutes) were observed, with intermediate intensities presumably resulting from a dynamic conversion between the oligomeric and monomeric states. The oligomer fraction in IgG1-005-ERGY was measured to be 58.4%, while 31.4% was monomeric; 10.2% eluted as intermediate species. The oligomer fraction in IgG1-005-DRGY was measured to be 78.8%, while the monomer fraction was 12.5%; 8.7% intermediate species were 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), a single molecular species with an apparent MW of approximately 150 kDa was shown for both IgG1-005-ERGY and IgG1-005-DRGY under non-reducing conditions, while 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 indicated that the behavior of IgG1-005-ERGY and IgG1-005-DRGY was similar to that of the WT monomeric IgG1 assay control antibody, showing that hexamerization was disrupted under denaturing CE-SDS conditions and consistent with non-covalent Fc-Fc interactions.

[0396] Native mass spectrometry analysis of 2 μM IgG1-005-DRGY was performed using a modified LCT time-of-flight (Waters, UK) mass spectrometer adjusted for optimal performance in high mass detection, buffered in 150 mM ammonium acetate, pH 7.5, in the absence or presence of excess C1q. Samples were sprayed from a borosilicate glass capillary placed in a standard fixed nano-spray source. Data analysis was performed using MassLynx (Waters, UK) and Origin Pro (Origin Lab, USA) software. IgG1-005-DRGY formed hexamers as observed with IgG1-005-RGY (Figure 9). No detectable C1q binding was obtained upon addition of C1q to the IgG1-005-DRGY hexamer, while IgG1-005-RGY readily bound to C1q under equivalent conditions (Figure 9C).

[0397] In summary, the biophysical analysis described in this example showed that the introduction of the C1q-binding inhibitory mutations P329D or K322E did not block the 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 variants 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 to induce killing by a functional DR5 antibody having enhanced Fc-Fc interaction and the P329D mutation The agonistic death receptor 5 (DR5) antibody can induce the death of DR5-positive tumor cells by activating the extrinsic apoptosis pathway through DR5 hyperclustering, which leads to the recruitment of the adapter protein Fas-associated protein with death domain (FADD) to the intracellular DR5 death domain, and further results in the binding and activation of caspase-8 and the formation of the DISC (death-inducing signaling complex) that initiates apoptosis. To show that the Fc-Fc interaction is involved in the killing by a combination of DR5 antibodies (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) containing the E430G mutation for enhanced Fc-Fc interaction, the inventors utilized a 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science 2000 Feb 18;287(5456):1279-83) that binds to the Fc within the region containing the core amino acids in the hydrophobic patch involved in the Fc-Fc interaction (Diebolder et al., Science.2014 Mar 14;343(6176):1260-3). A viability assay was 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. The cells were pelleted by centrifugation at 1,200 rpm for 5 minutes and resuspended in culture medium at 0.5×10 5Resuspended at a concentration of cells / mL in RPMI 1640 containing [25 mM Hepes and L-glutamine (Lonza catalog number BE12-115F) + 10% DBSI (Life Technologies catalog number 10371-029) + Pen / Strep (Lonza catalog number DE17-603E)]. 100 μL of single cell suspension (5,000 cells / well) was seeded into a 96-well flat-bottom polystyrene plate (Greiner Bio-One, catalog number 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 non-specific 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 examine maximum killing, samples were incubated with 5 μM staurosporine (Sigma Aldrich, catalog number S6942). The percentage of viable cells was examined using the CellTiter-Glo luminescent cell viability assay (Promega, catalog number G7571) which quantifies the ATP present (which is an indicator of metabolically active cells). From the kit, 20 μL of luciferin solution reagent was added per well and the plate was mixed by shaking at 500 rpm for 2 minutes. Next, the plate was incubated at 37°C for 1.5 hours. 100 uL of the supernatant was transferred to a white OptiPlate-96 (Perkin Elmer, catalog number 6005299) and luminescence was measured using an EnVision Multilabel Reader (PerkinElmer). The data was analyzed and plotted using non-linear regression (sigmoid 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)] * Calculated using 100.

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

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

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

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

[0403] Example 14: Glycosylation Profiling of IgG1-005 Variants with Enhanced Fc-Fc Interaction Containing K322E, P329D, or P329R Mutations The N-linked glycans of the purified antibodies IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G were analyzed by mass spectrometry.

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

[0405] The antibody variants IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G all showed a glycosylation profile similar to that commonly observed for IgG1 antibodies expressed in EXPI293 cells, with low levels of mannose-5 or charged species, high levels of fucosylation, and 10% - 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 of the IgG1-005-E430G variant TIFF0007695293000010.tif75142

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

[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 guidelines defined by FELASA. All experiments were conducted in accordance with the Dutch Animal Protection Act (WoD), 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, 11–12 weeks old (3 mice per group), were intravenously injected with 500 μg of antibody (25 mg / kg) in an injection volume of 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 min, 4 h, 1 day, 2 days, 7 days, 14 days, and 21 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged at 14,000 g for 10 min. 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 a 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 h. After washing, 100 μL of diluted plasma samples were added and incubated on a plate shaker at room temperature for 1 h.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 substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS; Roche, catalog number 11112 422001; 1 tablet in 50 mL of ABTS buffer (Roche, catalog number 11112 597001)] for 15 minutes at room temperature in the dark. 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 using a microplate reader (Biotek, Winooski, VT). The concentration was calculated by using the injected material as a reference curve. As a plate control, human myeloma protein containing IgG (The binding site, UK) was included. The human IgG concentration (unit: μg / mL) was plotted using Graphpad prism 6.0 (Figure 11A), and the area under the curve (AUC) was calculated. The clearance until the last day of blood sample collection (day 21) was determined by the formula D. * 1.000 / AUC (where D is the injection dose (25 mg / kg)) (Figure 11B).

[0409] All of the CDC-inhibitory mutant IgG1-005-K322E / E430G, IgG1-005-P329D / E430G, and IgG1-005-P329R / E430G showed 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 with enhanced Fc-Fc interaction was not affected by K322E (which inhibits CDC but retains ADCC efficacy) or P329D and P329R (which inhibit the efficacy of both CDC and ADCC).

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

[0411] The CDC efficacy of IgG1-005-E430G against Daudi cells was completely inhibited by substituting the proline at position 329 with methionine (M), aspartic acid (D), or arginine (R) (Figure 12). In contrast, substitution of the proline at position 329 with alanine (A) only partially decreased the CDC efficacy, with a shift in the EC50 from 0.01 μg / mL in IgG1-005-E430G to 0.10 μg / mL in IgG1-005-P329A / E430G, but no effect on maximum killing was observed. These data indicate that substitution of the 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 the CDC efficacy by IgG1-005-E430G.

[0412] Example 17: Effect of the P329R and P329D mutations on the in vitro CDC efficacy of Campath IgG isotype variants with enhanced Fc-Fc interaction The effects of the P329R and P329D mutations on in vitro CDC efficacy were 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 (final concentration range of 0.001 - 30.0 μg / mL) were tested in the in vitro CDC assay in Wien 133 cells using 20% NHS essentially as described in Example 2. The area under the dose-response curve of the triplicate experiments was calculated using GraphPad Prism 7.02 with a log-transformed concentration axis 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 of IgG1-Campath-E430G in Wien 133 cells increased approximately 3-fold compared to WT, but the CDC activity decreased to background levels by substituting proline at position 329 with arginine (R) or aspartic acid (D) (Figure 13). Similarly, CDC by IgG2-Campath-E430G also decreased to background levels upon introduction of the mutation P329R or P329D. Also, IgG3 and IgG4 isotype variants containing both the E430G mutation and either P329R or P329D did not show 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 the CDC efficacy by IgG1, IgG2, IgG3, and IgG4 isotype variants of IgG1-Campath-E430G.

[0415] Example 18: Effect of the Mutation K322E on the In Vitro CDC Efficacy of Campath IgG Isotype Variants with Enhanced Fc-Fc Interaction The effect of the mutation K322E 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 14). Different concentrations of purified antibody (final concentration range of 0.001 - 30.0 μg / mL) were tested in vitro in Wien 133 cells in a CDC assay essentially as described in Example 2 using 20% NHS. The area under the dose-response curve of the triplicate experiments was calculated using GraphPad Prism 7.02 with a log-transformed concentration axis 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 of IgG1-Campath-E430G in Wien 133 cells increased approximately 3-fold compared to WT but decreased by up to approximately 18% by substituting the lysine at position 322 with glutamic acid (E) (Figure 14). Similarly, CDC by IgG2-Campath-E430G decreased to background levels upon introduction of the mutation K322E. Also, IgG3 and IgG4 isotype variants containing both mutations E430G and K322E did not show CDC lysis above background levels. These data indicate that substitution of the lysine at position 322 with glutamic acid resulted in efficient inhibition of the CDC efficacy by the IgG1, IgG2, IgG3, and IgG4 isotype variants of IgG1-Campath-E430G.

[0417] Example 19: Effect of mutations P329R and K322E on in vitro CDC efficacy of Campath 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 the purified antibody (final concentration range of 0.001 - 30.0 μg / mL) were tested in vitro in Wien 133 cells in a CDC assay essentially as described in Example 2 using 20% NHS. The area under the dose-response curve of triplicate experiments was calculated using GraphPad Prism 7.02 with a log-transformed concentration axis 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 of IgG1-Campath-E345K, which contains the Fc-Fc interaction-promoting mutation E345K, increased ap...

Claims

1. A polypeptide comprising the Fc region of human IgG and an antigen-binding region, wherein the Fc region comprises CH2 and CH3 domains, and the Fc region has the following mutations corresponding to the following amino acid positions in human IgG1 according to EU numbering: (i) a first mutation and (ii) a second mutation: i. a first E430G mutation; and ii. a second P329R mutation and comprising the polypeptide.

2. The polypeptide according to claim 1, wherein the Fc region comprises one or more additional mutations.

3. The polypeptide according to claim 2, wherein the Fc region comprises an additional mutation corresponding to position K439 in the CH3 domain, or the additional mutation is present at position S440.

4. The polypeptide according to claim 3, wherein the additional mutation is selected from S440K or K439E.

5. At least 20%, at least 30%, at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced Fc effector function compared to a parental polypeptide that is identical to the polypeptide except for not containing the second mutation, the polypeptide according to any one of claims 1 to 4.

6. The polypeptide according to any one of claims 1 to 5, which does not induce Fc effector function.

7. The polypeptide according to claim 5 or 6, 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.

8. The polypeptide according to any one of claims 1 to 7, wherein the Fc region is a human IgG1 isotype.

9. The polypeptide according to any one of claims 1 to 8, wherein the antigen-binding region binds to a member of TNFR-SF.

10. The polypeptide according to claim 9, wherein the TNFR-SF does not contain an intracellular death domain.

11. The polypeptide according to claim 9, wherein the member of TNFR-SF is selected from the group consisting of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR.

12. The polypeptide according to claim 9, wherein the TNFR-SF is selected from the group consisting of OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR.

13. An antibody, monospecific antibody, bispecific antibody, or multispecific antibody comprising the polypeptide according to any one of claims 1 to 12.

14. The antibody according to claim 13, which is a human antibody, humanized antibody, or chimeric antibody.

15. A method for reducing the Fc effector function of a polypeptide comprising an Fc region and an antigen-binding region of a human immunoglobulin, wherein the Fc region comprises CH2 and CH3 domains, the Fc region comprises a first mutation corresponding to E430G in human IgG1 according to EU numbering, the method comprising the step of introducing a second mutation corresponding to P329R in human IgG1 according to EU numbering, the method.

16. The method according to claim 15, wherein the Fc region contains one or more additional mutations in the CH3 domain.

17. The method according to claim 16, wherein said further mutation is selected from S440K or K439E. **Claim 18** The method according to any one of claims 15 to 17, wherein the Fc effector function is reduced by at least 20%, at least 30%, 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 a parental polypeptide that is identical to said polypeptide except for not containing said second mutation. **Claim 19** The method according to any one of claims 15 to 18, wherein the Fc effector function is selected from the group consisting 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. **Claim 20** The method according to claim 19, 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% compared to a comparative antibody comprising a comparative polypeptide that is identical to said polypeptide except for not containing said second mutation. **Claim 21** A composition comprising at least one polypeptide according to any one of claims 1 to 12, or an antibody according to claim 13 or 14. **Claim 22** A first polypeptide according to any one of claims 1 to 12, and a second polypeptide according to any one of claims 1 to 12 The composition according to claim 21. **Claim 23** A first polypeptide comprising a first antigen-binding region and a first Fc region, and a second polypeptide 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, (iii) a further mutation, and these mutations correspond to the following amino acid positions in human IgG1 according to EU numbering: (i) a first E430G mutation; (ii) a second P329R mutation; (iii) a further mutation at K439 or S440, provided that the first and second Fc regions do not contain the further mutation at the same amino acid position. The composition according to claim 22.

24. The composition according to claim 22 or 23, wherein the first polypeptide and the second polypeptide are present in a molar ratio of 1:49 to 49:1 in the composition.

25. The composition according to claim 22 or 23, wherein the first polypeptide and the second polypeptide and / or a further polypeptide are present in an equimolar ratio in the composition.

26. The composition according to any one of claims 21 to 25, which is a pharmaceutical composition.

27. The polypeptide according to any one of claims 1 to 12, the antibody according to claim 13 or 14, or the composition according to any one of claims 21 to 26, for use as a medicament.

28. The polypeptide according to any one of claims 1 to 12, the antibody according to claim 13 or 14, or the composition according to any one of claims 21 to 27, for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

29. A kit of parts comprising the polypeptide according to any one of claims 1 to 12, the antibody according to claim 13 or 14, or the composition according to any one of claims 21 to 28, wherein the polypeptide, antibody, or composition is present in one or more containers.

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