Polypeptide variants and their uses

Abstract

This provides improved antibody treatments, particularly treatments with improved selectivity. [Solution] The present invention provides a combination therapy comprising two or more antigen-binding polypeptides containing Fc regions, such as antibodies. The polypeptides are modified such that heterooligomerization between polypeptides is significantly more favorable than homooligomerization when bound to their respective target antigens. Polypeptides, compositions, kits, and devices suitable for use in the combination therapy of the present invention are also provided.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a combination therapy comprising two or more Fc region-containing antigen-binding polypeptides, such as antibodies, wherein the polypeptides are modified such that heterooligomerization between polypeptides is significantly more favorable than homooligomerization when bound to their respective target antigens. The present invention also relates to polypeptides suitable for use in the combination therapy of the present invention, as well as the use, composition, kit, and device of such polypeptides. [Background technology]

[0002] Background of the Invention Antibodies are highly effective molecules that can influence target cells through a variety of mechanisms. In some cases, the mere binding of an antibody to a target antigen on the cell surface can produce an antagonist or agonist effect on the target antigen, and therefore on the target cell. Alternatively, or in addition to the above, the effect of an antibody on target cells is achieved by effector function, typically Fc-mediated effector function, such as the ability of the antibody to induce complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0003] ADCC and ADCP are initiated by the binding of an IgG Fc region to an Fcγ receptor on effector cells. WO2012 / 130831 (Patent Document 1) discloses an Fc region-containing polypeptide having altered ADCC function as a result of one or more amino acid substitutions in the Fc region of the polypeptide.

[0004] CDC is initiated by the binding of C1q to the Fc region of an antibody. C1q is a multimeric protein consisting of six globular binding heads attached to a stalk. Each globular binding head has low affinity for IgG, and for the classical complement pathway to be triggered, C1q must acquire avidity by binding to multiple IgG1 molecules on the cell surface. IgG hexamerization at target binding on the cell surface has been shown to support strong (avid) C1q binding. This hexamerization is mediated by non-covalent intermolecular Fc-Fc interactions. Fc-Fc interactions can be enhanced by point mutations in the CH3 domain, including E345R and E430G (see, e.g., WO2013 / 004842 and WO2014 / 108198 (Patent Documents 2 and 3)). WO2017 / 093447 (Patent Document 4) is directed towards antibodies that bind to death receptors, including an intracellular death domain. This application discloses that the K439E mutation in the Fc region of an antibody results in Fc-Fc repulsion, and therefore a weak Fc-Fc interaction between two antibody molecules having the mutation. This effect can be neutralized by introducing the S440K mutation into the other antibody molecule, which leads to the restoration of the Fc-Fc interaction. See also Diebolder et al. (2014) Science 343:126 (Non-Patent Literature 1).

[0005] While antibody therapy is often highly effective, antibody target antigens are frequently not expressed only in diseased cells or tissues, but are also found in other healthy cells or tissues. Therefore, antibody therapy may lack selectivity for the target tissue, and non-affected tissues may be affected by the antibody treatment, leading to toxicity.

[0006] Therefore, improved antibody treatments, particularly those with improved selectivity, are needed.

[0007] Therefore, an object of the present invention is to provide a method for treating a disease by increasing the selectivity of polypeptides or antibodies. Another object of the present invention is to provide a method for treating a disease by providing a first polypeptide and a second polypeptide that do not have monotherapy activity but exhibit activity only when bound together on the same target cells or the same target tissue. Therefore, an object of the present invention is to provide a method for treating a disease by administering a first polypeptide that can bind to a first antigen and a second polypeptide that can bind to a second antigen, wherein the first and second polypeptides have no effect or only a slight effect on tissues or target organs expressing either the first or second antigen, but provide effective treatment to tissues or target organs expressing both the first and second antigens. A further object of the present invention is to provide a method for treating a disease by administering a first polypeptide and a second polypeptide modified to favor heterooligomerization while preventing homooligomerization (autooligomerization). Another object of the present invention is to provide polypeptides that can be used in such a method of treatment, i.e., polypeptides having at least one autooligomerization inhibitory mutation. Another object of the present invention is to provide a first polypeptide having an auto-oligomerization inhibitory mutation and a second polypeptide having an auto-oligomerization inhibitory mutation, wherein the auto-oligomerization inhibitory mutation in the first polypeptide and the auto-oligomerization inhibitory mutation in the second polypeptide are complementary, enabling heterooligomerization of the first polypeptide and the second polypeptide when bound to target cells. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO2012 / 130831 [Patent Document 2] WO2013 / 004842 [Patent Document 3] WO2014 / 108198 [Patent Document 4] WO2017 / 093447 [Non-patent literature]

[0009] [Non-Patent Document 1] Diebolder et al.(2014)Science 343:126 [Overview of the project]

[0010] The present invention provides methods, polypeptides, and compositions that can be used to improve the selectivity of antibody treatment against a desired target cell population.

[0011] The method and use of the present invention relate to treatment with two antibodies (or antibody-like polypeptides), wherein the two antibodies bind to two different target antigens, and the Fc regions of the antibodies are modified so that heterooligomerization of the two antibodies is significantly more favorable than homooligomerization. As a result of these modifications, cells expressing both antigen targets (allowing efficient (hetero)oligomerization of the two antibodies) will undergo more antibody oligomerization than cells expressing only one of the targets (resulting in inefficient (homo)oligomerization or no (homo)oligomerization). Since oligomerization generally enhances the potency of antibodies, the antibody combination treatment will be more potent against cells co-expressing the targets than against cells expressing only one of the targets. Thus, this antibody combination treatment has improved selectivity against cells or tissues expressing both target antigens. Therefore, by selecting two antigens that are co-expressed in the desired target cell population but not in or are only co-expressed in low amounts in cell populations that should not be targeted, it is possible to design a combined antibody treatment that will have a selective effect on the desired target cell population.

[0012] The increased efficacy is thought to be obtained not only when two target antigens are co-expressed on the same cell, but also in other situations where target cells are in proximity. Furthermore, in addition to classical antibodies, antibody-like polypeptides can be used as long as they contain an Fc region and an antigen-binding region.

[0013] Thus, in a first aspect, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen, (a) the first polypeptide comprises an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide comprises an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa, or the first polypeptide comprises an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide comprises an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa, and / or (b) the first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation at the amino acid position corresponding to Q438 in human IgG, or vice versa, or the first polypeptide comprises a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Y436K or Y436R mutation at the amino acid position corresponding to Y4 in human IgG1, or vice versa, or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0014] In one embodiment of the method of the present invention, the first polypeptide comprises a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide comprises a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0015] In one aspect of the present invention, the first polypeptide contains an F436N, F436K, F436Q, or F436R mutation at the amino acid position corresponding to F436 in human IgG3, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains an F436N or F436Q mutation at the amino acid position corresponding to F436 in human IgG3, and the second polypeptide contains an F436K or F436R mutation at the amino acid position corresponding to F436 in human IgG3, or vice versa.

[0016] In a further aspect, the present invention relates to a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, for use as a pharmaceutical in combination with a second polypeptide comprising a second antigen-binding region capable of binding to a second antigen and a second Fc region of human IgG, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0017] In a further aspect, the present invention relates to a polypeptide comprising a human IgG Fc region and an antigen-binding region capable of binding to an antigen, wherein the polypeptide is (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1 and / or (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1. and / or (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 Includes, Here, the amino acid positions correspond to human IgG1 according to EU numbering. If the polypeptide contains the S440K mutation, then at least one of the other mutations specified in options (a) and (b) must also be present.

[0018] In another aspect, the present invention relates to compositions comprising one or more polypeptides of the present invention as described herein.

[0019] In a further aspect, the present invention relates to a pharmaceutical composition comprising one or more polypeptides of the present invention as described herein.

[0020] In a further aspect, the present invention relates to a kit comprising a first container containing a first polypeptide suitable for use in the invention described herein and a second container containing a second polypeptide suitable for use in the invention described herein.

[0021] In yet another aspect, the present invention relates to a device, such as a dual-chamber syringe, comprising a first compartment containing a first polypeptide suitable for use in the invention described herein and a second compartment containing a second polypeptide suitable for use in the invention described herein.

[0022] [Invention 1001] A method for treating a disease or disorder, comprising the step of administering to a subject in need of such treatment a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering. [Invention 1002] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The method of the present invention 1001. [Invention 1003] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. The method of the present invention 1001. [Invention 1004] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The method of the present invention 1001. [Invention 1005] (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The method of the present invention 1001. [Invention 1006] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The method of the present invention 1001. [Invention 1007] The method of the present invention, wherein the first polypeptide and the second polypeptide do not contain the mutation specified in option (c), the first polypeptide further contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide further contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. [Invention 1008] (i) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ii) The first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (iii) The first polypeptide contains the I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (iv) The first polypeptide contains the I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (vii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (viii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ix) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (x) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xvi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xvii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xviii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xix) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xx) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Any of the methods of the present invention described above. [Invention 1009] The first polypeptide further contains mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1, and / or the second polypeptide further contains mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1, or vice versa. However, if the first or second polypeptide contains a mutation at the amino acid position corresponding to K439 or S440, the further mutation in the polypeptide shall not be found at the amino acid position corresponding to S440. Any of the methods of the present invention described above. [Invention 1010] The first polypeptide contains one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or the second polypeptide contains one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or The first polypeptide contains the T437R mutation and the K248E mutation, and / or the second polypeptide contains the T437R mutation and the K248E mutation. The method of the present invention 1009. [Invention 1011] The method of the present invention 1010, wherein the first polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K, and / or the second polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K. [Invention 1012] The method of the present invention 1011, wherein the first polypeptide contains E430G and the second polypeptide also contains E430G. [Invention 1013] Any method of the present invention, wherein the first polypeptide and / or the second polypeptide are further modified such that their ability to induce effector functions, such as Fc-mediated effector functions, is altered compared to polypeptides that are identical except for the aforementioned further modifications. [Invention 1014] Any method of the present invention, wherein the first polypeptide and / or the second polypeptide are further modified such that their ability to induce antibody-dependent cell-mediated cytotoxicity is altered compared to polypeptides that are identical except for the aforementioned further modifications. [Invention 1015] Any method of the present invention, wherein the first polypeptide and / or the second polypeptide are further modified such that their ability to induce complement-dependent cytotoxicity is altered compared to a polypeptide that is otherwise identical. [Invention 1016] The method of the present invention, wherein the first polypeptide is an antibody such as a full-length antibody, and / or the second polypeptide is an antibody such as a full-length antibody. [Invention 1017] The method of the present invention, wherein the first polypeptide is an IgG1 antibody and / or the second polypeptide is an IgG1 antibody. [Invention 1018] The method of Invention 1016 or Invention 1017, wherein the first antibody is human, humanized, or chimeric, and / or the second antibody is human, humanized, or chimeric. [Invention 1019] The method according to any of items 1016 to 1018 of the present invention, wherein the first antibody is bispecific and / or the second polypeptide is bispecific. [Invention 1020] The present invention, any method wherein both the first antigen and the second antigen are molecules exposed on the cell surface. [Invention 1021] Any method of the present invention, wherein the first antigen and the second antigen coexist in a cell or tissue that is a target cell or target tissue for the disease or disorder to be treated. [Invention 1022] (a) The first and second antigens are not present in cells or tissues that are not target cells or tissues for the disease or disorder to be treated, or (b) The first and second antigens are present to a lower degree in cells or tissues that are not target cells or target tissues for the disease or disorder to be treated than in cells or tissues that are target cells or target tissues for the disease or disorder to be treated. The method of the present invention 1021. [Invention 1023] The method of the present invention, wherein the first antigen and the second antigen are not identical and neither is a death receptor containing an intracellular death domain. [Invention 1024] Any method of the present invention, wherein neither the first antigen nor the second antigen is a death receptor. [Invention 1025] The first polypeptide and the second polypeptide are combined in molar ratios of 1:50 to 50:1, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:5, 1:5, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:4 Any method of the present invention, wherein the drug is administered in a molar ratio of 0, 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, or 2:1. [Invention 1026] Any method of the present invention, wherein the first polypeptide and the second polypeptide are administered simultaneously. [Invention 1027] Any method of the present invention for treating cancer. [Invention 1028] A first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, for use as a pharmaceutical in combination with a second polypeptide comprising a second antigen-binding region capable of binding to a second antigen and a second Fc region of human IgG, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid position corresponds to human IgG1 according to EU numbering, representing the first polypeptide. [Invention 1029] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The first polypeptide of the present invention 1028. [Invention 1030] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. The first polypeptide of the present invention 1028. [Invention 1031] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The first polypeptide of the present invention 1028. [Invention 1032] (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The first polypeptide of the present invention 1028. [Invention 1033] (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. The first polypeptide of the present invention 1028. [Invention 1034] The first polypeptide according to any of the invention 1028 to 1033, wherein the first polypeptide and the second polypeptide do not contain the mutation specified in option (c), the first polypeptide further contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide further contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. [Invention 1035] (i) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ii) The first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (iii) The first polypeptide contains the I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (iv) The first polypeptide contains the I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (vii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (viii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ix) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (x) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; and the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (xiii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; and the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (xiv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xv) The first polypeptide contains the Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains the Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains the I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xvi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (xvii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (xviii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xix) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xx) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; and the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. A first polypeptide according to any of invention 1028 to 1034. [Invention 1036] The first polypeptide further contains mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1, and / or the second polypeptide further contains mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1, or vice versa. However, if the first or second polypeptide contains the K439E, K439D, S440K, S440R, or S440H mutation, the further mutation in the polypeptide shall not be located at position S440. A first polypeptide according to any of invention 1028 to 1035. [Invention 1037] The first polypeptide contains one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or the second polypeptide contains one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or The first polypeptide contains the T437R mutation and the K248E mutation, and / or the second polypeptide contains the T437R mutation and the K248E mutation. The first polypeptide of the present invention 1036. [Invention 1038] The first polypeptide of Invention 1037, wherein the first polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K, and / or the polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K. [Invention 1039] The first polypeptide of Invention 1038, wherein the first polypeptide contains E430G, and the second polypeptide also contains E430G. [Invention 1040] A first polypeptide according to any of the invention 1028 to 1039, wherein the first polypeptide and / or second polypeptide are further modified to alter the ability to induce effector functions such as Fc-mediated effector function compared to a polypeptide that is identical except for the aforementioned further modifications. [Invention 1041] A first polypeptide according to any of the inventions 1028 to 1040, wherein the first polypeptide and / or second polypeptide are further modified to alter their ability to induce antibody-dependent cell-mediated cytotoxicity compared to a polypeptide that is identical to the polypeptide except for the aforementioned further modifications. [Invention 1042] A first polypeptide according to any of the inventions 1028 to 1041, wherein the first polypeptide and / or second polypeptide are further modified to alter their ability to induce complement-dependent cytotoxicity compared to a polypeptide that is identical except for the aforementioned modifications. [Invention 1043] The first polypeptide according to any of invention 1028 to 1042, wherein the first polypeptide is an antibody such as a full-length antibody, and / or the second polypeptide is an antibody such as a full-length antibody. [Invention 1044] The first polypeptide according to any one of the invention 1028 to 1043, wherein the first polypeptide is an IgG1 antibody and / or the second polypeptide is an IgG1 antibody. [Invention 1045] The first polypeptide of Invention 1043 or Invention 1044, wherein the first antibody is human, humanized, or chimeric, and / or the second antibody is human, humanized, or chimeric. [Invention 1046] The first polypeptide according to any one of invention 1043 to 1045, wherein the first antibody is bispecific and / or the second polypeptide is bispecific. [Invention 1047] A first polypeptide according to any of items 1028 to 1046 of the present invention, wherein both the first antigen and the second antigen are molecules exposed on the cell surface. [Invention 1048] The first antigen and the second antigen are co-existing in cells or tissues that are target cells or target tissues for the disease or disorder to be treated, according to any first polypeptide of the present invention 1028 to 1047. [Invention 1049] (a) The first and second antigens are not present in cells or tissues that are not target cells or tissues for the disease or disorder to be treated, or (b) The first and second antigens are present to a lower degree in cells or tissues that are not target cells or target tissues for the disease or disorder to be treated than in cells or tissues that are target cells or target tissues for the disease or disorder to be treated. The first polypeptide of the present invention 1048. [Invention 1050] The first polypeptide according to any of the invention items 1028 to 1049, wherein the first antigen and the second antigen are not identical, and neither is a death receptor containing an intracellular death domain. [Invention 1051] A first polypeptide according to any of the invention items 1028 to 1050, wherein neither the first antigen nor the second antigen is a death receptor. [Invention 1052] The first polypeptide and the second polypeptide are in a molar ratio of 1:50 to 50:1, for example, a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:5, 1:5, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40. A first polypeptide according to any of the invention 1028 to 1051, administered in a molar ratio of 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, or 2:1. [Invention 1053] The first polypeptide according to any of invention 1028 to 1052, wherein the first polypeptide and the second polypeptide are administered simultaneously. [Invention 1054] A first polypeptide of any one of the invention 1028-1053, which is used to treat cancer. [Invention 1055] A composition comprising a first polypeptide and a second polypeptide according to any of the present invention 1001 to 1024. [Invention 1056] A pharmaceutical composition comprising a first polypeptide and a second polypeptide according to any of Invention 1001 to 1024 and a pharmaceutically acceptable carrier, wherein the first polypeptide and the second polypeptide are preferably present in the molar ratio specified in Invention 1025. [Invention 1057] A kit comprising a first container containing a first polypeptide of any of Invention 1001 to 1024, and a second container containing a second polypeptide of any of Invention 1001 to 1024. [Invention 1058] A device such as a dual-chamber syringe, comprising a first compartment containing a first polypeptide of any of the present invention 1001 to 1024, and a second compartment containing a second polypeptide of any of the present invention 1001 to 1024. [Invention 1059] A polypeptide comprising the Fc region of human IgG and an antigen-binding region capable of binding to an antigen, (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1 and / or (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1. and / or (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 Includes, Here, the amino acid positions correspond to human IgG1 according to EU numbering. However, if the polypeptide contains the S440K mutation, then at least one of the other mutations specified in options (a) and (b) must also be present. Polypeptide. [Invention 1060] (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1. A polypeptide according to the present invention 1059, comprising: [Invention 1061] (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 A polypeptide according to the present invention 1059, comprising: [Invention 1062] (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 A polypeptide according to the present invention 1059, comprising: [Invention 1063] (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 A polypeptide according to the present invention 1059, comprising: [Invention 1064] A polypeptide according to any of the invention 1059 to 1063, which does not contain the mutation specified in option (c), and further comprises a K439E mutation at the amino acid position corresponding to K439 in human IgG1 or an S440K mutation at the amino acid position corresponding to S440 in human IgG1. [Invention 1065] (i) I253G mutation at the amino acid position corresponding to I253 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (ii) I253R mutation at the amino acid position corresponding to I253 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (iii) H310R mutation at the amino acid position corresponding to H310 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (iv) H310D mutation at the amino acid position corresponding to H310 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (v) Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (vi) Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (vii) Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (viii) Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, and K439E mutation at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1. Includes, or (ix) Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and I253G mutation at the amino acid position corresponding to I253 in human IgG1, or H310R mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (x) Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and I253G mutation at the amino acid position corresponding to I253 in human IgG1, or H310R mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xi) Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and I253G mutation at the amino acid position corresponding to I253 in human IgG1, or H310R mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xii) Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, and I253G mutation at the amino acid position corresponding to I253 in human IgG1, or H310R mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xiii) Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and I253R mutation at the amino acid position corresponding to I253 in human IgG1, or H310D mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xiv) Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and I253R mutation at the amino acid position corresponding to I253 in human IgG1, or H310D mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xv) Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and I253R mutation at the amino acid position corresponding to I253 in human IgG1, or H310D mutation at the amino acid position corresponding to H310 in human IgG1. Includes, or (xvi) Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, and I253R mutation at the amino acid position corresponding to I253 in human IgG1, or H310D mutation at the amino acid position corresponding to H310 in human IgG1. including, A polypeptide according to any of invention 1059 to 1064. [Invention 1066] The polypeptide further comprises mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1, However, if the polypeptide contains the K439E, K439D, S440K, S440R, or S440H mutation, the further mutation in the polypeptide shall not be located at position S440. A polypeptide according to any of invention 1059 to 1065. [Invention 1067] It contains one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or Including the T437R mutation and the K248E mutation, Polypeptide of the present invention 1066. [Invention 1068] A polypeptide of the present invention 1067 comprising one or both mutations selected from the group consisting of E430G and E345K. [Invention 1069] Polypeptide 1068 of the present invention, containing the E430G mutation. [Invention 1070] A polypeptide according to any of the invention 1059 to 1069, further modified to alter its ability to induce effector functions such as Fc-mediated effector function compared to a polypeptide that is identical to the polypeptide except for the aforementioned further modifications. [Invention 1071] A polypeptide according to any of the Invention 1059 to 1070, further modified to alter its ability to induce antibody-dependent cell-mediated cytotoxicity compared to a polypeptide that is identical to the polypeptide except for the aforementioned further modifications. [Invention 1072] A polypeptide according to any of the invention 1059 to 1071, further modified to alter its ability to induce complement-dependent cytotoxicity compared to a polypeptide that is identical to the polypeptide except for the aforementioned further modifications. [Invention 1073] A polypeptide according to any of the Invention 1059-1072, such as a full-length antibody. [Invention 1074] A polypeptide according to any of the present invention 1059 to 1073, which is an IgG1 antibody. [Invention 1075] The polypeptide of Invention 1073 or Invention 1074, wherein the antibody is human, humanized, or chimeric. [Invention 1076] A polypeptide according to any of the present invention 1073 to 1075, wherein the antibody is bispecific. [Invention 1077] A polypeptide according to any of items 1059 to 1076 of the present invention, wherein the antigen is a molecule exposed on the cell surface. [Invention 1078] The antigen is not a death receptor, but a polypeptide according to any of the Invention 1059-1077. [Invention 1079] A pharmaceutical composition comprising any polypeptide according to invention 1059 to 1078 and a pharmaceutically acceptable carrier. These and other aspects of the present invention, in particular the various uses and therapeutic applications of the polypeptide or antibody, will be described in more detail below. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows the amino acid sequence alignment of human IgG1m(a), IgG1m(f), IgG2, IgG3, and IgG4 Fc backbones using an EU-based (IgG1-specific) numbering scheme (Edelman et al. 1969 Proc Natl Acad Sci USA 63,78-85). [Figure 2] Figure 2 shows the results of a CDC assay testing the effects of IgG1-Campath-E430G antibody variants with the indicated I253 or H310 mutations on manipulating Fc-Fc interactions and their CDC efficacy against Wien 133 cells. Wien 133 cells were incubated in 5% pooled normal human serum (NHS) with concentration series of all possible antibody combinations of single antibody variants and I253+H310 variant pairs. CDC efficacy is expressed in ng / mL as half maximal effective antibody concentration (EC50), determined by the percentage of TO-PRO-3 iodide-positive cells. Maximum cell lysis due to unspecified low EC50 values ​​is indicated as <15 ng / mL. [Figure 3] Figure 3 shows the results of a CDC assay testing the effects of IgG1-Campath-E430G antibody variants with the indicated Y436 or Q438 mutations on manipulating Fc-Fc interactions and their CDC efficacy against Wien 133 cells. Wien 133 cells were incubated in the presence of 5% pooled NHS with concentration series of all possible antibody combinations of single antibody variants and Y436+Q438 variant pairs. CDC efficacy is expressed in ng / mL as the 50% effective antibody concentration (EC50), determined by the percentage of TO-PRO-3 iodide-positive cells. Maximum cell lysis due to unspecified low EC50 values ​​is indicated as <15 ng / mL. [Figure 4]Figure 4 shows the results of a CDC assay testing the effects of IgG1-Campath-E430G antibody variants with the indicated K439 or S440 mutations on manipulating Fc-Fc interactions and their CDC efficacy against Wien 133 cells. Wien 133 cells were incubated in the presence of 5% pooled NHS with concentration series of all possible antibody combinations of single antibody variants and K439+S440 variant pairs. CDC efficacy is expressed in ng / mL as the 50% effective antibody concentration (EC50), determined by the percentage of TO-PRO-3 iodide-positive cells. Maximum cell lysis due to unspecified low EC50 values ​​is indicated as <15 ng / mL. [Figure 5A] Figure 5 shows the effect of Fc-Fc inhibitory mutations I253G and H310R (A), I253K and H310D (B), and I253R and H310D (C) on the CDC efficacy of IgG1-Campath-E430G. Wien 133 cells were incubated in the presence of 20% pooled NHS with concentration series of indicated IgG1-Campath-E430G antibody variants (single mAbs) and combinations thereof (mAb mixtures) containing single Fc-Fc inhibitory mutations. CDC efficacy is expressed as lysis percentage determined by PI-positive cell percentage. IgG-b12 antibody against HIV gp120 was used as an unbound control antibody. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 6A] Figure 6 illustrates the effect of Fc-Fc inhibitory mutation combinations on the CDC efficacy of IgG1-Campath-E430G. Wien 133 cells were incubated in the presence of 20% pooled NHS with concentration series of indicated IgG1-Campath-E430G antibody variants (single mAbs) and combinations thereof (mAb mixtures) containing one or two Fc-Fc inhibitory mutations in each antibody. CDC efficacy is expressed as lysis percentage determined by PI-positive cell percentage. IgG-b12 antibody against HIV gp120 was used as an unbound control antibody. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 6F] See the explanation in Figure 6A. [Figure 6G] See the explanation in Figure 6A. [Figure 7] Figure 7 shows the binding of anti-CD52 IgG1-CAMPATH-1H antibodies with auto-oligomerization inhibitory substitutions to FcRn. The binding of antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E and anti-CD52 IgG1-CAMPATH-1H-E430G-S440K with auto-oligomerization inhibitory substitutions I253G, I253K, I253R, H310D, H310R, Y436N, Y436K, Q438N and / or Q438R to human FcRn is shown using an antibody concentration of 40 μg / ml at (A) pH 7.4 and (B) pH 6.0. FcRn ELISA was performed using recombinant extracellular domains of human FcRn coated with 5 μg / mL (FcRnECDHis-B2M-BIO) and antibody dilution series. The amount of bound antibody was determined using HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm. [Figure 8A]Figure 8 shows FcγR binding of IgG1-CAMPATH-1H variants with the Fc-Fc-enhanced mutation E430G and auto-oligomerization inhibitory substitutions. Binding of immobilized IgG1-CAMPATH-1H-E430G variants with auto-oligomerization inhibitory substitutions K439E, S440K, Y436K, Y436N, Q438N, and Q438R to dimeric His-tagged biotinylated ECD, tested in ELISA assays, for (A) FcγRIIA allotype 131H, (B) FcγRIIA allotype 131R, (C) FcγRIIB, (D) FcγRIIIA allotype 158V, and (E) FcγRIIIA allotype 158F. We present the binding of a 20 μg / mL antibody sample to IgG1-CAMPATH-1H-E430G compared to a control without antibody (background) and IgG1-CAMPATH-1H-E430G (100%). Detection was performed using streptavidin-polyHRP and ABTS. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 8E] See the explanation in Figure 8A. [Figure 9A] Figure 9 shows the CDC efficacy of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody and anti-CD52 IgG1-CAMPATH-1H-E430G-S440K antibody, both as monotherapy and in combination, which carry autooligomerization inhibitory mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as (A) AUC normalized to unbound control antibodies IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%), and (B) lysis percentage determined by PI-positive cell percentage at an antibody concentration of 40 μg / ml. [Figure 9B] See the explanation in Figure 9A. [Figure 10]Figure 10 shows the CDC efficacy of anti-CD20-IgG1-11B8-E430G-K439E antibody and anti-CD20 IgG1-11B8-E430G-S440K antibody, both as monotherapy and in combination, carrying additional auto-oligomerization inhibitory mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G (CAMP-E430G) + IgG1-11B8-E430G (100%). [Figure 11] Figure 11 shows the CDC efficacy of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody and anti-CD20 IgG1-11B8-E430G-S440K antibody, both as monotherapy and in combination, which carry autooligomerization inhibitory mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 12A] Figure 12 shows the CDC efficacy of variants of anti-CD52 IgG1-CAMPATH-1H antibody and anti-CD20 IgG1-11B8 antibody, both monotherapy and in combination, possessing different Fc-Fc interaction-enhancing mutations. (A, B) Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to unbound control antibody IgG1-b12 (0%) and mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 12B] See the explanation in Figure 12A. [Figure 13]Figure 13 illustrates the selectivity of CDC activity by mixed antibody subclass variants (IgG1, IgG2, and hinge-stabilized IgG4) containing anti-CD52 CAMPATH-1H-E430G-K439E with additional auto-oligomerization inhibitory mutations and anti-CD20 11B8-E430G-S440K with additional auto-oligomerization inhibitory mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as normalized AUC of PI-positive cell percentage. Normalization was performed for unbound control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 14A] Figure 14 illustrates the effects of introducing the FcγR binding inhibitory mutation G237A into IgG1-CAMPATH-1H and IgG1-11B8 variants with Fc-Fc interaction enhancing mutations and autooligomerization inhibitory mutations on FcγR binding and CDC activity. The binding of immobilized IgG1-CAMPATH-1H and IgG1-11B8 variants with autooligomerization inhibitory mutations K439E or S440K to dimerized His-tagged biotinylated ECD (A) FcγRIIA allotype 131H, (B) FcγRIIA allotype 131R, (C) FcγRIIB, (D) FcγRIIIA allotype 158F, and (E) FcγRIIIA allotype 158V was tested by ELISA assay. Binding is expressed as absorbance at a wavelength of 405 nm for a 20 μg / mL antibody sample. Detection was performed using streptavidin-polyHRP and ABTS. (F, G) Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as normalized AUC of PI-positive cell percentage. Normalization was performed against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%; F) or a mixture of unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%; G). [Figure 14B] See the explanation in Figure 14A. [Figure 14C]See the explanation in Figure 14A. [Figure 14D] See the explanation in Figure 14A. [Figure 14E] See the explanation in Figure 14A. [Figure 14F] See the explanation in Figure 14A. [Figure 14G] See the explanation in Figure 14A. [Figure 15] Figure 15 illustrates the selectivity of CDC activity by mixed antibody variants of anti-CD52 CAMPATH-1H-E430G-K439E and anti-CD20 11B8-E430G-S440K, with or without auto-oligomerization inhibitory mutations, FcγR binding inhibitory mutation G237A, and / or C1q binding enhancement mutations E333S or K326W-E333S. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC potency is expressed as normalized AUC of PI-positive cell percentage. Normalization was performed against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 16] Figure 16 shows the CDC efficacy of the anti-CD37 IgG1-CD37-37-3-E430G antibody variant, which carries an auto-oligomerization inhibitory mutation, both as a monotherapy and in combination. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G + IgG1-CD37-37-3-E430G (100%). [Figure 17] Figure 17 shows the CDC efficacy of anti-CD37 IgG1-CD37-37-3-E430G and IgG1-11B8-E430G antibody variants, both as monotherapy and in combination, that possess auto-oligomerization inhibitory mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G + IgG1-CD37-37-3-E430G (100%). [Figure 18] Figure 18 shows the CDC efficacy of anti-CD52 IgG1-CAMPATH-1H-E430G and anti-CD37 IgG1-CD37-37-3-E430G antibody variants, both as monotherapy and in combination, that possess auto-oligomerization inhibitory mutations. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G + IgG1-CD37-37-3-E430G (100%). [Figure 19] Figure 19 shows the cytotoxicity of the anti-DR5 antibody variants IgG1-DR5-01-G56T-E430G and IgG1-DR5-05-E430G, which possess self-oligomerization inhibitory mutations. BxPC-3 cells were incubated with antibody concentration series in the presence of purified human C1q (final concentration 2.9 μg / mL). Cytotoxicity is expressed as cell viability at an antibody concentration of 20 μg / mL. 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)] × 100%. [Figure 20] Figure 20 shows the CDC efficacy of anti-CD37 IgG1-7D8-E430G antibody variants carrying autooligomerization inhibitory mutations, both as monotherapy and in combination. Raji cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G+IgG1-7D8-E430G (100%). [Figure 21A]Figure 21 shows the CDC efficacy of anti-CD52 IgG1-CAMPATH-1H-E430G and anti-CD20 IgG1-11B8-E430G antibody variants, each carrying an autooligomerization inhibitory mutation, either as monotherapy or mixed in various ratios. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as a percentage of cell lysis calculated from the number of PI-positive cells. (A) CDC efficacy of monotherapy IgG1-CAMPATH-1H-E430G-K439E-Q438N and IgG1-11B8-E430G-S440K-Y436K and mixtures thereof. (B) CDC efficacy of monotherapy IgG1-CAMPATH-1H-E430G-K439E-Q438N and IgG1-11B8-E430G-S440K-Q438R and mixtures thereof. [Figure 21B] See the explanation in Figure 21A. [Figure 22A] Figure 22 shows the CDC efficacy of anti-CD52 IgG1-CAMPATH-1H-E430G and non-antigen-binding IgG1-b12-E430G antibody variants, both monotherapy and in combination, that harbor auto-oligomerization inhibitory mutations. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is expressed as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%). (A) CDC and (B) maximum cell lysis induced by monotherapy antibody variants harboring mutation E430G in combination with either mutation K439E or S440K, and by mixtures thereof. (C)CDC and (D)maximal cell lysis induced by antibody variants carrying the E430G, K439E, and Y436N mutations, mixed with complementary mutation-carrying IgG1-CAMPATH-1H or IgG1-b12 antibody variants, compared to each monotherapy control response. (E)CDC and (F)maximal cell lysis induced by antibody variants carrying the E430G, K439E, and Q438N mutations, mixed with complementary mutation-carrying IgG1-CAMPATH-1H or IgG1-b12 antibody variants, compared to each monotherapy control response. [Figure 22B]See the explanation in Figure 22A. [Figure 22C] See the explanation in Figure 22A. [Figure 22D] See the explanation in Figure 22A. [Figure 22E] See the explanation in Figure 22A. [Figure 22F] See the explanation in Figure 22A. [Modes for carrying out the invention]

[0024] Detailed description of the invention definition In the context of this invention, the term "polypeptide comprising an Fc region and an antigen-binding region of IgG" refers to a polypeptide comprising the Fc region of an IgG isotype immunoglobulin and a binding region capable of binding to an antigen, which can be any type of molecule, such as a polypeptide present on a cell, bacterium, or virion. The Fc region of an immunoglobulin is typically defined as a fragment of the antibody that would result after digestion of the antibody with papain (known to those skilled in the art), which includes two CH2-CH3 regions and a connecting region, such as a hinge region. Thus, in the context of this invention, the term "Fc region of IgG" means the presence of the connecting region, such as a hinge region, as well as the CH2 and CH3 regions of the immunoglobulin. The constant domain of the antibody heavy chain defines the antibody isotype, which can be, for example, IgG1, IgG2, IgG3, or IgG4. The Fc region, together with cell surface receptors and complement system proteins called Fc receptors, mediates the effector function of the antibody. Polypeptides containing the Fc domain and antigen-binding region of IgG can be antibodies such as chimeric antibodies, humanized antibodies or human antibodies, or antibodies consisting only of the heavy chain, or ScFv-Fc fusions or Fc fusion proteins. Polypeptides are not limited to human origin and can have any origin, such as from mice, rats, rabbits or cynomolgus monkeys.

[0025] The term "immunoglobulin" or "Ig" refers to a structurally related group of glycoproteins consisting of two pairs of polypeptide chains, namely one pair of light (L) low molecular weight chains and one pair of heavy (H) high molecular weight chains, all four of which are potentially linked together by disulfide bonds. "IgG" refers to immunoglobulin G. The structure of immunoglobulins has been characterized in detail; see, for example, Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains CH1, CH2, and CH3. The heavy chains are linked together by disulfide bonds in 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 a single domain CL. The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with highly 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 carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or the context contradicts it, CDR sequences in this specification are identified using DomainGapAlign according to the rules of IMGT (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38,D301-307(2010); Internet http address www.imgt.org / See also). Unless otherwise stated or the context contradicts it, references to amino acid positions in this invention follow EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1): 78-85, Kabat et al., Sequences of Proteins of immunological interest. 5th Edition - 1991 NIH Publication No. 91-3242).

[0026] As used herein, the term “amino acid corresponding to position…” refers to the amino acid position number in the human IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Figure 1 shows the alignments of the IgG1, IgG2, IgG3, and IgG4 sequences, which indicate which positions in IgG2, IgG3, and IgG4 correspond to which positions in IgG1. Therefore, an amino acid or segment in one sequence that “corresponds” to an amino acid or segment in another sequence is one that, when using a standard sequence alignment program such as ALIGN or ClustalW, typically aligns with the other amino acid or segment by default and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning a sequence or a segment within a sequence, thereby determining the corresponding position in a sequence for a given amino acid position according to the present invention, are considered to be well known in the art.

[0027] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 in EU numbering.

[0028] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. Therefore, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 in EU numbering. However, the CH2 region may be any of the other isotypes described herein.

[0029] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. Therefore, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 in EU numbering. However, the CH3 region may be any of the other isotypes described herein.

[0030] The terms "Fc region" or "Fc domain" may be used interchangeably herein and refer to an antibody region that, when aligned from the amino terminus to the carboxyl terminus, includes at least a hinge region, a CH2 domain, and a CH3 domain. The Fc region of an IgG1 antibody can be generated, for example, by digestion of the IgG1 antibody with papain.

[0031] In the context of this invention, the term “antibody” (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which is capable of specifically binding to an antigen. The antibodies used in this invention include an immunoglobulin Fc domain and an antigen-binding region. Antibodies generally contain a CH2-CH3 region and a linking region such as a hinge region, for example, at least an Fc domain. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. Antibodies may be monospecific or multispecific antibodies, such as bispecific antibodies or similar molecules. The term “bispecific antibody” refers to an antibody that has specificity to at least two different, typically non-overlapping, epitopes. Such epitopes may be located on the same target or on different targets. If epitopes are located on different targets, such targets may be located on the same cell or on different cells or cell types. As stated above, unless otherwise stated or clearly inconsistent with the context, the term “antibody” as used herein encompasses a fragment of an antibody that includes at least a portion of the Fc region and retains the ability to specifically bind to an antigen. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, but are not limited to, the following: monovalent antibodies (described by Genmab in WO2007059782); heavy-chain antibodies, which consist of only two heavy chains and are naturally occurring in animals such as camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab (Roche, WO2011069104); and asymmetric bispecific antibody-like molecules such as strand-exchange engineered domains (SEED or Seed-body) (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, U.S. Patent No. 7,612,181); Dual domain double head antibody (Unilever, Sanofi Aventis, WO20100226923); Di-diabody (ImClone / Eli Lilly); Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); Electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004, Chugai, US201000155133, Oncomed, WO2010129304A2); CrossMAb (Roche, WO2011117329); LUZ-Y (Genentech), Biclonic (Merus, WO2013157953); Dual Targeting domain antibody (GSK / Domantis); Two-in-one antibody or dual-action Fab recognizing two targets (Genentech, NovImmune, Adimab); Cross-linked Mab (Karmanos Cancer Center);Covalently fused mAbs (AIMM), CovX-body (CovX / Pfizer); FynomAb (Covagen / Janssen cilag); DutaMab (Dutalys / Roche); iMab (MedImmune); IgG-like bispecific (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2):p.65-74); TIG-body, DIG-body and PIG-body (Pharmabcine); Dual-affinity retargeting molecules (molecule)(Fc-DART or Ig-DART, by Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538); BEAT (Glenmark); Zybodies (Zyngenia); Common light chain approach (Crucell / Merus, US7262028) or common heavy chain approach (κλBody by NovImmune, WO2012023053), as well as fusion proteins containing polypeptide sequences fused to antibody fragments containing Fc domains, e.g., scFv fusions, e.g., BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and ZymoGenetics / BMS, Ts2Ab (MedImmune / AZ(Dimasi, N., et al. J Mol Biol, 2009). 393(3):p.672-92), scFv fusions by Genentech / Roche, scFv fusions by Novartis, scFv fusions by Immunomedics, scFv fusions by Changzhou Adam Biotech Inc (CN 102250246), TvAbs by Roche (WO 2012025525, WO 2012025530), mAbs by f-Star; 2(WO2008 / 003116), and double scFv fusions. It should also be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals), those produced by technologies developed by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides such as chimeric antibodies and humanized antibodies. The antibodies produced can potentially be any isotype.

[0032] As used herein, the terms “antigen-binding region,” “antigen-binding site,” or “antigen-binding domain” refer to the region of a polypeptide, such as an antibody, that can bind to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further subdivided into hypervariable regions, also known as complementarity-determining regions (CDRs), which contain highly conserved regions called framework regions (FRs). These hypervariable regions (i.e., regions whose sequences can be hypervariable and / or which can form structurally distinct loops) can be hypervariable. The antigen can be any molecule, such as a polypeptide, present on a cell, bacterium, or virion, for example.

[0033] As used herein, the term "cell-associated antigen" refers to an antigen that is not soluble in circulation and is associated with a cell. In one embodiment, a cell-associated antigen is an antigen located on the cell surface, such as an antigen exposed on the cell surface. In another embodiment, a cell-associated antigen is an intrinsic membrane protein.

[0034] As used herein, the term "full-length antibody" refers to an antibody that contains all of the constant and variable heavy and light chain domains corresponding to those typically found in the wild-type antibody of that isotype.

[0035] As used herein, the term "human antibody" encompasses antibodies having a variable region and a constant region derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by the human germline immunoglobulin sequence (mutations, insertions, or deletions, for example, introduced by in vitro random mutagenesis or site-directed mutagenesis, or by in vivo somatic mutagenesis). However, as used herein, the term "human antibody" does not encompass antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence.

[0036] As used herein, the term "chimeric antibody" refers to an antibody in which both chain types are chimeric as a result of antibody engineering. A chimeric chain is a chain containing an exogenous variable domain (which may originate from a non-human species, be synthetic, or be engineered from any species, including humans) linked to a human-derived constant region. When analyzed as a whole, the variable domain of the chimeric chain has a V-region amino acid sequence that is closer to that of a non-human species than to that of a human species.

[0037] As used herein, the term "humanized antibody" refers to an antibody in which both chain types have been humanized as a result of antibody engineering. Typically, a humanized chain has a complementarity-determining region (CDR) of the variable domain that is exogenous (originating from a non-human species or being synthetic), while the rest of the chain is of human origin. Humanization is assessed based on the resulting amino acid sequence, not the method itself, and therefore, protocols other than transplantation can be used. 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 other species.

[0038] As used herein, terms such as “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” and “mAb” refer to preparations of Ab molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity to a particular epitope. Therefore, the term “human monoclonal antibody” refers to an Ab exhibiting a single binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human mAbs can be produced by hybridomas, which include B cells obtained from transgenic non-human animals such as transgenic mice or transchromosomal non-human animals having a genome containing human heavy-chain and light-chain transgene repertoires, and which have been rearranged to produce functional human antibodies and fused to immortalized cells.

[0039] As used herein, the term “isotype” refers to an immunoglobulin class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype can be combined with either a kappa (κ) light chain or a lambda (λ) light chain.

[0040] As used herein, the term “mixed isotype” refers to the Fc region of an immunoglobulin produced by generating a hybrid isotype by combining the structural features of one isotype with a similar region from another isotype. A mixed isotype may include an Fc region having a sequence composed of two or more isotypes selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM. This results in combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, or IgG2 / IgG4.

[0041] As used herein, the terms “antigen,” “target antigen,” or “antigen target” refer to molecules such as proteins to which the antigen-binding domain of a polypeptide binds. An antigen molecule may contain one or more epitopes.

[0042] The term "epitope" refers to a protein determinant that has the ability to specifically bind to an antibody variable domain. Epitopes typically consist of molecular surface arrangements such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural and specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. Epitopes may include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding.

[0043] As used herein, the term "affinity" refers to the strength of binding of one molecule, such as an antibody, to another molecule, such as a target or antigen, at a single site, for example, the strength of monovalent binding of individual antigen-binding sites of an antibody against an antigen.

[0044] As used herein, the term "avidity" refers to the combined strength of multiple binding sites between two structures, for example, between multiple antigen-binding sites of an antibody interacting simultaneously with a target, or between an antibody and C1q. When two or more binding interactions exist, the two structures will only dissociate if all binding sites dissociate, so the dissociation rate is slower than the rate for individual binding sites, thereby giving a stronger effective total binding strength (avidity) compared to the binding strength (affinity) of individual binding sites.

[0045] In the present invention, a "variant," "polypeptide variant," or "antibody variant" refers to a polypeptide molecule or antibody molecule that contains one or more mutations compared to a reference antibody. Exemplary reference antibody formats include, but are not limited to, wild-type antibodies, e.g., wild-type IgG1 antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof. Exemplary mutations include amino acid deletions, insertions, and substitutions in the parent amino acid sequence. Amino acid substitutions may involve replacing a native amino acid with another natural or non-natural amino acid derivative. Amino acid substitutions may be conserved or non-conservative. In the present invention, a conserved substitution may be defined as a substitution within an amino acid class represented in one or more of the following three tables.

[0046] Amino acid residue classes for conservative substitutions TIFF2026123171000001.tif52135 Alternative Conservative Amino Acid Substitution Classes TIFF2026123171000002.tif39135 Alternative physical and functional classification of amino acid residues TIFF2026123171000003.tif70139

[0047] In relation to the present invention, substitutions in variations are expressed as follows: Original amino acid - position - substituted amino acid. To indicate amino acid residues, a three-letter code containing the codes Xaa and X, or a one-letter code, is used. Therefore, the notation "I253G" or "Ile253Gly" means that the variant contains a glycine substitution of isoleucine at the variant amino acid position corresponding to the amino acid at position 253 in the reference antibody. If the position itself does not exist in the antibody, and the variant involves the insertion of an amino acid, for example, The notation "position-inserted amino acid," for example "253G," is used.

[0048] Such notation is particularly appropriate in relation to modifications of a series of homologous polypeptides or homologous antibodies.

[0049] Similarly, if the identity of the substituted amino acid residue is not important, the following applies: The original amino acid-position, i.e., "I253".

[0050] In the case of modifications in which the original amino acid and / or substituted amino acid may include two or more amino acids, but not all, such as the substitution of isoleucine with glycine, lysine, or arginine at position 253, "Ile253Gly,Lys,Arg", "I253G,K,R", "I253G / K / R", or "I253→G,K or R" can be used interchangeably in connection with the present invention.

[0051] Furthermore, the term “substitution” encompasses substitutions to any one of the other 19 natural amino acids, or to other amino acids, such as non-natural amino acids. For example, substitutions of amino acid E at position 345 include each of the following 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. These substitutions can also be expressed as E345A, E345C, etc., or E345A,C, etc., or E345A / C / , etc. The same applies to any example at any position mentioned herein, which specifically includes any one of such substitutions.

[0052] As used herein, the term "and / or" between options or embodiments shall encompass all possible alternatives and combinations. For example, "A and / or B and / or C" shall encompass all of the following embodiments: -A -B -C -A and B -A and C -B and C -A, B, and C.

[0053] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of the immune response, rather than the recognition and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (including B cells and cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells expressing FcRs are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system, or their binding to antigen-presenting cells. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation generates C3 fragments on target cells. These C3 cleavage products can directly promote complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. Expression of specific FcRs or complement receptors in effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-carrying cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells.In some embodiments, antibody-driven classical complement activation generates C3 fragments on target cells. These C3 cleavage products can either directly promote phagocytosis by effector cells or indirectly enhance antibody-mediated phagocytosis.

[0054] As used herein, the term “Fc-mediated effector function” refers to a function resulting from the binding of a polypeptide or antibody to its target, such as an antigen, on the cell membrane, where the Fc effector function is attributed to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cell-mediated cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor binding, (vi) antibody-dependent cell phagocytosis (ADCP), (vii) complement-dependent cell-mediated cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated binding of complement-opsonized antibodies to complement receptors, (x) opsonization, and (xi) any combination of (i) to (x).

[0055] As used herein, the term “vector” refers to a nucleic acid molecule capable of inducing the transcription of a nucleic acid segment ligated into the vector. One type of vector is a “plasmid,” which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment can be ligated into the viral genome.

[0056] The term "host cell" refers to cells into which an expression vector has been introduced, such as through transfection. It should be understood that such a term refers not only to the specific target cell but also to the offspring of such cells. Examples of host cells include CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphoid cells, as well as prokaryotic cells such as E. coli, and other eukaryotic hosts such as plant cells and fungi.

[0057] As used herein, the term "oligomer" refers to a molecule consisting of two or more but a limited number of monomeric units (e.g., antibodies), unlike polymers which consist of an unlimited number of monomers, at least in principle. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to specify the number of monomeric units in an oligomer; for example, a tetramer consists of four isolated units, and a hexamer consists of six units.

[0058] As used herein, the term “oligomerization” refers to the process of converting a monomer to a limited degree of polymerization. Here, it has been observed that antibodies containing the target-binding region of the present invention can form oligomers, such as hexamers, after target binding, for example, on a cell surface, through non-covalent association of the Fc region. In this application, the terms “self-oligomerization,” “auto-oligomerization,” or “homo-oligomerization” can be used interchangeably and refer to the process of oligomerization between antibody molecules having identical protein sequences, without considering post-translational modifications. As used herein, the term “hetero-oligomerization” refers to the process of oligomerization between antibody molecules having different protein sequences, without considering post-translational modifications. Different antibodies participating in hetero-oligomerization may bind to different antigens, such as different target proteins, different target glycoproteins, different target glycans, or different target glycolipids.

[0059] The term "auto-oligomerization inhibitory substitution" refers to a substitution in a polypeptide, including the Fc region and antigen-binding region of an immunoglobulin, that inhibits the process of oligomerization between antibody molecules having identical protein sequences, without regard to post-translational modifications. Inhibition of auto-oligomerization can be exemplified as an increase in the EC50 of the antibody's CDC activity or a decrease in its maximum CDC lysis activity, as measured according to the methods described in Examples 5 and 9.

[0060] As used herein, the term "clustering" refers to the oligomerization of antibodies, polypeptides, antigens, or other proteins by non-covalent interactions.

[0061] As used herein, the term “codependency” refers to a functional effect that depends on the simultaneous binding of two or more different polypeptides having self-oligomerization inhibitory substitutions to the same cell target. In relation to the present invention, functional effects such as CDC activity may depend on the simultaneous binding of the first and second polypeptides; that is, the effect is said to be codependent. Therefore, the effector function of the first polypeptide having a self-oligomerization inhibitory substitution, such as CDC activity, depends on the binding of the second polypeptide having a self-oligomerization inhibitory substitution, and a codependent effector function exists if the self-oligomerization substitutions are complementary.

[0062] As used herein, in relation to two antigens, “co-located” or its grammatical variation “co-located” refers, firstly, to a situation in which the two antigens are simultaneously expressed on the same cell. The antigens are either already adjacent to each other on the cell or are brought together by the binding polypeptide of the present invention, such as oligomerization of antibodies. Furthermore, “co-located” also refers to a situation in which the two antigens are expressed on different cells, but such cells are located in close proximity to each other.

[0063] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on its surface. C1 is a complex consisting of the recognition protein C1q, which is composed of six heterotrimeric subunits, and the serine protease heterotetramer C1r2C1s2. C1 is the first protein complex in the initial events of the classical complement cascade, which involves a series of cleavage reactions beginning with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b deposits together with C2a to form an enzymatically active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, forming C5 convertase. This C5 convertase cleaves C5 into C5a and C5b. The final component deposits on the membrane, which then triggers the final event of complement activation, in which terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane invasion complex (MAC). This complement cascade leads to pore formation, thereby causing cell lysis, also known as complement-dependent cell injury (CDC). Complement activation can be evaluated using the C1q efficacy or CDC kinetics CDC assay (described in WO2013 / 004842, WO2014 / 108198) or by the method of C3b and C4b cell deposition described in Beurskens et al April 1, 2012 vol.188 no.7 3532-3541.

[0064] As used herein, the term “complement-dependent cell injury” (“CDC”) refers to the antibody-mediated complement activation process that leads to the lysis of cells or virions as a result of membrane pores created by MAC assembly when an antibody binds to its target on a cell or virion. CDC can be evaluated by in vitro assays such as the CDC assay, using normal human serum as the complement source, in conjunction with antibody concentration series as described in Examples 2, 3, 4, 5, and 6, or in a C1q concentration series.

[0065] As used herein, the term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) refers to the mechanism by which antibody-coated target cells or target virions are killed by cells expressing an Fc receptor that recognizes the Fc region of the bound antibody. ADCC can be determined using in vitro methods such as a chromium-releasing ADCC assay or a luminescent ADCC reporter bioassay.

[0066] 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 to, for example, an antibody. The linker is cleavable or incleavable in the presence of malignant cells, and the antibody-drug conjugate kills the malignant cells.

[0067] As used herein, the term “antibody-drug conjugate uptake” refers to the process by which an antibody-drug conjugate binds to a target on a cell, followed by engulfment by the cell membrane, thereby being drawn into the cell. Antibody-drug conjugate uptake can be evaluated as described in WO 2011 / 157741, “Antibody-mediated internal transfer and cell killing by anti-TF ADC in an in vitro killing assay.”

[0068] As used herein, the term “death receptor” refers to members of the tumor necrosis factor receptor superfamily (TNFR-SF) that include an intracellular death domain, including DR1, DR2 (also known as FAS), DR3, DR4, DR5, DR6, EDAR, and NGFR. In humans, the DR1 protein is encoded by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot P19438, the DR2 protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot P25445, the DR3 protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot Q93038, the DR4 protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot 000220, the DR5 protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot 014763, the DR6 protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot 075509, the EDAR protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot Q9UNE0, and the NGFR protein by a nucleic acid sequence encoding the amino acid sequence UniprotKB / Swissprot P08138. Death domains (DDs) are well-known protein interaction modules belonging to the death domain superfamily (Park Apoptosis. 2011 Mar;16(3):209-20).

[0069] Further aspects and embodiments of the present invention As described above, the present invention is directed toward two variant Fc region-containing polypeptides, a first variant polypeptide and a second variant polypeptide, in which each Fc region is modified so that heterooligomerization is more favorable than homooligomerization, typically in combination treatments involving a variant antibody. That is, oligomerization between the first variant molecule and the second variant molecule is more favorable than oligomerization between the first variant molecule and the first variant molecule or between the second variant molecule and the second variant molecule. This can be achieved by introducing modifications to the positions corresponding to 253, 310, 436, 438, 439 and / or 440 in the Fc region of human IgG1, as further detailed herein.

[0070] Therefore, the present invention relates to a method for treating a disease or disorder, comprising the step of administering to a target in need a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, in combination with a second polypeptide comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen. (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0071] In one aspect of the present invention, the first polypeptide contains an F436N, F436K, F436Q, or F436R mutation at the amino acid position corresponding to F436 in human IgG3, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains an F436N or F436Q mutation at the amino acid position corresponding to F436 in human IgG3, and the second polypeptide contains an F436K or F436R mutation at the amino acid position corresponding to F436 in human IgG3, or vice versa.

[0072] In EU numbering, the amino acid at position 436 is not conserved between IgG1 and IgG3. Therefore, the amino acid at position 436 in IgG1 is tyrosine (Y), while the amino acid at position 436 in IgG3 is phenylalanine (F).

[0073] In one aspect of the method of the present invention, the first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, or the first polypeptide comprises a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, or the first polypeptide comprises a Q438R, Q438K or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide comprises a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where the amino acid position corresponds to human IgG1 according to EU numbering.

[0074] In one aspect of the method of the present invention, the first polypeptide comprises a Y436N, Y436K, Y436Q or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R, Q438K, Q438H, Q438G or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where the amino acid position corresponds to human IgG1 according to EU numbering.

[0075] In one aspect of the method of the present invention, the first polypeptide comprises a Y436N or Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where the amino acid positions correspond to human IgG1 according to EU numbering.

[0076] As explained above, the Fc region can be from human IgG1, but it can also be from a different human IgG, such as IgG2, IgG3, or IgG4. Figure 1 shows which positions in human IgG2, IgG3, and IgG4 correspond to which positions in IgG1.

[0077] The Fc region of the polypeptide used in this invention consists of two heavy chains, similar to that of an antibody. When a specific mutation in the Fc region is specified, it should be understood that the mutation is present on both chains of the Fc region.

[0078] In one embodiment of the method of the present invention, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0079] In one embodiment of the method of the present invention, the first polypeptide comprises a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide comprises a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0080] In one embodiment of the method of the present invention, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0081] In one embodiment of the method of the present invention, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0082] In one embodiment of the method of the present invention, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q436K mutation at the amino acid position corresponding to Q436 in human IgG1, or vice versa.

[0083] In one embodiment of the method of the present invention, the first polypeptide contains a Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0084] In one embodiment of the method of the present invention, the first polypeptide contains a Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0085] In one embodiment of the method of the present invention, the first polypeptide contains a Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0086] In one embodiment of the method of the present invention, the first polypeptide contains a Y438N mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0087] In another embodiment of the method of the present invention, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa, and (b) The first polypeptide contains a Y436N, Y436K, Y436Q or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, or The first polypeptide contains a Q438R, Q438K or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably, the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0088] In another aspect of the method of the present invention, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0089] In another embodiment of the method of the present invention, (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0090] In another embodiment of the method of the present invention, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0091] In a further embodiment of the method of the present invention, the first and second polypeptides do not contain the mutations specified in option (c), but the first polypeptide further contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide further contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0092] In another embodiment of the method of the present invention, (i) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ii) The first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (iii) The first polypeptide contains the I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (iv) The first polypeptide contains the I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (vii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (viii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (ix) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (x) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xvi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xvii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xviii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xix) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xx) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0093] In a preferred embodiment of the method of the present invention, (i) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (ii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (iii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (iv) The first polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (vii) The first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0094] In one embodiment of the method of the present invention, (i) The first polypeptide contains a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, and (ii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0095] In one embodiment of the method of the present invention, (i) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, and (ii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0096] In one embodiment of the method of the present invention, (i) The first polypeptide contains a Y438N mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, and (ii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0097] The following table is a non-limiting list of embodiments of the method of the present invention, describing combinations of a first polypeptide and a second polypeptide with specific mutations. For example, Embodiment 1 in the table below is a combination of a first polypeptide containing I253G and K439E mutations at positions corresponding to I253 and K439, respectively, in human IgG1, and a second polypeptide containing H310R and S440K mutations at positions corresponding to H310 and S440, respectively, in human IgG1. As described herein, all first and second polypeptides in Embodiments 1 to 288 may optionally contain further mutations, such as oligomerization-enhancing mutations, e.g., E430G. TIFF2026123171000004.tif213133TIFF2026123171000005.tif219133TIFF202 6123171000006.tif219133TIFF2026123171000007.tif219133TIFF20261231710 00008.tif219133TIFF2026123171000009.tif219133TIFF2026123171000010.t if219133TIFF2026123171000011.tif219133TIFF2026123171000012.tif117133

[0098] Any further modifications In some embodiments, one or both polypeptides used in the present invention include further mutations that enhance oligomerization, such as hexamerization. Such mutations are described, for example, in WO2013 / 004842 and WO2014 / 108198. By including such further mutations, the tendency of the first and second polypeptides to form heterooligomers, such as heterohexamers, will be further enhanced. Examples of amino acid mutations that enhance Fc-Fc interactions between polypeptides and thereby enhance oligomerization are E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y, T437R, and K248E. Thus, such mutations promote or enhance oligomerization, such as hexamerization, and they may also be described as Fc-Fc interaction enhancing mutations, hexamerization enhancing mutations, or self-oligomerization enhancing mutations.

[0099] Therefore, in some embodiments, the first polypeptide further comprises a mutation at an amino acid position corresponding to E430, E345, S440, T437, or K248 in human IgG1, and / or the second polypeptide further comprises a mutation at an amino acid position corresponding to E430, E345, S440, T437, or K248 in human IgG1, or vice versa. However, if the first or second polypeptide contains a K439E, K439D, S440K, S440R, or S440H mutation, such further mutation in the polypeptide shall not be at position S440.

[0100] In some of these responses, the further mutation in the first polypeptide is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

[0101] In some of these responses, the further mutation in the second polypeptide is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

[0102] In some of these responses, the further mutation in the first polypeptide is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and the further mutation in the second polypeptide is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. and / or The first polypeptide contains the T437R mutation and the K248E mutation, and / or the second polypeptide contains the T437R mutation and the K248E mutation.

[0103] In some embodiments, the further mutation in the first polypeptide is selected from the group consisting of E430G, E345K, and E345R, and the further mutation in the second polypeptide is selected from the group consisting of E430G, E345K, and E345K. The further mutations may be selected independently from the groups for the first polypeptide and the second polypeptide.

[0104] In some embodiments, the further mutation in the first polypeptide is selected from the group consisting of E430G and E345K, and the further mutation in the second polypeptide is selected from the group consisting of E430G and E345K. In a preferred embodiment, the further mutation in the first polypeptide is E430G, and the further mutation in the second polypeptide is E430G. In one embodiment, the further mutation in the first polypeptide is E345K, and the further mutation in the second polypeptide is E345K. In one embodiment, the further mutation in the first polypeptide is E345R, and the further mutation in the second polypeptide is E345R.

[0105] In one embodiment, the first polypeptide comprises the T437R mutation and the K248E mutation, and the second polypeptide comprises the T437R mutation and the K248E mutation.

[0106] In one embodiment of the present invention, the first polypeptide contains the E430G mutation, and the second polypeptide contains the E430G mutation.

[0107] In another embodiment of the present invention, (i) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (ii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (iii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (iv) The first polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (vii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. and (viii) The first polypeptide contains an E430G, E345K, or E345R mutation at the amino acid position corresponding to K430 or E345 in human IgG1, and the second polypeptide contains an E430G, E345K, or E345R mutation at the amino acid position corresponding to K430 or E345 in human IgG1, or vice versa.

[0108] The E430G, E345K, or E345R mutations can be independently selected for the first and second polypeptides. Therefore, the first and second polypeptides have the same or different mutations selected from the group consisting of E430G, E345K, or E345R.

[0109] In another embodiment of the present invention, (i) The first polypeptide contains a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, and (ii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa, and (iii) The first polypeptide contains an E430G or E345K mutation at the amino acid position corresponding to K430 or E345 in human IgG1, and the second polypeptide contains an E430G or E345R mutation at the amino acid position corresponding to K430 or E345 in human IgG1, or vice versa.

[0110] In another embodiment of the present invention, (i) The first polypeptide contains a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, and (ii) The first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa, and (iii) The first and second polypeptides contain the E430G mutation at the amino acid position corresponding to K430 in human IgG1.

[0111] In another embodiment of the present invention, the first polypeptide comprises Y436N, K439E, and E430G mutations, the amino acid positions of which correspond to Y436, K439, and E430 in human IgG1, respectively, and the second polypeptide comprises Q438R, S440K, and E430G mutations, the amino acid positions of which correspond to Q438, S440, and E430 in human IgG1, or vice versa.

[0112] In another embodiment of the present invention, the first polypeptide comprises Y436K, K439E, and E430G mutations, the amino acid positions of which correspond to Y436, K439, and E430 in human IgG1, respectively, and the second polypeptide comprises Q438N, S440K, and E430G mutations, the amino acid positions of which correspond to Q438, S440, and E430 in human IgG1, or vice versa.

[0113] In another embodiment of the present invention, the first polypeptide comprises Y436K, K439E, and E430G mutations, the amino acid positions of which correspond to Y436, K439, and E430, respectively, in human IgG1, and the second polypeptide comprises Q438R, S440K, and E430G mutations, the amino acid positions of which correspond to Q438, S440, and E430, or vice versa, in human IgG1.

[0114] In some embodiments, one or both polypeptides used in the present invention include further mutations that alter the polypeptide's ability to induce or mediate effector function, such as Fc-mediated effector function, such as CDC or ADCC. Such alterations may be an increase or decrease in the ability to induce effector function. In some embodiments, one or both polypeptides used in the present invention include further mutations that alter the polypeptide's ability to bind to the Fc gamma receptor. Mutations that alter the antibody's ability to induce effector function and / or the antibody's ability to bind to the Fc gamma receptor have been described in the Art. By including such further mutations, the tendency of the first and second variant polypeptides to induce effector function can be increased or decreased and thus adjusted according to what is desired in a given situation. For example, it may be desirable to introduce mutations that increase the polypeptide's ability to induce CDC in order to further enhance the potency of the heterooligomer. In some other situations, it may be a priority to further reduce the toxicity of the homooligomer by introducing mutations that reduce the ability to induce CDC.

[0115] Accordingly, in some aspects of the present invention, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce effector functions, such as Fc-mediated effector functions, is altered compared to polypeptides that are otherwise identical.

[0116] In some embodiments, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce antibody-dependent cell-mediated cytotoxicity is altered compared to polypeptides that are otherwise identical.

[0117] In another embodiment, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce complement-dependent cytotoxicity is altered compared to polypeptides that are otherwise identical.

[0118] polypeptide format As described above, in one preferred embodiment of the method of the present invention, the first polypeptide is an antibody. In another preferred embodiment of the present invention, the second polypeptide is an antibody. In a more preferred embodiment, the first polypeptide is an antibody and the second polypeptide is an antibody.

[0119] In a further embodiment, the first polypeptide is a full-length antibody, and / or the second polypeptide is a full-length antibody.

[0120] The Fc region or antibody may be of any IgG isotype, for example, IgG1, IgG2, IgG3, or IgG4. In one aspect of the present invention, the polypeptide or antibody has an Fc region that is a human IgG1, IgG2, IgG3, or IgG4 isotype. In one aspect of the present invention, the Fc region is a mixed isotype, for example, a mixed isotype selected from the group consisting of IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, and IgG3 / IgG4. In a mixed isotype, the Fc region is composed of amino acid sequences from two or more isotypes.

[0121] In a preferred embodiment, the first polypeptide is an IgG1 antibody and / or the second polypeptide is an IgG1 antibody.

[0122] In one embodiment of the present invention, the first polypeptide and / or the second polypeptide comprises a first Fc region and / or a second Fc region containing the sequence described in SEQ ID NO: 22, 23, 24, 25, 31, 32, and 33, wherein at least one mutation of the present invention is introduced into the sequence. The first Fc region and the second Fc region can be independently selected from the sequences described in SEQ ID NO: 22, 23, 24, 25, 31, 32, and 33. Thus, the first Fc region and the second Fc region may be from the same parent sequence or from different parent sequences.

[0123] In one embodiment of the present invention, the first polypeptide and / or the second polypeptide comprises a first Fc region and / or a second Fc region containing the sequences described in SEQ ID NO: 22, 23, 24 and 25, wherein at least one mutation of the present invention is introduced into the sequences.

[0124] In one embodiment of the present invention, the first polypeptide comprises a first Fc region containing a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, wherein at least one mutation of the present invention is introduced into the sequence. In one embodiment of the present invention, the second polypeptide comprises a second Fc region containing a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, wherein at least one mutation of the present invention is introduced into the sequence.

[0125] In one embodiment of the present invention, the first polypeptide comprises a first Fc region containing a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, and is introduced with at least two or at least three mutations of the present invention. In one embodiment of the present invention, the second polypeptide comprises a second Fc region containing a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, and is introduced with at least two or at least three mutations of the present invention.

[0126] In one embodiment of the present invention, the first polypeptide includes a first Fc region containing a sequence selected from the group consisting of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing a sequence selected from the group consisting of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110.

[0127] In one embodiment of the present invention, (i) The first polypeptide comprises a first Fc region containing a sequence selected from the group consisting of 74, 76, 79, and 81, and (ii) The second polypeptide contains a second Fc region comprising a sequence selected from the group consisting of 75, 77, 80, and 82, or vice versa. The first and second Fc regions have at most 5, for example, at most 4, for example, at most 3, for example, at most 2, for example, at most 1, further mutations.

[0128] In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 63. In one embodiment of the present invention, the second polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 64. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 65. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 66. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 67. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 68. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 69. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 70. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 71. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:72. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:74. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:75. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:76. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:77. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:79. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:80. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:81. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:82. In one embodiment of the present invention, the first polypeptide includes a first Fc region comprising the sequence described in SEQ ID NO:83.In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:84. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:85. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:86. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:87. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:88. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:89. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:90. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:91. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:92. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:93. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:94. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:95. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:96. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:97. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:98. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:99. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:100. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:101. In one embodiment of the present invention, the first polypeptide includes a first Fc region comprising the sequence described in SEQ ID NO:102.In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 103. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 104. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 105. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 106. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 107. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 108. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 109. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO: 110.

[0129] In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 63. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 64. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 65. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 66. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 67. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 68. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 69. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 70. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 71. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:72. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:74. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:75. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:76. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:77. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:79. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:80. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:81. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:82. In one embodiment of the present invention, the second polypeptide includes a second Fc region comprising the sequence described in SEQ ID NO:83.In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 84. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 85. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 86. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 87. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 88. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 89. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 90. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 91. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 92. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:93. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:94. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:95. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:96. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:97. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:98. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:99. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:100. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:101. In one embodiment of the present invention, the second polypeptide includes a second Fc region comprising the sequence described in SEQ ID NO:102.In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 103. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 104. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 105. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 106. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 107. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 108. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 109. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO: 110.

[0130] As described above, in some aspects of the present invention, further mutations can be introduced into the Fc region that alter the ability of the polypeptide or antibody to induce / mediate effector function or other properties of the polypeptide or antibody. Such other properties may be plasma clearance, and mutations associated with such modifications are well known to those skilled in the art.

[0131] In one embodiment of the present invention, the first polypeptide comprises a first Fc region containing a sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 31, 32, and 33, and having up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide comprises a second Fc region containing a sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 31, 32, and 33, and having up to 10 mutations introduced. The up to 10 mutations introduced into the sequence may include amino acid mutations introduced according to the present invention.

[0132] In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:22 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:23 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:24 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:25 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:31 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:32 and has up to 10 mutations introduced. In one embodiment of the present invention, the first polypeptide includes a first Fc region containing the sequence described in SEQ ID NO:32 and has up to 10 mutations introduced.

[0133] In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:22 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:23 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:24 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:25 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:31 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:32 and has up to 10 mutations introduced. In one embodiment of the present invention, the second polypeptide includes a second Fc region containing the sequence described in SEQ ID NO:32 and has up to 10 mutations introduced.

[0134] In one aspect of the present invention, up to 10 mutations are introduced, for example, up to 9 mutations, for example, up to 8 mutations, for example, up to 7 mutations, for example, up to 6 mutations, for example, up to 5 mutations, for example, up to 4 mutations, or for example, up to 3 mutations.

[0135] In one embodiment of the present invention, the first polypeptide and the second polypeptide each include a first Fc region and a second Fc region containing a sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25, 31, 32, and 33, and each contains at most 10 mutations, for example, at most 9 mutations, for example, at most 8 mutations, for example, at most 7 mutations, for example, at most 6 mutations, for example, at most 5 mutations, for example, at most 4 mutations, or for example, at most 3 mutations.

[0136] In one embodiment of the present invention, the first polypeptide and the second polypeptide each include a first Fc region and a second Fc region containing the sequence described in SEQ ID NO:22, and each has up to 10 mutations, for example, up to 9 mutations, for example, up to 8 mutations, for example, up to 7 mutations, for example, up to 6 mutations, for example, up to 5 mutations, for example, up to 4 mutations, or for example, up to 3 mutations.

[0137] In a further embodiment, the first antibody is a human antibody, a humanized antibody, or a chimeric antibody, and / or the second antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0138] The polypeptides of the present invention are not limited to natural polypeptides, such as antibodies having a human Fc domain, but may also be polypeptides having mutations other than those of the present invention, such as mutations that affect glycosylation, or mutations that enable the antibody to be a bispecific antibody. The term "natural antibody" means any antibody that does not contain any genetically introduced mutations that do not exist in nature. Therefore, antibodies containing naturally occurring modifications, such as different allotypes, can be understood as "natural antibodies" in the sense of the present invention and thus as parent antibodies. Such antibodies can serve as templates for one or more mutations according to the present invention, thereby providing variant antibodies of the present invention.

[0139] The polypeptide or antibody used in the present invention has the specified mutations, but may also have additional mutations to introduce additional functions into the polypeptide or antibody. In one embodiment, the Fc region may contain as many as 10 mutations, e.g., 9 mutations, e.g., 8 mutations, e.g., 7 mutations, e.g., 6 mutations, e.g., 5 mutations, e.g., 4 mutations, e.g., 3 mutations, or e.g., 2 mutations. Additional mutations also allow for variations in the Fc region at locations not involved in Fc-Fc interactions and locations not involved in Fc effector function. Furthermore, as mentioned above, additional mutations may be due to allelic variations.

[0140] Therefore, in one aspect of the present invention, the polypeptide or antibody has an Fc region which is an IgG1m(f), IgG1m(a), IgG1m(z), IgG1m(x) allotype or mixed allotype.

[0141] The polypeptides or antibodies used in the present invention may be monospecific or multispecific, for example, bispecific. Therefore, in one embodiment, the first antibody is bispecific and / or the second polypeptide is bispecific.

[0142] Target antigen, target cells, and disease being treated As described above, the present invention provides a method that can be used to improve the selectivity of antibody treatment for a desired target cell population.

[0143] The present invention relates to treatment with a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the two antibodies bind to two different target antigens (first antigen and second antigen), and the Fc region of the antibodies is modified so that heterooligomerization of the two antibodies is significantly more favorable than homooligomerization. As a result of these modifications, cells expressing both antigen targets (allowing efficient (hetero)oligomerization of the two antibodies) will undergo more antibody oligomerization than cells expressing only one of the targets (resulting in inefficient (homo)oligomerization or no (homo)oligomerization). Since oligomerization generally enhances the potency of antibodies, the antibody combination treatment will be more potent against cells co-expressing the targets than against cells expressing only one of the targets. Thus, this antibody combination treatment has improved selectivity for cells or tissues expressing both target antigens. Therefore, by selecting two antigens that are co-expressed in the desired target cell population but not in or are only co-expressed in low amounts in cell populations that should not be targeted, it is possible to design a combined antibody treatment that will have a selective effect on the desired target cell population.

[0144] Therefore, in a preferred embodiment of the method of the present invention, the first antigen and the second antigen are both molecules and ligands exposed on the cell surface. Target antigens that activate, inhibit, modulate, and / or regulate signaling pathways may be particularly suitable as targets of the present invention.

[0145] The following protein classes may also be particularly suitable as antigen-binding targets for the first and / or second polypeptide of the present invention: tumor necrosis receptor superfamily, GPI-anchored proteins, hematopoietic factor receptor family, cytokine receptor family, serine / threonine kinase receptor family, hydrolases and regulator superfamily, hormone receptor family, B7 family-related proteins, immunoglobulin superfamily, interleukin receptor family, integrins, Ig-like cell adhesion molecule family, receptor protein tyrosine phosphatases, C-type lectins, tetraspanins, membrane spanning 4-domains, interleukin receptors, Activating leukocyte immunoglobulin-like receptors, CC-motif chemokine receptors, G protein-coupled receptors, Toll-like receptors, and receptor tyrosine kinases. In one embodiment of the present invention, the first antigen-binding domain and the second antigen-binding domain may bind to a target antigen from the same protein class. In one embodiment of the present invention, the first antigen-binding region and the second antigen-binding region can bind to target antigens from different protein classes.

[0146] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of the GPI-anchored protein, and the second antigen-binding region can bind to a target antigen from the protein class of the tetraspanin.

[0147] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of the GPI-anchored protein, and the second antigen-binding region can bind to a target antigen from the protein class of the transmembrane 4-domain protein.

[0148] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen from a transmembrane 4-domain protein class, and the second antigen-binding region can bind to a target antigen from a tetraspanin protein class.

[0149] CD20 is an example of a transmembrane 4-domain protein class. The examples illustrate the use of the present invention in relation to transmembrane 4-domain proteins.

[0150] CD37 is an example of a tetraspanin protein class. The examples illustrate the use of the present invention in relation to tetraspanin protein classes.

[0151] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen from a protein class of the tumor necrosis receptor superfamily, and the second antigen-binding region can bind to a target antigen from a protein class of the tumor necrosis receptor superfamily.

[0152] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen from a protein class of the tumor necrosis receptor superfamily, and the second antigen-binding region can bind to a target antigen from a protein class of the immunoglobulin superfamily.

[0153] In one embodiment of the present invention, the first polypeptide and / or the second polypeptide comprises a first antigen-binding region and / or a second antigen-binding region, the antigen-binding region binding to a member of the tumor necrosis factor receptor superfamily (TNFR-SF), the G protein-coupled receptor (GPCR) superfamily, a transmembrane 4 domain, or a membrane tetraspanin.

[0154] Several TNFRSFs, such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR, are involved in apoptosis and contain intracellular death domains. Other TNFRSFs, such as DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BlySR, BCMA, GITR, and RELT, are involved in other signaling pathways, including proliferation, survival, and differentiation. TNF receptors are expressed in a wide variety of tissues in mammals, particularly in leukocytes.

[0155] DR5 is an example of the TNFRSF receptor class. Example 19 illustrates the use of the present invention in relation to the TNFRSF receptor class.

[0156] In one embodiment of the present invention, the first antigen-binding region and / or the second antigen-binding region bind to a member of TNFR-SF selected from the group consisting of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, NGFR, OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BlySR, BCMA, RELT, and GITR.

[0157] In one embodiment of the present invention, the first antigen-binding region binds to DR5. In one embodiment of the present invention, the second antigen-binding region binds to DR5.

[0158] In one embodiment of the present invention, the first antigen-binding domain and / or the second antigen-binding domain bind to a member of TNFR-SF that does not contain an intracellular death domain. In one embodiment of the present invention, TNFR-SF is selected from the group OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BlySR, BCMA, RELT, and GITR. In one embodiment of the present invention, TNFR-SF is selected from the group FAS, DR4, DR4, TNFR1, DR6, DR3, EDAR, and NGFR.

[0159] The polypeptides of the present invention can bind to any target. Examples of such targets or antigens of the present invention that may be targeted include 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, and RELT.

[0160] In one embodiment of the present invention, the first antigen-binding region binds to CAMPATH-1. In one embodiment of the present invention, the second antigen-binding region binds to CAMPATH-1.

[0161] In one embodiment of the present invention, the first antigen-binding region binds to CD20. In one embodiment of the present invention, the second antigen-binding region binds to CD20.

[0162] In one embodiment of the present invention, the first antigen-binding region binds to CD37. In one embodiment of the present invention, the second antigen-binding region binds to CD37.

[0163] In one embodiment of the present invention, the first antigen-binding region binds to CAMPATH-1 and the second antigen-binding region binds to CD20, or vice versa.

[0164] In one embodiment of the present invention, the first antigen-binding region binds to CD37 and the second antigen-binding region binds to CD20, or vice versa.

[0165] In a preferred embodiment of the method of the present invention, the first and second antigens coexist in cells or tissues that are target cells or target tissues for the disease or disorder to be treated. A preferred disease to be treated is cancer.

[0166] In a more preferred embodiment, (a) The first and second antigens are not present in cells or tissues that are not target cells or tissues for the disease or disorder to be treated, or (b) The first and second antigens are present to a lower degree in cells or tissues that are not target cells or target tissues for the disease or disorder to be treated than in cells or tissues that are target cells or target tissues for the disease or disorder to be treated.

[0167] In one embodiment of the method of the present invention, the first antigen and the second antigen are not identical, and neither is a death receptor containing an intracellular death domain. In another embodiment, neither the first antigen nor the second antigen is a death receptor.

[0168] The increased efficacy is likely to be obtained not only when the two target antigens are co-expressed on the same cell, but also in other situations where the target cells are in close proximity.

[0169] Dosage, mode of administration, and combination therapy The present invention provides a method for treating a disease or disorder, the method comprising the step of administering a polypeptide described herein to a subject in need thereof. In one embodiment, the subject is a human. In the method of the present invention, an effective amount of polypeptide is administered.

[0170] "Treatment" or "to treat" means administering an effective amount of the therapeutically active polypeptide of the present invention for the purpose of reducing, improving, stopping or eradicating (curing) a symptom or disease condition.

[0171] The "effective dose" or "therapeutic effective dose" refers to the amount of medication that is effective in achieving the desired therapeutic outcome, in the required dosage and duration. The therapeutic effective dose of polypeptides such as antibodies can vary depending on factors such as the individual's disease stage, age, sex, and weight, as well as the antibody's ability to elicit the desired response in the individual. Furthermore, the therapeutic effective dose is the amount in which the therapeutically beneficial effects of the antibody or antibody moiety outweigh any toxic or adverse effects of the antibody or antibody moiety.

[0172] Preferably, the first and second polypeptides are administered sequentially or simultaneously within a certain time interval, for example, within 5 days, 2 days, 1 day, 12 hours, 6 hours, 2 hours, or 1 hour. One polypeptide may be administered more frequently than the other.

[0173] Administration may be carried out via any appropriate route, but typically it will be parenteral administration, such as intravenous, intramuscular, or subcutaneous.

[0174] The effective dosage and regimen for polypeptides such as antibodies depend on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary, non-limiting range of the therapeutically effective dose of the antibody of the present invention is about 0.1 to 100 mg / kg, for example about 0.1 to 50 mg / kg, for example about 0.1 to 20 mg / kg, for example about 0.1 to 10 mg / kg, for example about 0.5, about 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0175] In the present invention, the molar ratio of the first polypeptide to the second polypeptide when administered may vary depending on the target antigen to which they bind and the degree of their selectivity for the target cell population. In one embodiment of the method of the present invention, the molar ratio of the first polypeptide to the second polypeptide is 1:50 to 50:1, for example, 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 It is administered in molar ratios of 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, or equimolar ratios.

[0176] In a preferred embodiment, the first polypeptide and the second polypeptide are in a molar ratio of 1:50 to 50:1, for example, a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:5, 1:5, 1:5, 1:10, 1:15, 1:20, 1:25, or 1:30. It is administered in molar ratios of 1:35, 1:40, 1:45, 1:50, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1.

[0177] In one embodiment of the present invention, the first polypeptide and the second polypeptide are administered in a molar ratio of about 1:50 to 50:1, for example, a molar ratio of about 1:40 to 40:1, for example, a molar ratio of about 1:30 to 30:1, for example, a molar ratio of about 1:20 to 20:1, for example, a molar ratio of about 1:10 to 10:1, for example, a molar ratio of about 1:9 to 9:1, for example, a molar ratio of about 1:5 to 5:1.

[0178] The polypeptide or antibody of the present invention may be administered as a combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition being treated. Therefore, in one embodiment, the antibody-containing pharmaceutical is a pharmaceutical intended for use in combination with one or more further therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents. Such combination administration can be simultaneous, individual, or sequential.

[0179] In a further embodiment, the present invention provides a method for treating or preventing a disease such as cancer, the method comprising administering a therapeutically effective amount of a variant or pharmaceutical composition of the present invention to a subject in need, in combination with radiotherapy and / or surgery.

[0180] In one aspect of the present invention, a method according to any aspect or embodiment disclosed herein relates to a step of further administering an additional therapeutic agent. In one aspect of the present invention, the additional therapeutic agent includes, but is not limited to, chemotherapeutic agents (including, but not limited to, paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, and pemetrexed), kinase inhibitors (including, but not limited to, sorafenib, sunitinib, or everolimus), apoptosis regulators (including, but not limited to, recombinant human TRAIL or virinapant), RAS inhibitors, and proteasome inhibitors (including, but not limited to, bortezomib). One or more anticancer agents selected from the group consisting of (but not limited to) histone deacetylase inhibitors (including, but not limited to, vorinostat), functional foods, 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), and antibody-drug conjugates.

[0181] Polypeptide As described above, in a further aspect, the present invention relates to a polypeptide that can be used in the method of the present invention in combination with an appropriate "counterpart" polypeptide such that, when combined, heterooligomerization is more favorable than homooligomerization.

[0182] Therefore, the present invention relates to a polypeptide comprising the Fc region of human IgG and an antigen-binding region capable of binding to an antigen. (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1 and / or (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1. and / or (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 This includes, where the amino acid position corresponds to human IgG1 according to EU numbering, and if the polypeptide contains the S440K mutation, then at least one of the other mutations specified in options (a) and (b) is also present.

[0183] In one embodiment, polypeptide is (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1. Includes.

[0184] In another embodiment, the polypeptide is (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 Includes.

[0185] In another embodiment, the polypeptide is (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 Includes.

[0186] In another embodiment, the polypeptide is (a) I253G, I253K, or I253R mutations at the amino acid position corresponding to I253 in human IgG1, or H310R or H310D mutations at the amino acid position corresponding to H310 in human IgG1, and (b) Y436N, Y436K, Y436Q, or Y436R mutations at the amino acid position corresponding to Y436 in human IgG1, Q438R, Q438K, Q438H, Q438G, or Q438N mutations at the amino acid position corresponding to Q438 in human IgG1, and (c) K439F, K439I, K439Y, K439T, K439V, K439W mutations at the amino acid position corresponding to K439 in human IgG1, or S440K mutation at the amino acid position corresponding to S440 in human IgG1 Includes.

[0187] In another embodiment, the polypeptide does not contain the mutation specified in option (c) and further comprises a K439E mutation at the amino acid position corresponding to K439 in human IgG1 or an S440K mutation at the amino acid position corresponding to S440 in human IgG1.

[0188] In another embodiment, (i) The polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (ii) The polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (iii) The polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, and a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (iv) The polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, and a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (v) The polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (vi) The polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (vii) The polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (viii) The polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, a K439E mutation at the amino acid position corresponding to K439 in human IgG1, or an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or (ix) The polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, an I253G mutation at the amino acid position corresponding to I253 in human IgG1, or an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or (x) The polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and an I253G mutation at the amino acid position corresponding to I253 in human IgG1, or an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or (xi) polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, an I253G mutation at the amino acid position corresponding to I253 in human IgG1, or an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or (xii) The polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, an I253G mutation at the amino acid position corresponding to I253 in human IgG1, or an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or (xiii) The polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and an I253R mutation at the amino acid position corresponding to I253 in human IgG1, or an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or (xiv) The polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, an I253R mutation at the amino acid position corresponding to I253 in human IgG1, or an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or (xv) polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, an I253R mutation at the amino acid position corresponding to I253 in human IgG1, or an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or The (xvi) polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, and an I253R mutation at the amino acid position corresponding to I253 in human IgG1, or an H310D mutation at the amino acid position corresponding to H310 in human IgG1.

[0189] In a further embodiment, the polypeptide further comprises mutations at amino acid positions corresponding to E430, E345, S440, T437, or K248 in human IgG1. However, if the polypeptide contains K439E, K439D, S440K, S440R, or S440H mutations, such further mutations in the polypeptide are not present at position S440.

[0190] In another embodiment, the polypeptide comprises one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y, and / or the polypeptide comprises the T437R mutation and the K248E mutation.

[0191] In another embodiment, the polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K.

[0192] In another embodiment, the polypeptide contains the E430G mutation.

[0193] In another embodiment, the polypeptide is further modified such that its ability to induce effector functions, such as Fc-mediated effector functions, is altered compared to a polypeptide that is otherwise identical to the polypeptide.

[0194] In one such embodiment, the polypeptide is further modified to alter its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) compared to a polypeptide that is otherwise identical. An example of an amino acid mutation that alters the ability of a polypeptide or antibody to induce ADCC is G237A. The G237A mutation would reduce the polypeptide's ability to bind to the Fc gamma receptor, thereby reducing its ability to induce ADCC. Polypeptides with reduced ADCC-inducing ability would be of particular interest when enhancing the control of effector function induced by the polypeptide is of interest, for example, when the target to which the antibody binds is ubiquitously expressed. Therefore, in one embodiment of the present invention, the polypeptide was modified by introducing a further G237A mutation.

[0195] In one embodiment, the polypeptide contains the G237A mutation.

[0196] In another such embodiment, the polypeptide is further modified such that its ability to induce complement-dependent cell-mediated cytotoxicity (CDC) is altered compared to a polypeptide that is otherwise identical to the polypeptide.

[0197] Examples of amino acid mutations that alter the ability of polypeptides or antibodies to induce CDC include E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y T437R, K248E, E333S, and K326W. Polypeptides with increased CDC-inducing ability would be of particular interest when the eradication or depletion of specific cell types or specific tissues is of concern. Accordingly, in one aspect of the present invention, polypeptides were modified by introducing one or more amino acid mutations from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y T437R, K248E, E333S, and K326W.

[0198] In one embodiment, the polypeptide comprises the E333S mutation and / or the K326W mutation.

[0199] In one embodiment, the polypeptide comprises E333S.

[0200] In one embodiment, the polypeptide contains the E333S mutation and the K326W mutation.

[0201] In one embodiment, the polypeptide is an antibody such as a full-length antibody. In one embodiment, the polypeptide is an IgG1 antibody. In one embodiment, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the antibody is bispecific.

[0202] In one embodiment of the polypeptide of the present invention, the antigen is a molecule exposed on the cell surface. In one embodiment, the antigen is not a death receptor.

[0203] The present invention further relates to a pharmaceutical composition comprising the polypeptide of the invention described herein and a pharmaceutically acceptable carrier.

[0204] Further aspects and embodiments of the present invention As described above, in a further aspect, the present invention relates to a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, for use as a pharmaceutical in combination with a second polypeptide comprising a second antigen-binding region capable of binding to a second antigen and a second Fc region of human IgG, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0205] In one aspect, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0206] In another embodiment, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0207] In another embodiment, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0208] In another embodiment, (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0209] In another embodiment, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa, where preferably the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where preferably the first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and (c) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, or K439W mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0210] In a further embodiment, the first and second polypeptides do not contain the mutations specified in option (c), the first polypeptide further contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide further contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0211] In a further flight, (i) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (ii) The first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or (iii) The first polypeptide contains the I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (iv) The first polypeptide contains the I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains the H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains the K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (v) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (vi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or (vii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or (viii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (ix) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (x) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiii) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xiv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xv) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xvi) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xvii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; or (xviii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xix) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; the first polypeptide contains an I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa; or (xx) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxi) The first polypeptide contains a Y436N mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. or (xxii) The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa; furthermore, the first polypeptide contains a K439E mutation at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains an S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa.

[0212] In a further embodiment, the first polypeptide further comprises a mutation at an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgG1, and / or the second polypeptide further comprises a mutation at an amino acid position corresponding to E430, E345, S440, T437 or K248 in human IgG1, or vice versa. However, if the first or second polypeptide contains the K439E, K439D, S440K, S440R, or S440H mutation, the further mutation in the polypeptide shall not be located at position S440.

[0213] In a further embodiment thereof, the first polypeptide comprises one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y, and / or the second polypeptide comprises one or more mutations selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y, and / or The first polypeptide contains the T437R mutation and the K248E mutation, and / or the second polypeptide contains the T437R mutation and the K248E mutation.

[0214] In a further embodiment thereof, the first polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K, and / or the polypeptide comprises one or both mutations selected from the group consisting of E430G and E345K.

[0215] In yet another embodiment, the first polypeptide comprises E430G, and the second polypeptide comprises E430G.

[0216] In another embodiment, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce effector functions, such as Fc-mediated effector functions, is altered compared to polypeptides that are otherwise identical.

[0217] In one embodiment, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce antibody-dependent cell-mediated cytotoxicity is altered compared to polypeptides that are otherwise identical.

[0218] In another embodiment, the first polypeptide and / or the second polypeptide are further modified such that their ability to induce complement-dependent cytotoxicity is altered compared to polypeptides that are otherwise identical.

[0219] In one embodiment, the first polypeptide is an antibody such as a full-length antibody, and / or the second polypeptide is an antibody such as a full-length antibody.

[0220] In one embodiment, the first polypeptide is an IgG1 antibody and / or the second polypeptide is an IgG1 antibody.

[0221] In one embodiment, the first antibody is a human antibody, a humanized antibody, or a chimeric antibody, and / or the second antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0222] In one embodiment, the first antibody is bispecific and / or the second polypeptide is bispecific.

[0223] In one embodiment, both the first antigen and the second antigen are molecules exposed on the cell surface.

[0224] In one embodiment, the first antigen and the second antigen coexist in cells or tissues that are target cells or target tissues for the disease or disorder to be treated.

[0225] In a further flight, (a) The first and second antigens are not present in cells or tissues that are not target cells or tissues for the disease or disorder to be treated, or (b) The first and second antigens are present to a lower degree in cells or tissues that are not target cells or target tissues for the disease or disorder to be treated than in cells or tissues that are target cells or target tissues for the disease or disorder to be treated.

[0226] In one embodiment, the first and second antigens are not identical, and neither is a death receptor containing an intracellular death domain. In a further embodiment, neither the first nor the second antigen is a death receptor.

[0227] In one embodiment, the first polypeptide and the second polypeptide are in a molar ratio of 1:50 to 50:1, for example, a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:5, 1:5, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1 It is administered in molar ratios of 35, 1:40, 1:45, 1:50, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1.

[0228] In one embodiment of the present invention, the first polypeptide and the second polypeptide are administered in a molar ratio of about 1:50 to 50:1, for example, a molar ratio of about 1:40 to 40:1, for example, a molar ratio of about 1:30 to 30:1, for example, a molar ratio of about 1:20 to 20:1, for example, a molar ratio of about 1:10 to 10:1, for example, a molar ratio of about 1:9 to 9:1, for example, a molar ratio of about 1:5 to 5:1.

[0229] In one embodiment, the first polypeptide and the second polypeptide are administered simultaneously.

[0230] In one embodiment of the present invention, the first polypeptide and the second polypeptide are administered simultaneously.

[0231] In one embodiment, the use is for the treatment of cancer.

[0232] In yet another aspect, the present invention relates to the use of a first polypeptide comprising a first antigen-binding region capable of binding to a first antigen, combined with a second polypeptide comprising a second antigen-binding region capable of binding to a second Fc region of human IgG, for the production of a pharmaceutical for cancer treatment, the first polypeptide comprising a first antigen-binding region capable of binding to a first antigen, the second polypeptide comprising a second antigen-binding region capable of binding to a second Fc region of human IgG, (a) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (b) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (c) The first polypeptide contains K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0233] composition As described above, in some embodiments of the method of the present invention, the first polypeptide and the second polypeptide are administered separately. However, in another embodiment, these polypeptides may be formulated into a single pharmaceutical composition.

[0234] In one main aspect, the present invention relates to a composition comprising the first polypeptide and the second polypeptide described herein.

[0235] Therefore, the present invention relates to a composition comprising a first polypeptide comprising a first antigen-binding region capable of binding to a first antigen, combined with a second polypeptide comprising a second antigen-binding region capable of binding to a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen, (d) The first polypeptide contains an I253G mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310R mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. or The first polypeptide contains an I253K or I253R mutation at the amino acid position corresponding to I253 in human IgG1, and the second polypeptide contains an H310D mutation at the amino acid position corresponding to H310 in human IgG1, or vice versa. and / or (e) The first polypeptide contains a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. and / or (f) The first polypeptide contains the K439F, K439I, K439Y, K439T, K439V, and K439W mutations at the amino acid position corresponding to K439 in human IgG1, and the second polypeptide contains the S440K mutation at the amino acid position corresponding to S440 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0236] In one embodiment of the present invention, the first polypeptide comprises a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Y436N or Y436Q mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide contains a Y436K or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, or vice versa. or The first polypeptide contains a Q438R, Q438K, or Q438H mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N or Q438G mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0237] In one embodiment of the present invention, the first polypeptide comprises a Y436N, Y436K, Y436Q, or Y436R mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R, Q438K, Q438H, Q438G, or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa, where the amino acid positions correspond to human IgG1 according to EU numbering.

[0238] In one embodiment of the present invention, the first polypeptide comprises a Y436N or Y436K mutation at the amino acid position corresponding to Y436 in human IgG1, and the second polypeptide comprises a Q438R or Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. or The first polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, and the second polypeptide contains a Q438N mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa. Here, the amino acid positions correspond to human IgG1 according to EU numbering.

[0239] In one embodiment of the present invention, the first polypeptide comprises a Y436N or Y436K mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide comprises a Q438N or Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0240] In one embodiment of the present invention, the first polypeptide contains a Y436N mutation at the amino acid position corresponding to Q436 in human IgG1, and the second polypeptide contains a Q438R mutation at the amino acid position corresponding to Q438 in human IgG1, or vice versa.

[0241] Therefore, in a further key aspect, the present invention relates to a pharmaceutical composition comprising the first and second polypeptides described herein and a pharmaceutically acceptable carrier.

[0242] In one embodiment, the first polypeptide and the second polypeptide are in a molar ratio of 1:50 to 50:1, 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 It is present in the composition in molar ratios of 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, or equimolar ratios.

[0243] In one embodiment of the present invention, the first polypeptide and the second polypeptide are present in the composition in a molar ratio of about 1:50 to 50:1, for example, a molar ratio of about 1:40 to 40:1, for example, a molar ratio of about 1:30 to 30:1, for example, a molar ratio of about 1:20 to 20:1, for example, a molar ratio of about 1:10 to 10:1, for example, a molar ratio of about 1:9 to 9:1, for example, a molar ratio of about 1:5 to 5:1.

[0244] In one embodiment of the present invention, the first polypeptide and the second polypeptide are present in the composition in a molar ratio of approximately 1:1.

[0245] Polypeptides for use in accordance with the present invention may be formulated using pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients in accordance with the prior art, such as those disclosed in Rowe et al., Handbook of Pharmaceutical Excipients, June 2012, ISBN 9780857110275. The pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients should be suitable for the polypeptide or antibody and the chosen mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined on the basis that they do not have a significant negative impact on the desired biological properties of the selected compound or pharmaceutical composition of the present invention (e.g., the impact on antigen binding is substantial (relative inhibition of 10% or less, relative inhibition of 5% or less)).

[0246] The pharmaceutical composition may also include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0247] In one embodiment of the present invention, the pharmaceutical composition comprises a polypeptide together with a pharmaceutical carrier. Pharmaceutically acceptable carriers include any suitable solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption retarders, etc., that are physiologically compatible with the compound of the present invention.

[0248] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline solution, phosphate-buffered saline solution, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters (e.g., ethyl oleate), and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0249] The pharmaceutical compositions of the present invention may also include pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0250] The pharmaceutical compositions of the present invention may also contain sugars, polyalcohols, isotonic agents such as mannitol, sorbitol, glycerol, or sodium chloride.

[0251] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for a selected route of administration, such as preservatives, humectants, emulsifiers, dispersants, buffers, etc., which can enhance the shelf life or efficacy of the pharmaceutical composition. The compounds of the present invention may be prepared using a carrier that protects the compound from rapid release, such as a controlled-release formulation including, for example, implants, transdermal patches, or microencapsulated delivery systems. Such carriers may contain gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, either alone or in combination with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.

[0252] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including factors well known in the medical field, such as the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, and the age, sex, weight, condition, overall health status, and medical history of the patient being treated.

[0253] Parts kit The present invention also relates to a parts kit for simultaneous, individual, or sequential use in therapy, comprising polypeptides or antibodies as described herein.

[0254] Therefore, in a further aspect, the present invention relates to a kit, i.e., a parts kit, comprising a first container containing the first polypeptide of the present invention as described herein and a second container containing the second polypeptide of the present invention as described herein.

[0255] In a further aspect, the present invention relates to a device, such as a dual-chamber syringe, comprising a first compartment containing the first polypeptide of the present invention as described herein and a second compartment containing the second polypeptide of the present invention as described herein. In one embodiment, the device is a dosing device, such as a dual-chamber syringe, i.e., a syringe comprising two compartments, one compartment containing the first polypeptide and the second compartment containing the second polypeptide.

[0256] Conjugate In one embodiment, the first and / or second polypeptide or antibody used in the present invention is optionally conjugated via a linker to one or more therapeutic moieties, such as cytotoxins, chemotherapeutic agents, cytokines, immunosuppressants, and / or radioisotopes. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates.” An immunoconjugate containing one or more cytotoxins is referred to as an “immunotoxin.”

[0257] Cytotoxic or cytotoxic agents include any active substance that is harmful to cells (e.g., kills cells). Suitable therapeutic agents for forming the immunoconjugate of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthracine dione. Dione), maytansine or its analogs or derivatives, enediyene, antitumor antibiotics such as neocartinostatin, calicheamycins, esperamicin, dynemycin, lidamycin, kedulcidin or their analogs or derivatives, anthracyclines, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-methotrexate) Lucaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives, e.g., carboplatin;Also, duocalmycin A, duocalmycin SA, CC-1065 (also known as rashelmycin) or analogs or derivatives of CC-1065), drastatin, 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, mitoxantrone, plicamycin, anthramycin (AMC)), mitotic inhibitors (e.g., tubulin inhibitors), e.g., monomethyl auristatin E, monomethyl a Ulistatin F, or other analogues or derivatives of drastatin 10; histone deacetylase inhibitors, such as hydroxamic acids trichostatin A, vorinostat (SAHA), bellinostat, LAQ824, and panobinostat, as well as benzamides, entinostat, CI994, mosetinostat, and aliphatic acid compounds, such as phenylbutyrate and valproic acid; proteasome inhibitors, such as danoprevir and bortezomib; amatoxin, such as alpha-amanthin; diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments, as well as hybrid molecules);Lysine toxin (e.g., lysine A or deglycosylated lysine A chain toxin), cholera toxin, Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman bark protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modesin, geranin, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria Examples of inhibitors that can be conjugated include geronin, mitogellin, restrictosin, phenomycin, and enomycin toxin. Other suitable molecules that can be conjugated include antimicrobial / lysing peptides such as CLIP, magainin 2, melittin, cecropin, and P18; ribonucleases (RNases), DNase I, Staphylococcus enterotoxin-A, pokeweed antiviral protein, diphtherin 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, as described in a separate section herein, such as anticancer cytokines or chemokines, are also candidate therapeutic moieties useful for conjugation to the antibodies of the present invention.

[0258] In one aspect, the polypeptides used in the present invention include conjugated nucleic acids or nucleic acid-related molecules. In such an aspect, the nucleic acids to be conjugated are cytotoxic ribonucleases, antisense nucleic acids, inhibitory RNA molecules (e.g., siRNA molecules) or immunostimulatory nucleic acids (e.g., immunostimulatory CpG motif-containing DNA molecules). In another aspect, the polypeptides used in the present invention are conjugated to aptamers or ribozymes.

[0259] In one aspect, polypeptides are provided that include one or more radiolabeled amino acids. Non-limiting examples of labels for polypeptides include 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 12 5I, 131 I, and 186 Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2 nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. 4,681,581, U.S. 4,735,210, U.S. 5,101,827, U.S. 5,102,990 (US RE35,500), U.S. 5,648,471 and U.S. 5,697,902). For example, radioisotopes can be conjugated by the chloramine-T method.

[0260] In one aspect, the polypeptides or antibodies used in the present invention are conjugated to radioisotopes or radioisotope-containing chelates. For example, the polypeptide can be conjugated to a chelator linker, such as DOTA, DTPA or thioxetan, that enables the polypeptide to form a complex with a radioisotope. Non-limiting examples of radioisotopes include3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 125 I, 111 In, 131 I, 186 Re, 213 Bs, 225 and 227 Th can be mentioned.

[0261] In one embodiment, the polypeptides or antibodies used herein can be conjugated to cytokines selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factors, ancestim, and TNFα.

[0262] Polypeptides or antibodies used in the present invention may be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching 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) (e.g., PEG with molecular weights of about 1,000 to about 40,000, e.g., about 2,000 to about 20,000).

[0263] Conjugation to the treatment site can be performed at the C-terminus of the polypeptide or at another site, typically a site that does not interfere with oligomer formation.

[0264] To conjugate the polypeptide or antibody used herein to the molecule to be conjugated, such as those mentioned above, refer to 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 Any method known in the art, such as the method described in 407 (1982), may be used. Such variants can be produced by chemically conjugating other parts to the N-terminal or C-terminal side of the variant or its fragment (e.g., the H chain or L chain of an antibody) (see, for example, Osamu Kanemitsu, "Antibody Engineering Handbook," Chijin Shokan (1994)). Such conjugated variant derivatives may be produced by conjugation with internal residues or sugars, as appropriate.

[0265] The active agent can be directly or indirectly coupled to the polypeptide or antibody used herein. An example of indirect coupling of the second active agent is coupling to a cysteine ​​or lysine residue in the antibody via a spacer or linker moiety. In one embodiment, the polypeptide or antibody can be conjugated to a prodrug molecule that can be activated into a therapeutic agent in vivo. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker in the intracellular environment releases a drug unit from the antibody. In some embodiments, the linker is cleavable by a cleavable active agent present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). For example, the spacer or linker can be cleaved by tumor cell-associated enzymes or other tumor-specific conditions, thereby forming an active drug. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 by Syntarga BV et al. Suitable antibody-prodrug technologies and duocalmycin analogs can also be found in U.S. Patent No. 6,989,452 (Medarex). The linker may also be a peptidyl linker that is simultaneously or alternatively cleaved by, for example, an intracellular peptidase or protease enzyme, but not limited to, a lysosomal protease or endosomal protease. In some embodiments, the peptidyl linker is at least 2 amino acid long or at least 3 amino acid long. Examples of cleavage agents include cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, leading to the release of active drugs within target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).In one specific embodiment, peptidyl linkers cleavable by intracellular proteases are Val-Cit (valine-citrulline) linkers or Phe-Lys (phenylalanine-lysine) linkers (see, for example, US6214345, which describes the synthesis of doxorubicin along with various examples of Val-Cit and Phe-Lys linkers). Examples of Val-Cit and Phe-Lys linker structures include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB, or MC-Phe-Lys-GABA, which are described below. Here, MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid.

[0266] Method for preparing polypeptides of the present invention, such as antibodies. The polypeptides of the present invention, such as antibodies, are typically produced recombinantly, i.e., by the expression of a nucleic acid construct encoding the polypeptide in a suitable host cell, after which the produced recombinant polypeptide is purified from cell culture. The nucleic acid construct can be produced by standard molecular biological techniques well known in the art. The construct is typically introduced into host cells using a vector.

[0267] Suitable nucleic acid constructs and vectors are known in the art and are described in the examples. In most embodiments, polypeptides include not only a heavy chain (or its Fc-containing fragment) but also a light chain. In such embodiments, the nucleotide sequences encoding the heavy chain and light chain portions are typically expressed in the same cell and may reside on the same or different nucleic acids or vectors.

[0268] Host cells suitable for recombinant antibody expression are well known in this art, and include CHO, HEK-293, Expi293F, PER-C6, NS / 0, and Sp2 / 0 cells.

[0269] In one embodiment, the host cell is a cell that possesses the ability to glycosylate proteins via Asn-binding, 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 engineered to produce glycoproteins having human-like glycosylation or human glycosylation. Examples of such cells are genetically modified Pichia pastoris (Hamilton et al., Science 301(2003)1244-1246, Potgieter et al., J. Biotechnology 139(2009)318-325) and genetically modified Lemna minor (Cox et al., Nature Biotechnology 12(2006)1591-1597).

[0270] In one embodiment, the host cell is a mammalian or non-mammalian cell that produces a homogeneous glycoform. In a further embodiment, the host cell is genetically engineered to produce glycosylated antibodies, such as antibodies lacking core fucose. Examples of CHO cells that produce defucosylated antibodies include Lec13 cells and genetically modified CHO cells, such as GDP-mannose-4,6-dehydratase (GMD) knockout cells; GDP-fucose transporter knockout cells; FUT8 knockout cells; RNAi of FUT8 and / or GMD; or cells overexpressing GlcNAc transferase III or RMD (GDP-6-deoxy-d-lyxo-4-hexose reductase) (see Li et al. 2017 Front Immunol 13;8:1554 for a review).

[0271] In one embodiment, the host cell is one that lacks the ability to 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 in which C-terminal lysine removal is only partially achieved, such as Sp2 / 0, NS / 0, or transgenic mammary gland (goat).

[0272] The present invention is further illustrated by the following examples, but these examples should not be construed as further limitations.

[0273] (Table 1) Sequence List TIFF2026123171000013.tif208122TIFF2026123171000014.tif221122TIFF2026123171000015.tif220122TIFF2026123171000016.t if221122TIFF2026123171000017.tif222122TIFF2026123171000018.tif220122TIFF2026123171000019.tif220122TIFF20261231710 00020.tif220122TIFF2026123171000021.tif220122TIFF2026123171000022.tif220122TIFF2026123171000023.tif220122TIFF202 6123171000024.tif220122TIFF2026123171000025.tif220122TIFF2026123171000026.tif218122TIFF2026123171000027.tif166128

[0274] (Table 2) Self-oligomerization inhibitory substitution* TIFF2026123171000028.tif192100 Table 2* Each column shows an inhibitory substitution for self-oligomerization, and each row shows complementary inhibition of self-oligomerization.

[0275] (Table 3) Substitutions tested in Examples 5-23 TIFF2026123171000029.tif129168 [Examples]

[0276] Example 1: Antibody preparation, production, and purification Antibody expression construct To express the human antibodies and humanized antibodies used herein, variable heavy (VH) chain and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into pcDNA3.3 expression vectors (ThermoFisher Scientific, USA) containing the constant region (HC) of the human IgG heavy chain (constant region human IgG1m(f)HC: SEQ ID NO: 22; constant region human IgG2 HC: SEQ ID NO: 31; constant region human IgG3 HC: SEQ ID NO: 32; or constant region human IgG4 HC: SEQ ID NO: 33) and / or the constant region (LC) of the human kappa light chain: SEQ ID NO: 34. The desired mutations were introduced by gene synthesis. The CD20 antibody variant in this application has VH and VL sequences derived from the previously described CD20 antibodies (WO2004 / 035607) IgG1-CD20-7D8 (VH:SEQ ID NO:35; VL:SEQ ID NO:39) and IgG1-CD20-11B8 (VH:SEQ ID NO:8; VL:SEQ ID NO:12). The CD52 antibody variant in this application has VH and VL sequences derived from the previously described CD52 antibody CAMPATH-1H (aremutuzumab; Crowe et al., 1992 Clin Exp Immunol. 87(1):105-110; VH:SEQ ID NO:1; VL:SEQ ID NO:5). The CD37 antibody variant in this application has VH and VL sequences derived from the previously described CD37 antibody IgG1-CD37-37.3 (WO2011 / 112978; VH:SEQ ID NO:42; VL:SEQ ID NO:46). The DR5 antibody variant in this application has VH and VL sequences derived from the previously described DR5 antibodies DR5-01-G56T (WO 2017 / 093447; VH:SEQ ID NO:49; VL:SEQ ID NO:53) and DR5-05 (WO2014 / 009358; VH:SEQ ID NO:56; VL:SEQ ID NO:60).In some experiments, human IgG1 antibody b12, an HIV gp120-specific antibody, was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-23;VH:SEQ ID NO:15;VL:SEQ ID NO:19).

[0277] Transient occurrence The antibody was expressed as IgG1κ. A plasmid DNA mixture encoding both the heavy and light chains of the antibody was transiently transfected into Expi293F cells (Gibco, catalog number A14635) using 293fectin (Life Technologies), essentially as described by Vink et al. (Vink et al., Methods, 65(1), 5-10 2014). The antibody concentration in the supernatant was measured by absorbance at 280 nm. The antibody was either used directly in the in vitro assay or purified as described below.

[0278] 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 and loaded onto a 5 mL MabSelect SuRe column (GE Healthcare). It was 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-changed with 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 using several bioanalytical assays, including capillary electrophoresis (CE-SDS) and high-speed size exclusion chromatography (HP-SEC) on a sodium dodecyl sulfate-polyacrylamide gel. The concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2–8°C.

[0279] Example 2: CDC activity of IgG1-Campath-E430G variant with mutation at position I253 or H310The inventors investigated the Fc-Fc interface in the crystal structure of IgG1 antibody b12 (Protein Data Bank 1HZH; Ollman Sapphire et al, Science 2001, 293(5532):1155-1159) for intermolecular amino acid pairs that exhibit steric proximity and side-chain orientation on both sides of the Fc-Fc interface. Mutation pairs were tested for their complementarity in regulating intermolecular Fc-Fc interactions between cell surface target-binding antibodies by interfering with Fc-Fc interactions between antibodies with the same mutation and restoring Fc-Fc interactions by a mixture of two antibodies, each retaining one mutation and the other. The amino acid pair I253+H310 was selected for detailed mutation introduction and functional characterization. An antibody variant library was constructed based on positions I253 and H310 by introducing mutations into IgG1-Campath-E430G (i.e., antibody Campath-1H (WO2013004842) containing the heavy chain constant domain SEQ ID:26, including a Glu→Gly mutation at position 430 that enhances the Fc-Fc interaction) by substituting isoleucine at position 253 and histidine at position 310 with any other amino acid except cysteine ​​or proline. Next, the effects of individual mutations at positions 253 and 310, as well as all possible I253 and H310 mutation pairs, on the CDC potency of each IgG1-Campath-E430G variant and mixtures thereof were tested in an in vitro CDC assay using Wien 133 cells (courtesy of Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK). Cells were collected and resuspended in medium [RPMI (Lonza, catalog no. BE12-115F) containing 0.2% bovine serum albumin (BSA; Roche, catalog no. 10735086001)]. 5,000 cells per well were incubated with a concentration series of single antibodies and antibody combinations (final antibody concentrations of 15.6–2000 ng / mL at 2-fold dilutions; diluted supernatant of transient transfection described in Example 1) in the presence of 5% normal human serum (NHS; Sanquin, reference no. M0008) as a human complement source.Simultaneously, TO-PRO-3 iodide (ThermoFischer Scientific, catalog number T3605, final concentration 1 μM) was added as a cell survival marker, and SYBR Green I (ThermoFischer Scientific, catalog number S7563; 12,500-fold dilution from the original storage concentrate) was added to detect the presence of cells. The assay plates were incubated at room temperature for 1 hour, and cell elimination was calculated as the percentage of TO-PRO-3 iodide-positive cells determined by flow cytometry using a Celigo Imaging Cytometer (Brooks Life Science Systems).

[0280] The introduction of several I253 and H310 amino acid substitutions tested resulted in inhibition of the CDC efficacy of IgG1-Campath-E430G, as indicated by an increase in EC50 values ​​(summarized in Figure 2; the EC50 value of IgG1-Campath-E430G was <15 ng / μL). Mixtures of IgG1-Campath-E430G variants, each containing a mutation at either position 253 or 310, mostly failed to overcome the inhibition of CDC efficacy mediated by a single antibody. However, the mutation pair I253G (Ile253→Gly) + H310R (His310→Arg) was an exception. When introduced and tested as individual IgG1-Campath-E430G variants (containing either the I253G or H310R mutation), they each showed CDC inhibition. However, when tested as a mixture of two IgG1-Campath-E430G variants containing either I253G or H310R, they showed complete restoration of CDC efficacy. Furthermore, for the mutation pairs I253K(Ile253→Lys)+H310D(His310→Asp) and I253R(Ile253→Arg)+H310D, CDC inhibition was observed in a single IgG1-Campath-E430G variant (containing the I253K mutation, I253R mutation, or H310D mutation), while partial restoration of CDC efficacy was observed in a mixture of two IgG1-Campath-E430G variants, one containing the I253K mutation or I253R mutation and the other containing H310D (Figure 2).

[0281] These results suggest that it is unpredictable which Fc mutations at positions I253 and H310 in human IgG1 antibodies with the E430G Fc-Fc enhancing mutation inhibit the Fc-Fc interaction and CDC efficacy when a single variant containing either the I253 or H310 mutation is produced, and whether mixing two variants containing one of the two complementary I253 or H310 mutations will create complementary mutation pairs that restore this interaction. Using CDC assays with IgG1-Campath-E430G antibody variants at positions I253 and H310 in Wien 133 cells, I253G+H310R, I253K+H310D, and I253R+H310D were identified as complementary mutant pairs that control the CDC activity of antibodies with E430G Fc-Fc enhancing mutations, i.e., inhibition of CDC efficacy by a single variant (I253G, I253K, H310D, or H310R) and restoration by their complementary mixtures (I253G+H310R, I253K+H310D, or I253R+H310D).

[0282] Example 3: CDC activity of IgG1-Campath-E430G variant with mutation at position Y436 or Q438 Similar to the amino acid pair I253+H310 described in Example 2, the amino acid pair Y436+Q438 was also selected for detailed mutagenesis and functional characterization. Antibody variant libraries were constructed based on positions Y436 and Q438 by substituting tyrosine at position 436 and glutamine at position 438 in IgG1-Campath-E430G with any other amino acid except cysteine ​​or proline. The effects of individual mutations at positions 436 and 438, as well as the effects of all possible Y436 and Q438 mutation pairs on the CDC potency of each IgG1-Campath-E430G variant and mixtures thereof, were then tested using in vitro CDC assays with Wien 133 cells, as described in Example 2.

[0283] The introduction of several Y436 and Q438 amino acid substitutions tested resulted in inhibition of the CDC efficacy of IgG1-Campath-E430G, as indicated by an increase in EC50 values ​​(summarized in Figure 3; the EC50 value of IgG1-Campath-E430G was <15 ng / mL). Mixtures of IgG1-Campath-E430G variants, each containing a mutation at either position 436 or 438, mostly failed to overcome the inhibition of CDC efficacy mediated by a single antibody. However, the mutation pairs Y436K+Q438G, Y436K+Q438H, Y436K+Q438K, Y436K+Q438N, Y436K+Q438R, Y436N+Q438G, Y436N+Q438H, Y436N+Q438K, Y436N+Q438N, Y436N+Q438R, Y436Q+Q438G, Y436Q+Q438H, Y4 For mixtures of IgG1-Campath-E430G variants that combine 36Q+Q438K, Y436Q+Q438N, Y436Q+Q438R, Y436R+Q438G, Y436R+Q438H, Y436R+Q438K, Y436R+Q438N, or Y436R+Q438R, partial restoration of CDC efficacy was observed (Figure 3).

[0284] From these results, we can conclude that it is unpredictable which Fc mutations at positions Y436 and Q438 in human IgG1 antibodies with the E430G Fc-Fc enhancement mutation will inhibit the Fc-Fc interaction by a single variant, and whether a specific mixture of two variants can restore it. Using a CDC assay with IgG1-Campath-E430G antibody variants at positions Y436 and Q438 in Wien 133 cells, Y436K, Y436N, Y436Q, Y436R, Q438G, Q438H, Q438K, Q438N, and Q438R mutations were identified that can inhibit the Fc-Fc interaction and CDC activity of antibodies with E430G Fc-Fc enhancing mutations. Any mixture of one of these mutations at position 436 and one of these mutations at position 438 was identified as a complementary Y436;Q438 mutation pair capable of restoring the inhibition of Fc-Fc interaction and CDC efficacy of the single mutant.

[0285] Example 4: CDC activity of IgG1-Campath-E430G variant with mutation at position K439 or S440 Similar to the amino acid pairs I253+H310 (described in Example 2) and Y436+Q438 (described in Example 3), the amino acid pair K439+S440 was also selected for detailed mutagenesis and functional characterization. Antibody variant libraries were constructed based on positions K439 and S440 by substituting lysine at position 439 and serine at position 440 in IgG1-Campath-E430G with any other amino acid except cysteine ​​or proline. The effects of individual mutations at positions 439 and 440, as well as the effects of all possible K439 and S440 mutation pairs on the CDC potency of each IgG1-Campath-E430G variant a...

Claims

1. A composition comprising a first polypeptide comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, and a second polypeptide comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen, The first polypeptide contains the Y436N mutation, and the second polypeptide contains the Q438R mutation, or vice versa. Furthermore, the first polypeptide contains the K439E mutation, and the second polypeptide contains the S440K mutation, or vice versa. Furthermore, the first polypeptide contains the E430G mutation, and the second polypeptide contains the E430G mutation. Here, the amino acid positions correspond to human IgG1 according to EU numbering. composition.

2. A pharmaceutical composition comprising a first polypeptide and a second polypeptide according to claim 1, and a pharmaceutically acceptable carrier, wherein the first polypeptide and the second polypeptide are preferably in a molar ratio of 1:50 to 50:1, for example, a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:5, 1:5, 1:5, 1:10, 1:15, 1:20 Pharmaceutical compositions existing in molar ratios of 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, and 2:

1.

3. The composition according to claim 1 or 2, wherein the first polypeptide is an antibody such as a full-length antibody, and / or the second polypeptide is an antibody such as a full-length antibody.

4. The composition according to claim 3, wherein the first polypeptide is an IgG1 antibody and / or the second polypeptide is an IgG1 antibody.

5. The composition according to claim 3 or 4, wherein the first antibody is human, humanized, or chimeric, and / or the second antibody is human, humanized, or chimeric.

6. The composition according to any one of claims 3 to 5, wherein the first antibody is bispecific and / or the second polypeptide is bispecific.

Images