Combinations of antibody variants and their use

Abstract

The present invention relates to combination therapies involving two or more antibodies in which the Fc regions of the two antibodies have been modified such that hetero-oligomerization between the antibodies is strongly favored over self-oligomerization when the two antibodies bind to their corresponding target antigens, and such that hetero-oligomerization-independent effector function of one or both antibodies is eliminated or strongly reduced. The present invention also relates to antibodies, compositions, and kits suitable for use in the combination therapies of the invention.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a combination therapy involving two or more antibodies, wherein when the two antibodies bind to their corresponding target antigens, hetero-oligomerization between the antibodies is more strongly preferred over self-oligomerization, and the hetero-oligomerization-independent effector functions of one or both antibodies are eliminated or strongly reduced, and the Fc regions of the two antibodies are modified. The present invention also relates to antibodies, compositions, and kits suitable for use in the combination therapy of the present invention.

Background Art

[0002] Background of the Invention Antibodies are extremely effective molecules that can affect target cells through various mechanisms. In some cases, simply binding of an antibody to a target antigen on the cell surface may result in an antagonist or agonist effect on the target antigen and thus on the target cell. Instead or in addition, the effect of an antibody on a target cell is achieved by the ability of the antibody to induce effector functions, typically Fc-mediated effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). Fc region-mediated effector functions such as CDC, ADCC, and ADCP contribute to the therapeutic concentration range of antibody treatment defined by efficacy and toxicity.

[0003] 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. The affinity of an individual globular binding head for IgG is low, and C1q must increase its avidity by binding to multiple IgG1 molecules on the cell surface in order to induce the classical complement pathway. ADCC and ADCP are initiated by the binding of the IgG Fc region to Fcγ receptors (FcγR) on effector cells.

[0004] IgG hexamerization upon target binding to the cell surface has been shown to support strong C1q binding (Diebolder (2014) Science 343:1260 (Non-Patent Literature 1)). Hexamerization is mediated by intermolecular non-covalent Fc-Fc interactions, and point mutations in the CH3 domain, including E345R and E430G, can enhance the Fc-Fc interaction.

[0005] The utilization of effector function by IgG antibodies depends on their glycosylation status. In other words, if the IgG Fc region is not glycosylated, the ability of IgG antibodies to mediate effector function is greatly reduced. The Fc region of IgG antibodies contains a highly conserved N-glycosylation site at amino acid position N297. Wang et al. ((2016) Mol Cell 63:135) (Non-patent Literature 2) studied IgG variants that can form hexamers and bind to C1q in the soluble state. They found that deglycosylation of the Fc region of these variants inhibited both hexamer formation and the C1q binding avidity of the hexamers in the soluble state.

[0006] WO2013 / 004842 (Patent Document 1) discloses an antibody or polypeptide comprising a variant Fc region having one or more amino acid modifications that alter the effector function of CDC, etc.

[0007] WO2014 / 108198 (Patent Document 2) discloses a polypeptide, such as an antibody, that includes a variant Fc region having one or more amino acid modifications resulting in an increase in CDC.

[0008] WO2012 / 130831 (Patent Document 3) relates to an Fc region-containing polypeptide in which effector function is altered as a result of one or more amino acid substitutions in the Fc region.

[0009] Enhancing Fc-Fc interactions between antibodies may increase the efficacy of the antibody by improving effector functions such as CDC and / or ADCC. This may lead to cell death of target cells to which the antibody is bound. However, if the target antigen is widely expressed on the surface of both healthy cells and disease-causing cells in the body, the antibody may be toxic by killing healthy cells. Therefore, antibody therapy may not be selective for target tissues, and non-disease tissues may be affected and toxic by antibody treatment.

[0010] WO2019 / 145455 (Patent Document 4) and Oostindie et al. 2019 Haematologica 104:1841 (Non-Patent Document 3) relate to medical treatments using two antibodies that bind to two different target antigens, wherein the Fc regions of these antibodies are modified so that heterooligomerization of the two antibodies is strongly preferred over autooligomerization (or auto-oligomerization or homo-oligomerization). As a result of these modifications, more antibody oligomerization occurs on cells expressing both antigen targets (enabling efficient (hetero)oligomerization of the two antibodies) than on cells expressing only one of the targets (resulting in inefficient (auto)oligomerization or no (auto)oligomerization). Since oligomerization generally enhances the potency of antibodies, the potency of antibody combination treatments on cells expressing both targets is greater than the potency on cells expressing only one of the targets. Therefore, antibody combination therapy exhibits improved selectivity for cells or tissues expressing both target antigens. Thus, by selecting two antigens that are co-expressed in the desired target cell population but not in the undesirable cell population, or that are co-expressed at a lower rate than the desired target cell population, antibody combination therapy can be designed to have improved selective efficacy against the desired target cell population. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] WO2013 / 004842 [Patent Document 2] WO2014 / 108198 [Patent Document 3] WO2012 / 130831 [Patent Document 4] WO2019 / 145455 [Non-patent literature]

[0012] [Non-Patent Document 1] Diebolder (2014) Science 343:1260 [Non-Patent Document 2] Wang et al.((2016) Mol Cell 63:135) [Non-Patent Document 3] Oostindie et al. 2019 Haematologica 104:1841 [Overview of the Initiative]

[0013] While antibody combination therapy strategies, such as those described in WO2019 / 145455 and Oostindie et al. 2019 Haematologica 104:1841, can significantly enhance the selectivity of antibody therapy toward a desired target cell population, undesirable residual activity of each antibody in the combination remains, which may manifest toward healthy cells expressing only one of the targets.

[0014] Therefore, there is a need for further improved forms of antibody treatment, particularly treatments with an improved therapeutic concentration range.

[0015] Surprisingly, it has now been found that by blocking or removing antibody glycosylation, without strongly affecting the activity of the hetero-oligomer, the residual activity of the individual antibodies used in the above-described antibody combination treatments can be strongly reduced. Therefore, it is possible to further reduce the unwanted toxicity to healthy cells without strongly affecting the desired efficacy against the desired target cells.

[0016] Therefore, a first antibody and a second antibody that are engineered to produce maximum activity on a target cell to which both the first antibody and the second antibody are simultaneously bound, wherein the first antibody does not produce activity or produces minimal activity on a target cell to which only the first antibody is bound, as compared to the activity on a cell to which both antibodies are simultaneously bound, and the second antibody produces minimal activity or reduced activity on a target cell to which only the second antibody is bound, is an object of the present invention.

[0017] Therefore, in a first aspect, the present invention provides a second antibody comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen for use as a medicament in combination with a first antibody comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, wherein the first Fc region comprises a. a substitution at position E430, or a substitution at position E345, or a combination of substitutions K248E and T437R, and b. a K439E or S440K substitution and the second Fc region comprises c. a substitution at position E430, or a substitution at position E345, or a combination of substitutions K248E and T437R, and d. a K439E or S440K substitution and The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody does not contain N-linked glycosylation at position N297; The amino acid positions correspond to human IgG1 according to the EU numbering system, A first antibody for use as a medicament in combination with a second antibody is provided.

[0018] In a further aspect, the invention relates to an antibody comprising an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, which does not contain N-linked glycosylation at position N297, wherein the Fc region is a. a substitution at position E430, or a substitution at position E345, or a combination of substitutions K248E and T437R, and b. a K439E or S440K substitution and relates to an antibody

[0019] In a further aspect, the invention relates to a method for producing an antibody according to the invention, as well as a composition and a kit comprising an antibody according to the invention.

[0020] In one aspect, the invention relates to a method for producing an antibody, comprising the step of producing the antibody in a recombinant host cell capable of glycosylating asparagine at position N297 of the antibody, followed by the step of removing N-linked glycosylation from the produced antibody.

[0021] In another aspect, the invention relates to a method for producing an antibody, comprising the step of producing the antibody in a recombinant host cell incapable of glycosylating asparagine at position N297 of the antibody. ​​​

[0022] In another aspect, the present invention is A composition comprising a first antibody and a second antibody, The first antibody comprises a first antigen-binding region capable of binding to a first antigen, and a first Fc region of human IgG; The second antibody comprises a second antigen-binding region capable of binding to a second antigen, and a second Fc region of human IgG; The first Fc region described above is a. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and b. Replacement with K439E or S440K Includes; The aforementioned second Fc region is c. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and d. Replacement with K439E or S440K Includes; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody do not contain N-linked glycosylation at position N297; The amino acid positions correspond to human IgG1 according to the EU numbering system. A composition comprising a first antibody and a second antibody. Regarding.

[0023] In another aspect, the present invention is A method for treating an individual having a disease, comprising the step of administering to the individual an effective amount of first and second antibodies or compositions according to any aspect or embodiment described herein. Regarding.

[0024] In another aspect, the present invention is A method for depleting a cell population expressing a first antigen and a second antigen, comprising the step of contacting the cell population with first and second antibodies or compositions according to any aspect or embodiment described herein. Regarding.

[0025] In another aspect, the present invention is A method for inducing proliferation in a cell population expressing a first antigen and a second antigen, comprising the step of contacting the cell population with a first antibody and a second antibody according to any aspect or embodiment described herein. Regarding.

[0026] In another aspect, the present invention is A kit comprising a first container containing a first antibody as described in any aspect or aspect of this specification, and a second container containing a second antibody as described in any aspect or aspect of this specification. Regarding.

[0027] In another aspect, the present invention relates to nucleic acids encoding antibodies or a first antibody or a second antibody according to any aspect or embodiment of this specification.

[0028] In another aspect, the present invention relates to a nucleic acid encoding the heavy chain of a first antibody or a second antibody according to any aspect or embodiment of this specification.

[0029] In another aspect, the present invention relates to an expression vector. In yet another aspect, the present invention relates to a delivery vehicle.

[0030] [Invention 1001] A second antibody comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen. For use as a medical drug in combination with, A first antibody comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, The first Fc region is a. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and b. Replacement with K439E or S440K Includes; The second Fc region is c. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and d. Replacement with K439E or S440K Includes; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody do not contain N-linked glycosylation at position N297; The amino acid positions correspond to human IgG1 according to the Eu numbering system. The first antibody for use as a medical drug in combination with the second antibody. [Invention 1002] A first antibody for use as a pharmaceutical agent in combination with a second antibody of Invention 1001, wherein the first antibody and / or second antibody include an amino acid substitution, deletion, or insertion that inhibits N-linked glycosylation at position N297. [Invention 1003] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and / or the second Fc region contain an amino acid substitution at position N297 or position T299, wherein the substitution at position T299 is not T299S. [Invention 1004] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and / or the second Fc region comprises a substitution selected from the group consisting of N297A, N297G, N297Y, N297Q, N297D, N297S, N297T, T299A, and T299G. [Invention 1005] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and / or the second Fc region include an N297A substitution or an N297G substitution. [Invention 1006] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antibody and / or second antibody does not contain N-linked glycosylation at any position of the antibody. [Invention 1007] a. The first Fc region includes K439E substitution and Y436N substitution, and the second Fc region includes S440K substitution and Q438R substitution, or b. The first Fc region includes K439E substitution and Q438R substitution, and the second Fc region includes S440K substitution and Y436N substitution, or c. The first Fc region includes S440K substitution and Y436N substitution, and the second Fc region includes K439E substitution and Q438R substitution, or d. The first Fc region includes S440K substitution and Q438R substitution, and the second Fc region includes K439E substitution and Y436N substitution. A first antibody for use as a medical agent in combination with any of the second antibodies of the present invention described above. [Invention 1008] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and the second Fc region include substitutions selected from the group consisting of E345K, E430G, E345R, E430Y, E345Q, E345Y, E430S, E430T, and E430F, and / or combinations of substitution K248E and T437R. [Invention 1009] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and the second Fc region include substitutions selected from the group consisting of E345K, E430G, E345R, and E430Y. [Invention 1010] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and the second Fc region include substitutions selected from the group consisting of E430G and E345K. [Invention 1011] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and the second Fc region include substitutions selected from the group consisting of E345R and E430Y. [Invention 1012] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and the second Fc region contain a combination of substitution K248E and T437R. [Invention 1013] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and / or the second Fc region comprises one or more substitutions selected from the group consisting of E333S, K326A, E333A, and K326W. [Invention 1014] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first Fc region and / or the second Fc region contain E333S and K326A substitutions. [Invention 1015] A first antibody for use as a medical agent in combination with a second antibody according to any of the present inventions, wherein the first antibody and / or second antibody is a human antibody, humanized, or a chimeric antibody. [Invention 1016] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antibody and / or second antibody is a monoclonal antibody. [Invention 1017] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antibody and / or second antibody is a human IgG1, IgG2, IgG3, or IgG4 subclass. [Invention 1018] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antibody and / or second antibody is a human IgG1, IgG2, or IgG4 subclass. [Invention 1019] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antibody and / or second antibody is a human IgG1 subclass. [Invention 1020] A first antibody for use as a medical agent in combination with any second antibody of the present invention, wherein both the first antigen and the second antigen are cell surface expression molecules. [Invention 1021] A first antibody for use as a pharmaceutical agent in combination with any second antibody of the present invention, wherein the first antigen and the second antigen are co-expressed in cells or tissues that are target cells or target tissues of the disease or disorder to be treated. [Invention 1022] A first antibody for use as a medical agent in combination with any second antibody of the present invention, wherein the first antigen and the second antigen are not identical. [Invention 1023] A first antibody for use as a medical agent in combination with any of the second antibodies of the present invention, wherein the combination of the first antibody and the second antibody depletes a population of cells that simultaneously express the first antigen and the second antigen. [Invention 1024] A first antibody for use as a medical agent in combination with any of the second antibodies of the present invention, wherein the combination of the first antibody and the second antibody induces cell death in a cell population that simultaneously expresses the first antigen and the second antigen. [Invention 1025] A first antibody for use as a medical agent in combination with any of the second antibodies of the present invention, wherein the combination of the first antibody and the second antibody induces proliferation in a cell population expressing the first antigen and the second antigen. [Invention 1026] A first antibody for use as a medical agent in combination with a second antibody according to any of the present inventions 1023 to 1024, wherein the cell population is a tumor cell population. [Invention 1027] A first antibody for use as a pharmaceutical agent in combination with a second antibody according to any of the inventions 1023-1024 and 1026, wherein the cell population is a hematological tumor cell population or a solid tumor cell population. [Invention 1028] A first antibody for use as a pharmaceutical agent in combination with a second antibody according to any of the Invention 1023 to 1027, wherein the cell population is a population of leukocytes, lymphocytes, B cells, T cells, regulatory T cells, NK cells, myeloid-derived suppressor cells, or tumor-associated macrophage cells. [Invention 1029] An antibody comprising the Fc region of human IgG and an antigen-binding region capable of binding to an antigen, and without N-linked glycosylation at position N297, The Fc region is c. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and d. Replacement with K439E or S440K Antibodies containing antibodies. [Invention 1030] The antibody of the present invention 1029, comprising an amino acid substitution, deletion, or insertion that inhibits N-linked glycosylation at position N297. [Invention 1031] The antibody of Invention 1029 or 1030, wherein the Fc region contains an amino acid substitution at position N297 or position T299, and the substitution at position T299 is not T299S. [Invention 1032] An antibody according to any of Invention 1029 to 1031, wherein the Fc region includes a substitution selected from the group consisting of N297A, N297G, N297Y, N297Q, N297D, N297S, N297T, T299A, and T299G. [Invention 1033] An antibody according to any of the present invention 1029 to 1032, which does not contain N-linked glycosylation at any position on the antibody. [Invention 1034] The Fc region is a. K439E substitution and Y436N substitution, b. K439E substitution and Q438R substitution, or c. Replacement of S440K and Y436N, or d. Replacement of S440K and Q438R An antibody according to any of the present invention 1029 to 1033, including the above. [Invention 1035] An antibody according to any of Invention 1029 to 1034, wherein the Fc region comprises substitutions selected from the group consisting of E345K, E430G, E345R, E430Y, E345Q, E345Y, E430S, E430T, and E430F, and / or combinations of substitution K248E and T437R. [Invention 1036] An antibody according to any of Invention 1029 to 1035, wherein the Fc region includes a substitution selected from the group consisting of E345R, E430G, E345K, and E430Y. [Invention 1037] An antibody according to any of Invention 1029 to 1036, wherein the Fc region contains one or more substitutions selected from the group consisting of E333S, K326A, E333A, and K326W. [Invention 1038] An antibody according to any of Invention 1029 to 1037, wherein the Fc region contains E333S and K326A substitutions. [Invention 1039] An antibody according to any of the present invention 1029-1038, which is a human IgG1, IgG2, IgG3, or IgG4 subclass. [Invention 1040] An antibody according to any of the present invention 1029-1039, which is a human IgG1, IgG2, or IgG4 subclass. [Invention 1041] An antibody of any of the human IgG1 subclasses 1029 to 1040 of this invention. [Invention 1042] The process includes the step of producing antibodies in recombinant host cells, The host cell is unable to glycosylate the asparagine located at position N297 of the antibody. A method for producing any of the antibodies of the present invention described above. [Invention 1043] A method for producing any of the antibodies of the present invention, comprising the steps of producing an antibody in recombinant host cells capable of glycosylation of asparagine at position N297 of the antibody, and subsequently removing N-linked glycosylation from the produced antibody. [Invention 1044] A composition comprising a first antibody and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region according to any of invention 1001 to 1041, and the second antibody comprises a second antigen-binding region and a second Fc region according to any of invention 1001 to 1041. [Invention 1045] below: The first antibody comprises a first antigen-binding region capable of binding to a first antigen, and a first Fc region of human IgG; The second antibody comprises a second antigen-binding region capable of binding to a second antigen, and a second Fc region of human IgG; The first Fc region is e. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and f. Replacement of K439E or S440K Includes; The second Fc region is g. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and h. Replace K439E or S440K Includes; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody do not contain N-linked glycosylation at position N297; The first and second antibodies have amino acid positions corresponding to human IgG1 according to the Eu numbering system. A composition containing the following: [Invention 1046] The first antibody and the second antibody are present in the composition in a molar ratio of approximately 1:50 to 50:1, for example, approximately 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, and 1:40. A composition of the present invention 1044 or 1045, existing in molar ratios of approximately 1:45, approximately 1:50, approximately 50:1, approximately 45:1, approximately 40:1, approximately 35:1, approximately 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. [Invention 1047] A composition according to any one of the present invention 1044 to 1046, wherein the first antibody and the second antibody are present in the composition in a molar ratio of 1:50 to 50:1, for example, a molar ratio of 1:40 to 40:1, for example, a molar ratio of 1:30 to 30:1, for example, a molar ratio of 1:20 to 20:1, for example, a molar ratio of 1:10 to 10:1, for example, a molar ratio of 1:9 to 9:1, for example, a molar ratio of 1:5 to 5:1, for example, a molar ratio of 1:2 to 2:1. [Invention 1048] A composition according to any one of the present inventions 1044 to 1047, wherein the first antibody and the second antibody are present in the composition in a 1:1 molar ratio. [Invention 1049] A composition according to any one of the present invention 1044 to 1048, further comprising a pharmaceutical carrier or excipient. [Invention 1050] A pharmaceutical composition, which is any of the compositions described in invention 1044 to 1049. [Invention 1051] A composition according to any of Invention 1044 to 1050 for use as a medical drug. [Invention 1052] A first antibody or second antibody according to any of Invention 1001 to 1028, an antibody according to any of Invention 1029 to 1041, a method according to any of Invention 1042 to 1044, or a composition according to any of Invention 1045 to 1051, wherein the antigen-binding region can bind to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, PD1, and FAS. [Invention 1053] The antigen-binding region is a. The VH region containing the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10, and the CDR3 sequence shown in SEQ ID NO:11, and the VL region [CD20, 11B8] containing the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown in DAS, and the CDR3 sequence shown in SEQ ID NO:14; b. The VH region containing the CDR1 sequence shown in SEQ ID NO:43, the CDR2 sequence shown in SEQ ID NO:44, and the CDR3 sequence shown in SEQ ID NO:45, and the VL region [CD37] containing the CDR1 sequence shown in SEQ ID NO:47, the CDR2 sequence shown in VAT, and the CDR3 sequence shown in SEQ ID NO:48; c. The VH region containing the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, and the VL region [CD52, CAMPATH-1H] containing the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown in NTN, and the CDR3 sequence shown in SEQ ID NO:7; d. The VH region containing the CDR1 sequence shown in SEQ ID NO:118, the CDR2 sequence shown in SEQ ID NO:119, and the CDR3 sequence shown in SEQ ID NO:120, and the VL region [CD52, h2E8] containing the CDR1 sequence shown in SEQ ID NO:122, the CDR2 sequence shown in SEQ ID NO:123, and the CDR3 sequence shown in SEQ ID NO:124; e. The VH region containing the CDR1 sequence shown in SEQ ID NO:104, the CDR2 sequence shown in SEQ ID NO:105, and the CDR3 sequence shown in SEQ ID NO:106, and the VL region [Fas, Fas-E09] containing the CDR1 sequence shown in SEQ ID NO:108, the CDR2 sequence shown in YNN, and the CDR3 sequence shown in SEQ ID NO:109; f. The VH region containing the CDR1 sequence shown in SEQ ID NO:111, the CDR2 sequence shown in SEQ ID NO:112, and the CDR3 sequence shown in SEQ ID NO:113, and the VL region containing the CDR1 sequence shown in SEQ ID NO:115, the CDR2 sequence shown in DAS, and the CDR3 sequence shown in SEQ ID NO:116 [4-1BB, BMS-663513]; g. The VH region containing the CDR1 sequence shown in SEQ ID NO:90, the CDR2 sequence shown in SEQ ID NO:91, and the CDR3 sequence shown in SEQ ID NO:92, as well as the VL region [DR5, hDR5-01-G56T] containing the CDR1 sequence shown in SEQ ID NO:94, the CDR2 sequence shown in FAS, and the CDR3 sequence shown in SEQ ID NO:95; h. The VH region containing the CDR1 sequence shown in SEQ ID NO:97, the CDR2 sequence shown in SEQ ID NO:98, and the CDR3 sequence shown in SEQ ID NO:99, and the VL region [DR5, hDR5-05] containing the CDR1 sequence shown in SEQ ID NO:101, the CDR2 sequence shown in RTS, and the CDR3 sequence shown in SEQ ID NO:102. A composition comprising any first or second antibody according to Invention 1001 to 1028, any antibody according to Invention 1029 to 1041, any method according to Invention 1042 to 1044, or any composition according to Invention 1045 to 1051. [Invention 1054] A method for treating an individual having a disease, comprising the step of administering to the individual an effective amount of a first antibody and a second antibody according to Invention 1001-1028, 1052, or 1053, an antibody according to any of Invention 1029-1041, 1052, or 1053, or a composition according to any of Invention 1045-1053. [Invention 1055] The method of the present invention 1054, wherein the disease is selected from the group of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. [Invention 1056] A method according to any one of the present invention 1054 to 1055, comprising the step of administering a further therapeutic agent. [Invention 1057] A method for depleting a population of cells expressing a first antigen and a second antigen, The step of contacting the cell population with a first antibody and a second antibody according to any of Invention 1001-1028, 1052, or 1053, or with any of the compositions according to Invention 1045-1053. Methods that include... [Invention 1058] The method of the present invention 1057, wherein the cell population is a tumor cell population, for example, a hematological tumor cell population or a solid tumor cell population. [Invention 1059] A method for inducing proliferation in a cell population expressing a first antigen and a second antigen, The step of contacting the cell population with a first antibody and a second antibody according to any of Invention 1001-1028, 1052, or 1053, or with any of the compositions according to Invention 1045-1053. Methods that include... [Invention 1060] A method according to any of the present invention 1057 to 1059, wherein a population of cells is present in the blood. [Invention 1061] A method according to any of the present invention 1057 to 1060, wherein the cell population is leukocytes. [Invention 1062] A method according to any one of the present invention 1057 to 1061, wherein the cell population is a subset of the leukocyte population. [Invention 1063] A method according to any of the present invention 1057 to 1062, wherein the cell population is a lymphocyte population. [Invention 1064] The method of the present invention 1063, wherein the cell population is a B cell population, a T cell population, an NK cell population, a regulatory T cell population, and a myeloid-derived suppressor cell population. [Invention 1065] Any method of the present invention 1054 to 1064, wherein the first antigen and / or the second antigen is a member of TNFR-SF. [Invention 1066] A first container comprising a first antibody according to any one of the present invention 1001-1028, 1052, or 1053, A second container comprising a second antibody according to any one of the present invention 1001-1028, 1052, or 1053, and A kit that includes the following: [Invention 1067] A first antibody or a second antibody according to any of Invention 1001 to 1028, or an antibody according to any of Invention 1029 to 1041 or 1052 to 1053. Nucleic acids that code for [something]. [Invention 1068] A first antibody or a second antibody according to any of Invention 1001 to 1028, or an antibody according to any of Invention 1029 to 1041 or 1052 to 1053. A nucleic acid that codes for the heavy chain of a molecule. [Invention 1069] An expression vector comprising the nucleic acid of the present invention 1067 or 1068. [Invention 1070] A delivery vehicle comprising the nucleic acid of the present invention 1067 or 1068. [Invention 1071] A delivery vehicle and a pharmaceutically acceptable carrier according to the present invention 1070. [Invention 1072] A delivery vehicle according to the present invention 1070 or 1071, which is a particle. These and other aspects of the present invention, in particular the various uses and therapeutic applications of the antibodies of the present invention, will be described in more detail below. [Brief explanation of the drawing]

[0031] [Figure 1A] Figure 1 shows that deglycosylated antibody variants with Fc-Fc interaction-enhancing mutations retain potent CDC activity. Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. Antibodies used in this experiment were either untreated, enzymatically deglycosylated ("deg"), or mocked ("trt"). Mass spectrometry spectra show the enzymatic deglycosylation efficiencies of (A) wild-type IgG1-CAMPATH-1H, (B) IgG1-CAMPATH-1H-E430G-K439E, and (C) IgG1-CAMPATH-1H-E430G-S440K. CDC efficacy was indicated by (D) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and (E) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 10 μg / mL. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 1C] Refer to the explanation in Figure 1A. [Figure 1D] Refer to the explanation in Figure 1A. [Figure 1E] Refer to the explanation in Figure 1A. [Figure 2A]Figure 2 shows that deglycosylation increases the selectivity of antibody variants containing Fc-Fc interaction-enhancing mutations and autooligomerization-inhibiting mutations. Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. (A, B) Cell lysis via CDC with a mixture of IgG1-CAMPATH-1H antibody variants. (C, D) Cell lysis via CDC with a mixture of IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants. CDC efficacy was shown as (A, C) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and as (B, D) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 10 μg / mL. [Figure 2B] Refer to the explanation in Figure 2A. [Figure 2C] Refer to the explanation in Figure 2A. [Figure 2D] Refer to the explanation in Figure 2A. [Figure 3A] Figure 3 shows that glycosyl antibody variants containing Fc-Fc interaction-enhancing mutations and autooligomerization-inhibiting mutations demonstrate high selectivity. Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. (A) CDC-mediated cell lysis by a mixture of IgG1-CAMPATH-1H antibody variant and IgG1-11B8 antibody variant is shown as AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). (B) Percentage of CDC-mediated cell lysis at the highest concentration tested with a mixture of IgG1-CAMPATH-1H antibody variant and IgG1-11B8 antibody variant, determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. [Figure 3B] Refer to the explanation in Figure 3A. [Figure 4A]Figure 4 shows the CDC activity of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants having mutations that enhance Fc-Fc interaction and mutations that disrupt the N-linked glycosylation site. Ramos cells (A, B, E, F) or Wien133 cells (C, D, G, H) were incubated with IgG1-CAMPATH-1H (A, B, C, D) or IgG1-11B8 (E, F, G, H) antibody variant concentration series in the presence of 10% NHS (A, B, E, F) or 20% NHS (C, D, G, H). CDC efficacy was expressed as (A, C, E, G) AUC normalized against unbound control antibody IgG1-b12 (0%) and positive control IgG1-CAMPATH-1H-E430G (100%), (B) percentage cytotoxicity normalized against positive control wells lysed with 0.02% Triton X-100, or (D, F, H) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 4E] See the explanation in Figure 4A. [Figure 4F] See the explanation in Figure 4A. [Figure 4G] See the explanation in Figure 4A. [Figure 4H] See the explanation in Figure 4A. [Figure 5A]Figure 5 shows that the non-glycosyl antibody variant with an Fc-Fc interaction-enhancing mutation retained partial CDC activity compared to the glycosylated parent molecule. (A-B) Daudi cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was indicated by (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G (100%), and (B) cytotoxicity determined at an antibody concentration of 40 μg / mL normalized against a positive control well lysed in 0.02% Triton X-100. (C-D) Raji cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was indicated by (C) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G (100%), and (D) percent cytotoxicity at an antibody concentration of 40 μg / mL, normalized against positive control wells dissolved in 0.02% Triton X-100. [Figure 5B] Refer to the explanation in Figure 5A. [Figure 5C] Refer to the explanation in Figure 5A. [Figure 5D] Refer to the explanation in Figure 5A. [Figure 6A]Figure 6 shows that disruption of the N297 glycosylation site improves the selectivity of CD52 target-directed antibody variants, which include Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. (A-B) Ramos cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was expressed as (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%), and (B) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL normalized against positive control wells lysed in 0.02% Triton X-100. (C-D) Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy was indicated by (C) AUC normalized against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%), and (D) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. [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 7A]Figure 7 shows that disruption of the N297 glycosylation site improves the selectivity of CD52-targeted antibody variants and CD20-targeted antibody variants, which include Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. (A-B) Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC potency was indicated by (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and (B) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. (C-D) Ramos cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was expressed as (C) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and as (D) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL, normalized against positive control wells dissolved in 0.02% Triton X-100. [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8A]Figure 8 shows that disruption of the N297 glycosylation site improves the selectivity of CD37-targeted antibody variants and CD20-targeted antibody variants, which include Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. (A-B) Raji cells were incubated with antibody concentration series in the presence of 10% NHS. CDC potency was expressed as (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G (100%), and (B) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL normalized against positive control wells lysed in 0.02% Triton X-100. (C-D) Daudi cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was expressed as (C) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G (100%), and as (D) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL, normalized against positive control wells dissolved in 0.02% Triton X-100. [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 9]This study shows that non-glycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, possessing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations, were suppressed in terms of ADCP induction ability when confirmed by an FcγRIIa activation reporter cell assay. Luciferase production was quantified by luminescence reading. Relative AUC: Area under the normalized dose-response curve (at 4-fold dilution, final concentration 0.15-40,000 ng / mL). (A) Raji cells were incubated with dose-specified IgG1-CAMPATH-1H antibody variants and FcγRIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-specified IgG1-CAMPATH-1H antibody variants, IgG1-11B8 antibody variants, and FcγRIIa-transduced Jurkat cells. [Figure 10] This study demonstrates that when an IgG1-CAMPATH-1H antibody variant containing an Fc-Fc interaction-enhancing mutation also has a mutation that inhibits glycosylation, antibody-dependent cell-mediated cytotoxicity is inhibited, as confirmed by an FcγRIIIa-activated reporter cell assay. Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and FcγRIIIa-expressing Jurkat cells. Luciferase production was quantified by luminescence reading using a Tecan Spark luminescence plate reader. [Figure 11A]Figure 11 shows that disruption of the N297 glycosylation site in IgG1-CAMPATH-1H-K248E-T437R has only a limited effect on its CDC activity. (A-B) Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy was shown as (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G positive control (100%), and (B) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. (C-D) Ramos cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was expressed as (C) AUC normalized against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G positive control (100%), and as (D) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL normalized against positive control wells dissolved in 0.02% Triton X-100. [Figure 11B] Refer to the explanation in Figure 11A. [Figure 11C] Refer to the explanation in Figure 11A. [Figure 11D] Refer to the explanation in Figure 11A. [Figure 12A]Figure 12 shows that disruption of the N297 glycosylation site improves the selectivity of antibody variants having the K248E-T437R Fc-Fc interaction enhancing mutation and the K439E or S440K autooligomerization inhibitory mutation. (A-D) Wien133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC potency was shown as AUC normalized against (A) unbound control antibody IgG1-b12-K439E+IgG1-b12-S440K(A) or IgG1-b12(C) (0%) and IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G positive control (100%), and (B, D) percentage lysis determined by percentage PI-positive cells at an antibody concentration of 40 μg / mL. (E-F) Ramos cells were incubated with antibody concentration series in the presence of 10% NHS. CDC efficacy was expressed as (E) AUC normalized against unbound control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G positive control (100%), and as (F) percentage cytotoxicity determined at an antibody concentration of 40 μg / mL, normalized against positive control wells dissolved in 0.02% Triton X-100. [Figure 12B] Refer to the explanation in Figure 12A. [Figure 12C] Refer to the explanation in Figure 12A. [Figure 12D] Refer to the explanation in Figure 12A. [Figure 13] This study demonstrates that non-glycosyl CD37-targeted antibody variants and CD20-targeted antibody variants, which include E430G or E345R Fc-Fc interaction-enhancing mutations and K439E or S440K auto-oligomerization-inhibiting mutations, interdependently induce CDC. (A-B) Daudi cells were incubated with antibody concentration series in the presence of 20% NHS. CDC potency was expressed as (A) AUC normalized against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G (100%), and (B) percentage cytotoxicity determined at an antibody concentration of 20 μg / mL. [Figure 14]This study demonstrates the selectivity of glycosylated and unglycosylated IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, possessing autooligomerization inhibitory mutations and E430Y Fc-Fc interaction enhancing mutations, for CDC activity in Wien133 cells, compared to variants with E430G and E345R mutations. CDC-inducing efficacy was normalized against unbound antibody control IgG1-b12 (0%) and against IgG1-CAMPATH-1H-E430G (100%; A) or IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%; B). (A) Wien133 cells were incubated with concentration series of IgG1-CAMPATH-1H antibody variants or mixtures thereof in the presence of 20% NHS. (B) Wien133 cells were incubated with concentration series of IgG1-CAMPATH-1H, IgG1-11B8, and / or IgG1-b12 antibody variants or mixtures thereof in the presence of 20% NHS. [Figure 15] This study demonstrates the selectivity for CDC induction in Wien133 cells by unglycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing the Fc-Fc interaction-enhancing mutation E345K, the auto-oligomerization-inhibiting mutation K439E or S440K, and the C1q-binding-enhancing mutations K326A and E333S. The efficacy in inducing CDC was normalized against an unbound antibody control IgG1-b12 (0%) and against IgG1-CAMPATH-1H-E430G (100%; A) or IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%; B). (A) Wien133 cells were incubated with concentration series of IgG1-CAMPATH-1H antibody variants or mixtures thereof in the presence of 20% NHS. (B) Wien133 cells were incubated with concentration series of IgG1-CAMPATH-1H antibody variants and / or mixtures thereof in the presence of 20% NHS. [Figure 16]This study demonstrates the selectivity for CDC induction in Wien133 cells by unglycosyl CAMPATH-1H antibody variants and 11B8 antibody variants of IgG2 or IgG4 subclasses, possessing the Fc-Fc interaction-enhancing mutation E345R and the auto-oligomerization-inhibiting mutation K439E or S440K. The efficacy in inducing CDC was normalized against unbound antibody control IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). (A) Wien133 cells were incubated with concentration-series IgG2-CAMPATH-1H antibody variants and IgG2-11B8 antibody variants or mixtures thereof in the presence of 20% NHS. (B) Wien133 cells were incubated with concentration-series IgG4-CAMPATH-1H antibody variants and IgG4-11B8 antibody variants or mixtures thereof in the presence of 20% NHS. [Figure 17A] Figure 17 shows the selectivity for CDC induction in Wien133 cells by unglycosyl IgG1-CAMPATH-1H antibody variants containing Fc-Fc interaction-enhancing mutations E430G, E430Y, E345K, or E345R and auto-oligomerization-inhibiting mutations K439E or S440K. The efficacy in inducing CDC was normalized against unbound antibody control IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%). Wien133 cells were incubated with concentration-series IgG1-CAMPATH-1H antibody variants or mixtures thereof in the presence of 20% NHS. (A) Relative CDC efficacy induced in Wien133 cells incubated with concentration-series IgG1-CAMPATH-1H antibody variants or mixtures thereof in the presence of 20% NHS. (B) Percentage of cell lysis of Wien133 cells incubated with concentration series of IgG1-CAMPATH-1H antibody variants or mixtures thereof in the presence of 20% NHS. [Figure 17B] Refer to the explanation in Figure 17A. [Figure 18]This document describes the ADCP-inducing ability of non-glycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, confirmed by an FcγRIIa activation reporter cell assay, which contain Fc-Fc interaction-enhancing mutations, auto-oligomerization-inhibiting mutations, and C1q binding-enhancing mutations K326A-E333S. Luciferase production was quantified by luminescence reading. Data are presented as the relative AUC (0.2 to 40,000 ng / mL at 4-fold dilution) of the area under the antibody dose-response curve, normalized to the AUC value (0%) measured against the unbound negative control IgG1-b12, the AUC value (100%; A) measured against the positive control IgG1-CAMPATH-1H-E430G, or the AUC value (100%; B) measured against IgG1-CAMPATH-E430G + IgG1-11B8-E430G. (A) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and FcγRIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, as well as FcγRIIa-transduced Jurkat cells. [Figure 20]This report describes the ADCC-inducing ability of non-glycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, each possessing an Fc-Fc interaction-enhancing mutation and an auto-oligomerization-inhibiting mutation, as confirmed by an FcγRIIIa-activating reporter cell assay. Luciferase production was quantified by luminescence reading. Data are presented as the relative AUC (0.2–40,000 ng / mL at 4-fold dilution) of the area under the antibody dose-response curve, normalized to the AUC value (0%) measured against the unbound negative control IgG1-b12, the AUC value (100%; A) measured against the positive control IgG1-CAMPATH-1H-E430G, or the AUC value (100%; B) measured against IgG1-CAMPATH-E430G + IgG1-11B8-E430G. (A) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and FcγRIIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants and FcγRIIIa-transduced Jurkat cells. [Figure 21]This report describes the ADCC-inducing ability of non-glycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, confirmed by an FcγRIIIa-activating reporter cell assay, using variants containing Fc-Fc interaction-enhancing mutations, auto-oligomerization-inhibiting mutations, and C1q binding-enhancing mutations. Luciferase production was quantified by luminescence reading. Data are presented as the relative AUC (0.2 to 40,000 ng / mL at 4-fold dilution) of the area under the antibody dose-response curve, normalized to the AUC value (0%) measured against the unbound negative control IgG1-b12, the AUC value (100%; A) measured against the positive control IgG1-CAMPATH-1H-E430G, or the AUC value (100%; B) measured against IgG1-CAMPATH-E430G + IgG1-11B8-E430G. (A) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and FcγRIIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-controlled IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants and FcγRIIIa-transduced Jurkat cells. [Figure 22]This shows the ADCP-inducing ability of unglycosyl CAMPATH-1H antibody variants and 11B8 antibody variants of IgG2 and IgG4 subclasses, possessing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations, as confirmed by an FcγRIIa activation reporter cell assay. Luciferase production was quantified by luminescence reading. Data are shown as relative AUC (0.6 to 40,000 ng / mL at 4-fold dilution) of the area under the antibody dose-response curve, normalized to the AUC value (0%) measured against the unbound negative control IgG1-b12 and the AUC value (100%) measured against the positive control IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G. (A) Raji cells were incubated with dose-specified IgG2-CAMPATH-1H antibody variants and / or IgG2-11B8 antibody variants, as well as FcγRIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-controlled IgG4-CAMPATH-1H and / or IgG4-11B8 antibody variants and FcγRIIa-transduced Jurkat cells. [Figure 23]This shows the ADCC-inducing ability of unglycosyl CAMPATH-1H antibody variants and 11B8 antibody variants of IgG2 and IgG4 subclasses, possessing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations, as confirmed by an FcγRIIIa activation reporter cell assay. Luciferase production was quantified by luminescence reading. Data are shown as the relative AUC (0.2 to 40,000 ng / mL at 4-fold dilution) of the area under the antibody dose-response curve, normalized to the AUC value (0%) measured against the unbound negative control IgG1-b12 and the AUC value (100%) measured against the positive control IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G. (A) Raji cells were incubated with dose-specified IgG2-CAMPATH-1H antibody variants and / or IgG2-11B8 antibody variants, as well as FcγRIIIa-transduced Jurkat cells. (B) Raji cells were incubated with dose-controlled IgG4-CAMPATH-1H and / or IgG4-11B8 antibody variants and FcγRIIIa-transduced Jurkat cells. [Figure 24] This study demonstrates the relative efficacy of a non-glycosyl anti-Fas-E09 antibody variant, which contains mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding, in inducing programmed cell death (PCD) in WIL2S-SF B cell lymphoblast target cells. (A) PCD efficacy is shown as the normalized cell death efficacy [AUC(IgG1-b12)-AUC(sample)] relative to IgG1-Fas-E09-E345R(100%). (B) Induction of cell death in WIL2S-SF cells induced at an antibody variant concentration of 20 μg / mL. [Figure 25A]Figure 25 shows the relative efficacy of two non-competitive, nonglycosyl anti-DR5 antibody variants (IgG1-hDR5-01-G56T and IgG1-hDR5-05), and mixtures thereof, in inducing programmed cell death (PCD) in BxPC-3 and COLO205 cancer cells. These variants contain mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding. PCD efficacy is shown as the normalized cell death efficacy [AUC(IgG1-b12)-AUC(sample)] against IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G(100%). (A) Relative efficacy in inducing cell death in BxPC-3 cancer cells. (B) Maximum cell death in BxPC-3 cells induced by the antibody variant and mixtures shown at a concentration of 20 μg / mL. (C) Relative efficacy in inducing cell death in COLO205 cancer cells. (D) Maximum cell death of COLO205 cells induced by antibody variants and mixtures at concentrations of 20 μg / mL. [Figure 25B] Refer to the explanation in Figure 25A. [Figure 25C] Refer to the explanation in Figure 25A. [Figure 25D] Refer to the explanation in Figure 25A. [Figure 26]This study demonstrates the selective induction of 4-1BB-mediated activation in reporter cells engineered to stably express 4-1BB and an activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, by IgG1-BMS663513 antibody variants containing mutations that enhance Fc-Fc interactions, mutations that inhibit self-oligomerization, and mutations that produce non-glycosyl antibodies. 4-1BB-expressing Jurkat cells were incubated with concentration series of IgG1-BMS663513 antibody variants or mixtures thereof in the presence of C1q. (A) The efficacy in inducing 4-1BB-mediated reporter cell activation is shown as relative AUC, normalized against the unbound antibody control IgG1-b12 (0%) and IgG1-BMS663513-E345R (100%). (B) The efficacy of inducing 4-1BB-mediated reporter cell activation at an antibody concentration of 1.25 μg / mL was demonstrated by normalized luminescence measurements compared to the unbound antibody control IgG1-b12 (0%) and IgG1-BMS663513-E345R (100%). [Figure 27] This study demonstrates the selective induction of 4-1BB-mediated activation in reporter cells engineered to stably express 4-1BB and an activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, by an IgG1-BMS663513 antibody variant containing mutations that enhance Fc-Fc interaction, inhibit autooligomerization, produce non-glycosyl antibodies, and enhance C1q binding. 4-1BB-expressing Jurkat cells were incubated with concentration series of IgG1-BMS663513 antibody variants or mixtures thereof in the presence of C1q. (A) The efficacy in inducing 4-1BB-mediated reporter cell activation is shown as a normalized relative AUC against the unbound antibody control IgG1-b12 (0%) and IgG1-BMS663513-E345R (100%). (B) The efficacy of inducing 4-1BB-mediated reporter cell activation at an antibody concentration of 1.25 μg / mL was demonstrated by normalized luminescence measurements compared to the unbound antibody control IgG1-b12 (0%) and IgG1-BMS663513-E345R (100%). [Modes for carrying out the invention]

[0032] Detailed description of the invention definition The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low-molecular-weight light (L) chains and one pair of heavy (H) chains, where all four chains may be interconnected by disulfide bonds. The structure of immunoglobulins is well-characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The heavy chains are interconnected via 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 one domain: CL. The VH and VL regions can be further divided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise specified or unless inconsistent with the context, CDR sequences in this specification are identified using DomainGapAlign in accordance with the IMGT rules (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.

[0033] Unless otherwise specified or unless inconsistent with the context, the description of the amino acid positions of the Fc region / Fc domain in this invention follows 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).

[0034] The Fc region of an immunoglobulin is typically defined as an antibody fragment that arises after digestion of an antibody with papain (known to those skilled in the art) and includes two CH2-CH3 regions of the immunoglobulin and a connecting region, such as a hinge region. The constant domain of the antibody heavy chain defines the antibody isotype / subclass, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates the effector function of the antibody together with cell surface receptors and complement system proteins called Fc receptors.

[0035] As used herein, the term “hinge region” is intended to refer 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 according to Eu numbering.

[0036] As used herein, the terms “CH2 region” or “CH2 domain” are intended to refer to the CH2 region of an immunoglobulin heavy chain. For example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to Eu numbering. However, the CH2 region may also be any of the other subtypes described herein.

[0037] As used herein, the terms “CH3 region” or “CH3 domain” are intended to refer to the CH3 region of an immunoglobulin heavy chain. For example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to Eu numbering. However, the CH3 region may also be any of the other subtypes described herein.

[0038] In the context of the present invention, the term “antibody” (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen. The antibodies of the present invention include an Fc region and an antigen-binding region of the immunoglobulin. Antibodies generally contain two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least an Fc region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. As described above, the constant region or “Fc” region of the antibody can mediate the binding of the immunoglobulin to various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., host tissue or factors containing C1q, the first component in the classical pathway of complement activation. The antibody may also be a multispecific antibody, e.g., a bispecific antibody or a similar molecule. The term “bispecific antibody” refers to an antibody that is specific to at least two different, typically non-overlapping epitopes. Such epitopes may be on the same target or on different targets. If the epitopes are located on different targets, such targets may be located on the same cell, on different cells, or on different cell types. Unless otherwise specified or unless clearly inconsistent with the context, the term antibody as used herein includes antibody fragments that include at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include monovalent antibodies (described by Genmab in WO2007059782); heavy-chain antibodies consisting of only two heavy chains, e.g., naturally occurring in camelid animals (e.g., Hamers-Casterman (1993) Nature 363:446); and ThioMab (Roche,WO2011069104), asymmetric and bispecific antibody-like molecules, such as strand-exchange engineered domain-body (SEED or seed-body molecule) (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), Dual variable domain (domain) Immunoglobulin (Abbott, DVD-Ig, US Patent No. 7,612,181); Dual domain double head antibody (Unilever; Sanofi Aventis, WO20100226923), Di-diabody molecule (ImClone / Eli Lilly), Knobs-into-holes antibody form (Genentech, WO9850431); DuoBody molecule (Genmab, WO2011 / 131746); DuetMab (MedImmune, US2014 / 0348839), Electrostatic steering antibody form (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat Neurosciences Corporation, WO11143545), CrossMAb (Roche,WO2011117329), LUZ-Y (Genentech), Biclonic molecule (Merus, WO2013157953), Dual Targeting domain antibody (GSK / Domantis), Two-in-one antibody or Dual action Fab (Genentech, NovImmune, Adimab) that recognizes two targets, Cross-linked mAb (Karmanos Cancer Center), Covalently fused mAb (AIMM), CovX body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecific molecule (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007). 318(1-2): p.65-74), TIG bodies, DIG bodies, and PIG body (PIG-body) molecules (Pharmabcine), dual-affinity retargeting molecules by Macrogenics (Fc-DART or Ig-DART, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybodoy (Zyngenia), approaches using common light chains (Crucell / Merus, US7262028) or approaches using common heavy chains (κλ bodies by NovImmune, WO2012023053), and fusion proteins containing polypeptide sequences fused with antibody fragments containing Fc regions such as scFv fusions, e.g., BsAb by ZymoGenetics / BMS, Biogen HERCULES (US007951918) by Idec, 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 fusion by Genentech / Roche, scFv fusion by Novartis, scFv fusion by Immunomedics, scFv fusion by Changzhou Adam Biotech Inc (CN102250246), TvAb by Roche (WO2012025525, WO2012025530), mAb by f-Star, 2 (WO2008 / 003116), as well as dual scFv fusions, are included but not limited thereto. The term antibody should also be understood to include, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), antibody mixtures (recombinant polyclonals), antibody mixtures (recombinant polyclonals) produced by techniques used by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. The antibodies produced may potentially have any isotype.

[0039] Where the term "full-length antibody" is used herein, it refers to an antibody (e.g., parent antibody) that contains all the constant and variable heavy and light chain domains, corresponding to those typically found in wild-type antibodies of the above isotypes.

[0040] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by in vitro random mutagenesis or site-directed mutagenesis, or by in vivo somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been transplanted into a human framework sequence.

[0041] As used herein, the term “chimeric antibody” refers to an antibody in which both chain types, i.e., the heavy chain and the light chain, are chimeric as a result of antibody engineering. A chimeric chain is a chain containing an exogenous variable domain (derived from a non-human species, synthetic, or engineered from any species including humans) linked to a human-derived constant region.

[0042] As used herein, the term “humanized antibody” refers to an antibody in which both chain types have been humanized as a result of antibody engineering. A humanized chain is typically one in which the complementarity-determining region (CDR) of the variable domain is exogenous (derived from a non-human species or synthesized), while the rest of the chain is derived from humans. The evaluation of humanization is based on the resulting amino acid sequence and not on the methodology itself; therefore, protocols other than grafting can be used.

[0043] As used herein, terms such as “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” and “mAb” refer to preparations of Ab molecules with only one molecular composition. Monoclonal antibody compositions exhibit only one binding specificity and affinity to a particular epitope. Therefore, the term “human monoclonal antibody” refers to an Ab exhibiting only one binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human mAbs can be produced by a hybridoma obtained by fusing B cells from a transgenic non-human animal or a transchromosomal non-human animal, such as a transgenic mouse, in which the genome containing the human heavy-chain transgene repertoire and light-chain transgene repertoire has been rearranged to produce a functional human antibody.

[0044] As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG, IgD, IgA1, IgGA2, IgE, or IgM). IgG may be further classified as subclasses IgG1, IgG2, IgG3, or IgG4. As used herein, the term “subclass” refers to the IgG1, IgG2, IgG3, and IgG4 subclasses of the IgG isotype. The IgG1 subclass may be further classified according to its allotype, e.g., IgG1m(za) and IgG1m(f)), encoded by a heavy chain constant region gene. Furthermore, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain. As used herein, the term “mixed isotype” or “mixed subclass” refers to the Fc region of an immunoglobulin created by combining structural features of one isotype with a similar region derived from another isotype, thereby producing a hybrid isotype. The mixed isotype consists of two or more isotypes selected from the following: IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, and may include an Fc region having a sequence that produces combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0045] As used herein, the terms “antigen-binding region,” “binding region,” or “antigen-binding domain” refer to the antibody region capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which may be further divided into a hypervariable region, also called a complementarity-determining region (CDR), which is interspersed with conserved regions called framework regions (FRs) (or a hypervariable region whose sequence is hypervariable and / or which may take the form of a structure-defined loop). The antigen may be any molecule, e.g., a polypeptide, e.g., a polypeptide present on the surface of a cell, bacterium, or virion.

[0046] As used herein, the terms “target” or “target antigen” refer to the molecule to which the antigen-binding domain of an antibody binds. A target includes any antigen to which an antibody is directed. The terms “antigen” and “target” are used synonymously with respect to an antibody and may constitute the same meaning and purpose in any aspect or embodiment of the present invention.

[0047] The term "epitope" refers to a molecular determinant that can specifically bind to an antibody variable domain. Epitopes typically consist of a molecular surface group, such as an amino acid, a sugar side chain, or a combination thereof, and usually possess specific three-dimensional structural and 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 contain an amino acid residue directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding.

[0048] The term "parent antibody" should be understood as the same antibody as the antibody according to the present invention, but the parent antibody does not have one or more of the specified mutations. A "variant," "antibody variant," or "variant of parent antibody" in the present invention is an antibody molecule containing one or more mutations compared to the "parent antibody." Different terms may be used synonymously and constitute the same meaning and purpose for any aspect or embodiment of the present invention. Exemplary parent antibody forms include, but are not limited to, wild-type antibodies, full-length antibodies or antibody fragments containing Fc, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof. Different terms may be used synonymously and constitute the same meaning and purpose for any aspect or embodiment of the present invention. Amino acid substitutions may involve the exchange of a native amino acid with another natural or non-natural amino acid derivative. Amino acid substitutions may be conserved or non-conservative. In the context of the present invention, a conserved substitution may be defined as a substitution within a class of amino acids reflected in one or more of the following three tables. Amino acid residue classes for conservative substitutions TIFF0007880817000001.tif58136 Another Conservative Amino Acid Residue Substitution Class TIFF0007880817000002.tif39136 Another physical and functional classification of amino acid residues TIFF0007880817000003.tif70142

[0049] In the context of the present invention, substitutions in the variants are Original amino acid - position - substituted amino acid This is indicated.

[0050] To indicate amino acid residues, three-letter or one-letter notations containing Xaa and X are used. Therefore, the notation "E345R" or "Glu345Arg" means that the variant includes a substitution from glutamic acid to arginine at the variant amino acid position corresponding to the amino acid at position 345 of the parent antibody.

[0051] Therefore, if a certain position is not present in the antibody, but the variant involves the insertion of an amino acid, for example, For position-substituted amino acids, the notation, for example, "448E," is used.

[0052] Such notation is particularly important with respect to modifications in a series of homologous polypeptides or antibodies.

[0053] Similarly, if the identity of the substituted amino acid residue is not important, The original amino acid position; or "E345".

[0054] In modifications where the original amino acid and / or substituted amino acid may contain multiple amino acids but not all of them, the substitution of glutamic acid at position 345 with arginine, lysine, or tryptophan may be expressed in the context of this invention as "Glu345Arg,Lys,Trp", "E345R,K,W", "E345R / K / W", or "E345→R,K, or W".

[0055] Furthermore, the term “substitution” includes substitutions to any one of the other 19 natural amino acids, or substitutions to other amino acids, such as non-natural amino acids. For example, substitutions of amino acid E at position 345 include the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345P, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y, respectively. This is equivalent to the name 345X, where X specifies any amino acid. These substitutions can also be specified as E345A, E345C, etc., or E345A,C, etc., or E345A / C / , etc. The same applies to any similarity to any position described herein so as to specifically include any one of such substitutions.

[0056] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of the immune response, as opposed to the recognition and activation phases of the immune response. Exemplary immune cells include cells derived from bone marrow or the lymphoid system, such as lymphocytes (e.g., T cells including B cells and cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, multinucleated cells, or granulocytes, such as neutrophils, mast cells, eosinophils, 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 that express FcRs are involved in the specific death 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-mediated classical complement activation, thereby depositing 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 may promote Fc receptor-mediated enhancement of ADCC. In some embodiments, antibody-mediated classical complement activation leads to C3 fragments on target cells. These C3 cleavage products may promote direct complement-dependent cell-mediated cytotoxicity (CDCC). In some embodiments, effector cells may phagocytose target antigens, target particles, or target cells. Expression of certain FcRs or complement receptors on effector cells may 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. Such increased expression leads to increased cytotoxic activity of FcγRI-containing cells against targets. Effector cells can phagocytose target antigens, or they can phagocytose or lyse target cells.In some embodiments, antibody-mediated classical complement activation leads to C3 fragments on target cells. These C3 cleavage products may promote direct phagocytosis by effector cells, or they may indirectly promote phagocytosis by enhancing antibody-mediated phagocytosis.

[0057] As used herein, the terms “Fc effector function” or “Fc-mediated effector function” are intended to refer to a function resulting from a polypeptide or antibody binding to its target on the cell membrane, e.g., an antigen, and 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-γ receptor binding, (vi) antibody-dependent cell-mediated phagocytosis (ADCP), (vii) complement-dependent cell-mediated cytotoxicity (CDCC), (viii) complement-enhanced cell-mediated cytotoxicity, (ix) binding of a complement receptor to an opsonized antibody mediated by the antibody, (x) opsonization, and (xi) any combination of (i) to (x).

[0058] As used herein, the term “clustering-dependent function” is intended to refer to a function resulting from the formation of an antigen complex after oligomerization of a polypeptide or antibody bound to an antigen, optionally an antigen on a cell, an antigen on a cell membrane, an antigen on a virion, or an antigen on another particle. Examples of clustering-dependent effector functions include (i) antibody oligomerization, (ii) antibody oligomer stability, (iii) antigen oligomerization, (iv) antigen oligomer stability, (v) apoptosis induction, (vi) regulation of proliferation, e.g., reduction, inhibition, or stimulation of proliferation, and (vii) any combination of (i) to (vi).

[0059] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of inducing the transcription of a nucleic acid segment linked to a vector. One type of vector is a “plasmid,” which takes the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, which can link a nucleic acid segment to a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be integrated into the host cell genome and thereby replicated together with the host genome. Furthermore, certain vectors can induce the expression of a gene functionally linked to the vector. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA methods often take the form of plasmids. Since plasmids are the most commonly used form of vector, “plasmid” and “vector” are sometimes used synonymously herein. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses with replication defects).

[0060] As used herein, the term “delivery vehicle” refers to a composition or formulation intended to protect nucleic acids, peptides, polypeptides, such as antibodies, small molecules, or other pharmacologically active substances from degradation upon administration. A delivery vehicle for nucleic acids may also be a vector, which should be understood as a nucleic acid molecule capable of inducing the transcription of a nucleic acid segment linked to the vector. Such a vector may contain nucleic acid sequences encoding heavy and light chains of polypeptide variants. Furthermore, a delivery vehicle may be a lipid formulation composed of components, such as lipids, ionizable aminolipids, PEG-lipids, cholesterol, or any combination thereof. Nucleic acids may be encapsulated within the delivery vehicle. Such a delivery vehicle may be modified, for example, by external attachment of a target-specific antibody, by adjustment of the vehicle size, or by tuning the physicochemical properties of the vehicle, so that delivery to the relevant tissue is optimally achieved.

[0061] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which an expression vector has been introduced. Such terminology should be understood to refer not only to specific target cells but also to the offspring of such cells. Because mutations or environmental influences can cause certain changes in later generations, such offspring may not actually be identical to their parent cells, but they are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli, and other eukaryotic hosts such as plant cells and fungi.

[0062] As used herein, the term “transfectoma” includes recombinant eukaryotic host cells expressing Ab or target antigen, such as yeast cells, CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi.

[0063] The term "preparation" refers to preparations of antibody variants and mixtures of different antibody variants that may have a high ability to form oligomers when interacting with antigens associated with cells (e.g., antigens expressed on the cell surface), cell membranes, virions, or other structures, and as a result may enhance antigen-mediated signaling and / or activation.

[0064] As used herein, the term "affinity" refers to the strength of the binding of one molecule, such as an antibody, to another molecule, such as a target or antigen, at a single site, such as the strength of the monovalent bond between an individual antigen-binding site of an antibody and an antigen.

[0065] As used herein, the term "avidity" refers to the binding strength of multiple binding sites between two structures, for example, the binding strength between multiple antigen-binding sites of an antibody that interact simultaneously with a target, or, for example, the binding strength between an antibody and C1q. When multiple binding interactions are present, the two structures dissociate only when all binding sites have dissociated. Therefore, the dissociation rate is slower than the dissociation rate of individual binding sites, thereby providing a considerably effective overall binding strength (avidity) compared to the binding strength (affinity) of individual binding sites.

[0066] As used herein, the term “oligomer” refers to a molecule consisting of a limited number of monomer units (e.g., an antibody), as opposed to a polymer, which consists of, at least in principle, countless monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to specify the number of monomer units in an oligomer. For example, a tetramer consists of 4 units, and a hexamer consists of 6 units.

[0067] As used herein, the term “oligomerization” is intended to refer to the process of converting a monomer into a finite degree of polymerization. In this specification, it is observed that antibodies containing a target-binding domain according to the present invention can form oligomers, such as hexamers, after binding to a target, for example, on a cell surface, via non-covalent bonds in the Fc domain. In the context of this application, “auto-oligomerization,” “homo-oligomerization,” or “auto-oligomerization” is intended to refer to the oligomerization process between antibody molecules having identical protein sequences, without considering post-translational modifications. As used herein, the term “hetero-oligomerization” is intended to refer to the oligomerization process between antibody molecules having different protein sequences, without considering post-translational modifications. Different antibodies involved in hetero-oligomerization may, for example, bind to different antigens, such as different target proteins, glycoproteins, glycans, or glycolipids.

[0068] The terms "self-oligomerization inhibiting substitution" or "self-oligomerization inhibiting substitution" are intended to refer to substitutions in antibodies, including the Fc region and antigen-binding region of an immunoglobulin, that inhibit the oligomerization process between antibody molecules having identical protein sequences, without considering post-translational modifications. Inhibiting self-oligomerization can, for example, increase the EC50 of CDC activity as measured according to the methods described herein, e.g., Examples 2 and 5, or decrease the maximum CDC lysis activity of a polypeptide. Examples of self-oligomerization inhibiting substitutions are K439E and S440K. For example, a first antibody with the K439E substitution has a weak tendency to oligomerize with another antibody with the K439E substitution, while an antibody with the K439E substitution has a strong tendency to oligomerize with another antibody with the S440K substitution. Antibodies containing the S440K substitution have a weak tendency to form oligomers with other antibodies containing the S440K substitution.

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

[0070] As used herein, the term "Fc-Fc strengthening" is intended to refer to increasing the binding strength between the Fc regions of two Fc-region-containing antibodies or polypeptides, or stabilizing the interaction between the Fc regions of two Fc-region-containing antibodies or polypeptides, so that they form an oligomer when bound to a target. As used herein, Fc-Fc strengthening substitutions refer to substitutions at E430 and E345, or the combination of K248E and T437R substitutions (WO2018 / 031258), and the amino acid numbering corresponds to human IgG1 by Eu numbering.

[0071] When used herein in the context of two antigens, the term “co-expressed” or its grammatical variation is intended, on the one hand, to refer to a situation in which two antigens are co-expressed on the same cell. The two antigens may already be adjacent to each other on the cell, or they may be combined by the binding polypeptide of the present invention, for example, by oligomerization of antibodies. Furthermore, the term “co-expressed” is also intended to refer to a situation in which two antigens are expressed on different cells, but such cells are close to each other.

[0072] As used herein, the term “codependent” is intended to refer to a functional effect that depends on the co-binding of two or more different Fc region-containing antibodies with self-oligomerization inhibitory substitutions to the same target, cell, or virion. In the context of the present invention, possible codependent functional effects include clustering-dependent function, Fc-mediated effector function, and binding of effector molecules such as FcγR or C1, but do not necessarily include the individual binding of Fc region-containing antibodies to their target antigens. As used herein, different Fc region-containing antibodies with self-oligomerization inhibitory substitutions may each bind individually to different targets, cells, or virions, but the codependent functional outcome depends on the co-binding of two or more different components to the same target, cell, or virion. As used herein, the codependent functional effect is specifically restored by two or more different Fc region-containing antibodies with self-oligomerization inhibitory substitutions by restoring non-covalent Fc-Fc interactions between different components in a codependent Fc-containing polypeptide mixture.

[0073] As used herein, the term "C1q binding" is intended to refer to a direct interaction between C1q and an antibody. Direct C1q binding can be evaluated, for example, by using an immobilized antibody on an artificial surface. Multivalent interactions resulting in high avidity binding between C1q and antibody oligomers can be evaluated when bound to a predetermined antigen on a cell surface or virion surface.

[0074] The binding of C1q to polypeptides or antibodies in an ELISA assay involves the following steps: (i) coating a 96-well Microlon ELISA plate overnight at 4°C with 1 μg / mL polypeptide or antibody dissolved in 100 μl of PBS; (ii) incubating the plate at 37°C for 1 hour with a 3-fold diluted serial dilution series of 100 μL / well C1q with a final C1q concentration range of 30 to 0.01 μg / mL; (iii) incubating the plate with 100 μl / well rabbit anti-human C1q at RT for 1 hour; (iv) incubating the plate with 100 μl / well porcine anti-rabbit IgG-HRP at RT for 1 hour; (v) incubating the plate with 100 μL / well substrate and 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) at RT for 15 minutes; (vi) 100 μL This can be verified by using a step to stop the reaction by adding 2% oxalic acid / well. Absorbance was measured at 405 nm using a BioTek EL808 microplate reader.

[0075] As used herein, the term “C1q-binding substitution” is intended to refer to substitutions in polypeptides, including the Fc region and antigen-binding region of an immunoglobulin, that enhance direct interaction with C1q. Enhanced C1q binding can lower the EC50 of the interaction between C1q and the antibody, for example, as measured according to the methods for verifying C1q binding described above.

[0076] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on its surface. C1 is a complex consisting of six recognition proteins, Clq, and a serine protease heterotetramer, C1r2C1s2. C1 is the first protein complex in the initial event of the classical complement cascade, involving a series of cleavage reactions starting with the cleavage of C4 to C4a and C4b, and the cleavage of C2 to C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase. C3 convertase cleaves complement component C3 into C3b and C3a, forming C5 convertase. This C5 convertase separates C5 into C5a and C5b, the last component of which is deposited on the membrane, resulting in the induction of the later event of complement activation. In the late event, terminal complement components C5b, C6, C7, C8, and C9 assemble to form the membrane invasion complex (MAC). The complement cascade creates pores in the cell membrane, leading to cell lysis, also known as complement-dependent cell-mediated cytotoxicity (CDC). Complement activation can be evaluated, for example, using C1q potency, CDC kinetics, or CDC assays (as described in WO2013 / 004842, WO2014 / 108198), or by cell deposition methods for C3b and C4b as described by Beurskens et al. in Journal of Immunology, 2012 vol. 188 no. 7, April 1, 3532-3541.

[0077] As used herein, the term “complement-dependent cell injury” (“CDC”) is intended to refer to the antibody-mediated complement activation process in which a cell or virion lyses as a result of pores created in the membrane by MAC assembly when an antibody binds to an antibody target on a cell or virion.

[0078] As used herein, the term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) is intended to refer to the mechanism by which cells expressing an Fc receptor that recognizes the constant region of a bound antibody kill antibody-coated target cells or virions. As used herein, the term “antibody-dependent cell phagocytosis” (“ADCP”) is intended to refer to the mechanism by which antibody-coated target cells or virions are expelled by phagocytic cell infiltration. The internally migrated antibody-coated target cells or virions are contained within vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes. ADCP can be evaluated using an in vitro cytotoxicity assay with macrophages as effector cells and video microscopy, as described in van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, pp. 677-685.

[0079] As used herein, the term “complement-dependent cell-mediated cytotoxicity” (“CDCC”) refers to a mechanism by which cells expressing complement receptors that recognize complement 3 (C3) cleavage products covalently bound to target cells or virions as a result of antibody-mediated complement activation kill the target cells or virions. CDCC can be evaluated in a manner similar to that described for ADCC.

[0080] As used herein, the term “plasma half-life” refers to the time it takes for the concentration of a polypeptide in plasma to decrease to half of its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase is typically 1 to 3 days, during which the plasma concentration decreases by approximately 50% due to redistribution between plasma and tissues. The plasma half-life can be measured by methods well known in the art.

[0081] As used herein, the term "plasma clearance rate" is a quantitative measure of the rate at which a polypeptide is removed from the blood when administered to an organism. Plasma clearance rate can be calculated as dose / AUC (mL / day / kg), and the AUC (area under the curve) value is obtained from the concentration-time curve.

[0082] As used herein, the term “antibody-drug conjugate” refers to an antibody or Fc-containing polypeptide that is specific to at least one type of malignant cell, a drug, and a linker that conjugates the drug, for example, a linker that conjugates the drug to the antibody. The linker may or may not be cleavable in the presence of malignant cells. The antibody-drug conjugate kills the malignant cells.

[0083] 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, is then taken up / phagocytosed by the cell membrane, thereby drawing the antibody-drug conjugate into the cell. Antibody-drug conjugate uptake can be evaluated as described in WO2011 / 157741, “antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay.”

[0084] As used herein, the term "apoptosis" refers to the process of programmed cell death (PCD) that can occur within cells. Characteristic cellular changes (morphological changes) and death are caused by biochemical events. These changes include bleving, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis can be induced when an antibody binds to a specific receptor.

[0085] As used herein, the term “programmed cell death” or “PCD” refers to any form of cell death mediated by an intracellular program. Various types of PCD exist, and these different types of PCD share the commonality of being carried out by active cellular processes that can be blocked by interfering with intracellular signaling. In certain embodiments, the occurrence of any type of PCD in cells or tissues can be confirmed by staining the cells or tissues with conjugated annexin V, which correlates with phosphatidylserine exposure.

[0086] As used herein, the term "annexin V" refers to proteins in the annexin group that bind to phosphatidylserine (PS) on the cell surface.

[0087] As used herein, the term “FcRn” is intended to refer to the fetal Fc receptor, an Fc receptor. “FcRn” was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk across the epithelium of the neonatal digestive tract into the neonatal bloodstream. Further research revealed the presence of a similar receptor in humans. However, in humans, “FcRn” has been found to assist in the transport of maternal IgG from the placenta to the developing fetus and has also been shown to play a role in monitoring IgG turnover. FcRn binds to IgG at an acidic pH of 6.0–6.5 but not at neutral or higher pH. Therefore, FcRn can bind IgG from the slightly acidic pH of the intestinal lumen (inside the digestive tract) and ensure efficient unidirectional transport to the neutral to basic pH (pH 7.0–7.5) of the basolateral side (inside the body). This receptor also plays a role in adult IgG salvage by appearing in the endocytosis pathway in endothelial cells. FcRn receptors within acidic endosomes bind to IgG that has been internalized via pinocytosis, recirculating IgG to the cell surface. They then release IgG at the basic pH of the blood, thereby preventing IgG from being lysosomal degraded. This mechanism may explain the longer half-life of IgG in the blood compared to other isotypes.

[0088] As used herein, the term "Protein A" is intended to refer to a 56 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. "Protein A" is encoded by the spa gene, and its regulation is controlled by DNA topology, the osmolarity of the cell's volume, and a two-component system called ArlS-ArlR. "Protein A" has been used in biochemical research because of its ability to bind to immunoglobulins. "Protein A" is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain can bind to proteins from many mammalian species, particularly IgG. "Protein A" binds to the Fc region of most immunoglobulins (overlapping the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. These interactions in serum cause IgG molecules to bind to bacteria not only through their Fab regions but also through their Fc regions, thereby disrupting opsonization, complement activation, and phagocytosis by the bacteria.

[0089] As used herein, the term "Protein G" is intended to refer to an immunoglobulin-binding protein expressed in group C and G streptococcal bacteria that is very similar to Protein A but has different specificities. "Protein G" is a 65 kDa (G148 Protein G) or 58 kDa (C40 Protein G) cell surface protein that has been used in antibody purification by binding to the Fc region.

[0090] "Treatment" refers to administering an effective amount of a therapeutically active compound of the present invention for the purpose of alleviating, remitting, preventing, or eradicating (curing) a symptom or disease state.

[0091] "Effective amount" or "therapeutically effective amount" refers to the dosage and time required to obtain a desired therapeutic result and an amount that exhibits an effect effective for obtaining the desired therapeutic result. The therapeutically effective amount of an antibody may vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the antibody to induce a desired response in the individual. The therapeutically effective amount is also an amount at which the beneficial effects of treating an antibody or antibody portion outweigh the toxic or detrimental effects.

[0092] As used herein, the term "glycosylation" refers to a post-translational modification involving the selective binding of a glycan to a polypeptide, such as an antibody, to stabilize the structure of the polypeptide or to transfer a function to the polypeptide. As used herein, the term "deglycosylation" refers to the removal of a glycan from a polypeptide, such as an antibody, for example, enzymatically. As used herein, the term "aglycosyl" refers to the characteristic that a glycan is not bound to a polypeptide, such as an antibody, achieved by genetic engineering, by recombinant techniques, or by deglycosylation such as enzymatic deglycosylation.

[0093] Certain aspects of the present invention As described above, in a first aspect, the present invention A second antibody comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen For use as a medicament in combination with A first antibody comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, wherein Said first Fc region is a. Substitution at position E430, or substitution at position E345, or a combination of substitutions K248E and T437R, and b. K439E or S440K substitution Comprising; Said second Fc region is c. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and d. Replacement with K439E or S440K Includes; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody do not contain N-linked glycosylation at position N297; The amino acid positions correspond to human IgG1 according to the Eu numbering system (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). The first antibody for use as a medical drug in combination with the second antibody. Regarding.

[0094] Substitutions at the positions corresponding to E430 and E345, or the combination of substitutions K248E and T437R, are considered Fc-Fc-enhancing substitutions according to the present invention. That is, such substitutions introduce enhanced Fc-Fc interaction and oligomerization effects into the polypeptide or antibody. When the antigen-binding region of the antibody binds to the corresponding target antigen, enhanced oligomerization occurs. Because oligomerization is enhanced, oligomers such as hexamers are generated. The generation of oligomer structures such as hexamers has the effect of increasing Fc effector function, such as CDC, by increasing the C1q binding avidity of the polypeptide.

[0095] In one embodiment, the first antibody contains at most one substitution at the position corresponding to E430 or E345. In one embodiment, the second antibody contains at most one substitution at the position corresponding to E430 or E345. Thus, in one embodiment, the Fc region contains at most one substitution at the position corresponding to E430 or E345.

[0096] In one embodiment of the present invention, the first Fc region and the second Fc region include substitutions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E430Y, E345Q, E345R, and E345Y, or a combination of substitutions K248E and T437R.

[0097] In one embodiment of the present invention, the first Fc region and the second Fc region include substitutions selected from the group consisting of E345K, E430G, E345R, E430Y, E345Q, E345Y, E430S, E430T, and E430F, and / or combinations of substitution K248E and T437R.

[0098] In one embodiment, the first Fc region and the second Fc region include substitutions selected from the group consisting of E430G and E345K.

[0099] In one embodiment, the first Fc region and the second Fc region include substitutions selected from the group consisting of E345R and E430Y.

[0100] In one embodiment, the first Fc region and the second Fc region include a combination of substitution K248E and T437R.

[0101] In one embodiment, the first Fc region may have an E430G substitution, and the second Fc region may have an E345K substitution. In another embodiment, the first Fc region may have an E345K substitution, and the second antibody may have an E430G substitution. The substitutions in the first and second Fc regions may be independently selected from the group of Fc-Fc reinforcing substitutions.

[0102] In one embodiment of the present invention, the first Fc region and the second Fc region include substitutions selected from the group consisting of E345K, E430G, E345R, and E430Y. In one embodiment of the present invention, the first Fc region and the second Fc region include substitutions selected from the group consisting of E430G, E345K, and E345R. In one embodiment of the present invention, the first Fc region and the second Fc region include an E430G substitution. In one embodiment of the present invention, the first Fc region and the second Fc region include an E345K substitution. In one embodiment of the present invention, the first Fc region and the second Fc region include an E345R substitution. In one embodiment of the present invention, the first Fc region and the second Fc region include an E430Y substitution.

[0103] The first and second Fc regions further comprise K439E or S440K substitutions, which are considered complementary oligomerization-inhibiting substitutions according to the present invention. Thus, for example, a first antibody having a K439E substitution has a weak tendency to form oligomers with another antibody having a K439E substitution, while an antibody having a K439E substitution has a strong tendency to form oligomers with another antibody having an S440K substitution. An antibody having an S440K substitution has a weak tendency to form oligomers with another antibody having an S440K substitution. Thus, in one embodiment of the present invention, the first Fc region comprises a K439E substitution, and the second Fc region comprises an S440K substitution. In one embodiment of the present invention, the first Fc region comprises an S440K substitution, and the second Fc region comprises a K439E substitution.

[0104] In a further manner, The first Fc region includes K439E substitution and Y436N substitution, and the second Fc region includes S440K substitution and Q438R substitution, or The first Fc region includes K439E substitution and Q438R substitution, and the second Fc region includes S440K substitution and Y436N substitution, or The first Fc region includes S440K substitution and Y436N substitution, and the second Fc region includes K439E substitution and Q438R substitution, or · The first Fc region contains S440K substitution and Q438R substitution, and the second Fc region contains K439E substitution and Y436N substitution. Y436N and Q438R are also considered complementary oligomerization-inhibiting substitutions according to the present invention.

[0105] In one aspect, neither the first antibody nor the second antibody contains an amino acid substitution at position G237 and does not contain one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E.

[0106] In one aspect, neither the first antibody nor the second antibody contains an amino acid substitution at position P329 and does not contain K322E substitution.

[0107] In one aspect, neither the first antibody nor the second antibody contains one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0108] In one aspect, neither the first antibody nor the second antibody contains an amino acid substitution at position G237 or P329 and does not contain one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E.

[0109] Furthermore, the first antibody and / or the second antibody does not contain N-linked glycosylation at position N297.

[0110] IgG antibodies, when produced in eukaryotic cells capable of glycosylation of polypeptides, are typically glycosylated at position N297. At least one of the antibodies used in this invention is not glycosylated at position N297. This can be achieved, for example, by mutating the N-glycosylation consensus sequence (NX / S / T) surrounding N297, thereby producing the antibody in a system where N-linked glycosylation at N297 does not occur, such as a prokaryotic expression system or a bacterial expression system, or by removing glycosylation, for example, enzymatically. The effect of eliminating glycosylation at position N297 is a strong reduction in the antibody's ability to perform effector functions as a single agent. Surprisingly, the inventors found that even without glycosylation, the ability of the antibody to form a highly active heterooligomer with the other antibody in a pair is not strongly affected.

[0111] In one embodiment, the first antibody and / or the second antibody include an amino acid substitution, deletion, or insertion that inhibits N-linked glycosylation at position N297.

[0112] In one embodiment, the first Fc region and / or the second Fc region include an amino acid substitution at position N297 or position T299, wherein the substitution at position T299 is not T299S.

[0113] In one embodiment, the first Fc region and / or the second Fc region includes substitutions selected from the group consisting of N297A, N297G, N297Y, N297Q, N297D, N297S, N297T, T299A, and T299G.

[0114] In one embodiment, the first Fc region and / or the second Fc region include N297A substitution or N297G substitution. In one embodiment, the first Fc region and the second Fc region include N297A substitution. In one embodiment, the first Fc region and the second Fc region include N297G substitution. In one embodiment, the first Fc region and the second Fc region include N297Y substitution. In one embodiment, the first Fc region and the second Fc region include N297Q substitution. In one embodiment, the first Fc region and the second Fc region include N297D substitution. In one embodiment, the first Fc region and the second Fc region include N297S substitution. In one embodiment, the first Fc region and the second Fc region include N297T substitution. In one embodiment, the first Fc region and the second Fc region include T299A substitution. In one embodiment, the first Fc region and the second Fc region include T299G substitution.

[0115] In one embodiment, the first antibody and / or the second antibody do not contain N-linked glycosylation at any position of the antibody.

[0116] The following table provides a non-limiting list of embodiments describing combinations of a first polypeptide and a second polypeptide with specific substitutions. For example, Embodiment 1 in the first table below is a combination of a first antibody containing substitutions at positions corresponding to E430G and K439E and lacking glycosylation (Aglyc) at position N297, and a second antibody containing E430G and S440K substitutions. As described herein, all first and second antibodies in the enumerated embodiments may optionally contain further substitutions. TIFF0007880817000004.tif43128TIFF0007880817000005.tif219126TIFF0007880817000006.tif211131 TIFF0007880817000007.tif217131TIFF0007880817000008.tif217131TIFF0007880817000009.tif243154

[0117] In one embodiment of the present invention, the first antibody and / or the second antibody are human antibodies, humanized, or chimeric. In one embodiment of the present invention, the first antibody and the second antibody are human antibodies, humanized, or chimeric. In one embodiment of the present invention, the first antibody and the second antibody are human antibodies. In one embodiment of the present invention, the first antibody is a human antibody and the second antibody is humanized. In one embodiment of the present invention, the first antibody is humanized and the second antibody is a human antibody.

[0118] In one embodiment of the present invention, the first antibody and / or the second antibody are monoclonal antibodies. In one embodiment of the present invention, the first antibody and the second antibody are monoclonal antibodies. In one embodiment of the present invention, the first antibody and the second antibody are bispecific antibodies. In one embodiment, the first antibody is a monoclonal antibody and the second antibody is a bispecific antibody. In one embodiment, the first antibody is a bispecific antibody and the second antibody is a monoclonal antibody.

[0119] The embodiments of antibodies described herein should be understood to refer to antibodies comprising an immunoglobulin Fc region and an antigen-binding region. Antibodies may also be multispecific antibodies, for example, bispecific antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin and a second polypeptide or antibody having a second Fc region and a second antigen-binding region of an immunoglobulin.

[0120] In one aspect of the present invention, the first antibody and / or the second antibody are IgG1, IgG2, IgG3, or IgG4 subclasses. In one aspect of the present invention, the first antibody and the second antibody are IgG1, IgG2, IgG3, or IgG4 subclasses. In one aspect of the present invention, the first antibody and the second antibody are human IgG1, IgG2, IgG3, or IgG4 subclasses. In one aspect of the present invention, the first antibody and the second antibody are IgG1, IgG2, or IgG4 subclasses. In one aspect of the present invention, the first antibody and the second antibody are human IgG1, IgG2, or IgG4 subclasses. In one aspect of the present invention, the first antibody and / or the second antibody are IgG1 subclasses. In one aspect of the present invention, the first antibody and / or the second antibody are human IgG1 subclasses. In one aspect of the present invention, the first antibody and / or the second antibody are human IgG1 subclasses. In one aspect of the present invention, the first antibody and the second antibody are IgG1 subclasses. In one aspect, the first antibody and the second antibody are IgG2 subclasses. In one embodiment of the present invention, the first antibody and the second antibody are of the IgG4 subclass. In one embodiment of the present invention, the first antibody is of the IgG1 subclass and the second antibody is of the IgG2 subclass. In one embodiment of the present invention, the first antibody is of the IgG2 subclass and the second antibody is of the IgG1 subclass.

[0121] In one embodiment of the present invention, the first antibody comprises a heavy chain of the IgG1 subclass. In one embodiment of the present invention, the second antibody comprises a heavy chain of the IgG1 subclass. In one embodiment of the present invention, the first antibody comprises a heavy chain of the IgG2 subclass. In one embodiment of the present invention, the second antibody comprises a heavy chain of the IgG2 subclass. In one embodiment of the present invention, the first antibody comprises a heavy chain of the IgG3 subclass. In one embodiment of the present invention, the second antibody comprises a heavy chain of the IgG3 subclass. In one embodiment of the present invention, the first antibody comprises a heavy chain of the IgG4 subclass. In one embodiment of the present invention, the second antibody comprises a heavy chain of the IgG4 subclass.

[0122] In one embodiment of the present invention, the first antibody comprises a heavy chain of the IgG1 subclass, and the second antibody comprises a heavy chain of the IgG1 subclass.

[0123] In a preferred embodiment, the first antibody comprises a first Fc region of a human IgG1 subclass, and / or the second antibody comprises a second Fc region of a human IgG1 subclass.

[0124] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a first constant region and / or a second constant region, the first constant region including a sequence selected from Table 1.

[0125] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a first constant region and / or a second constant region comprising a sequence selected from the group consisting of SEQ ID NO: 42-53, 57, 58, 61, or 62.

[0126] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a first constant region and / or a second constant region containing a sequence selected from the group consisting of SEQ ID NO: 42-49, 61-62, 64-68, 79-86, 127-128, and 132-133.

[0127] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 42 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 43 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 44 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 45 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 46 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 47 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 48 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 49 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 61 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 62 or a first constant region and / or a second constant region.In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 64 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 65 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 66 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 67 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 68 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:79 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:80 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:81 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:82 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:83 or a first constant region and / or a second constant region.In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 84 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 85 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 86 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 127 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO: 128 or a first constant region and / or a second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:132 or the first constant region and / or the second constant region. In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region containing the sequence shown in SEQ ID NO:133 or the first constant region and / or the second constant region.

[0128] In one embodiment of the present invention, the first antibody and / or the second antibody include a first heavy chain constant region and / or a second heavy chain constant region, each containing a sequence selected from the group consisting of SEQ ID NO: 42-53, 57, 58, 61, or 62, wherein the first heavy chain sequence and the second heavy chain sequence are independently selected from the group.

[0129] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a first constant region and / or a second constant region comprising a sequence selected from the group consisting of SEQ ID NO: 24-53, 57, 58, 61, or 62, and is introduced with up to five further substitutions, e.g., up to four further substitutions, e.g., up to three further substitutions, e.g., up to two further substitutions, e.g., up to one further substitution.

[0130] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody includes a constant region or a first constant region and / or a second constant region containing a sequence selected from the group consisting of SEQ ID NO: 42-49, 61-62, 64-68, 79-86, 127-128, and 132-133, from which the C-terminal lysine has been removed. The C-terminal lysine may be removed from the expression sequence, for example from the nucleic acid vector, or it may be removed by post-translational modification, for example by carboxypeptidase treatment.

[0131] In one embodiment of the present invention, both the first antigen and the second antigen are molecules exposed on the cell surface. In one embodiment of the present invention, both the first antigen and the second antigen are cell surface-expressed molecules. In one embodiment, the first antigen and the second antigen are co-expressed in cells or tissues that are target cells or target tissues of the disease or disorder to be treated.

[0132] In one embodiment of the present invention, the first antigen and the second antigen are not identical.

[0133] In one embodiment, the combination of the first antibody and the second antibody depletes a population of cells that co-express the first antigen and the second antigen. In one embodiment, the combination of the first antibody and the second antibody depletes a population of cells that co-express the first antigen and the second antigen. In one embodiment, the combination of the first antibody and the second antibody induces cell death in a population of cells that co-express the first antigen and the second antigen.

[0134] In one embodiment, the cell population is a tumor cell population. In a further embodiment, the cell population is a hematological tumor cell population or a solid tumor cell population.

[0135] In one embodiment, the cell population is a population of white blood cells.

[0136] In one embodiment, the cell population is lymphocytes, for example, a population of lymphocyte cells.

[0137] In one embodiment, the cell population is a B cell population, a T cell population, an NK cell population, a regulatory T cell population, and a myeloid-derived suppressor cell population.

[0138] In one embodiment, the cell population is a B cell population, for example, a subset of a B cell population.

[0139] In one embodiment, the cell population is T cells, for example, a T cell population, for example, a subset of a T cell population. In one embodiment of the present invention, the cell population is regulatory T cells.

[0140] In one embodiment, the cell population is an NK cell population. In one embodiment, the cell population is a myeloid-derived suppressor cell population. In one embodiment, the cell population is a tumor-associated macrophage population.

[0141] This describes how the first and second antibodies according to the present invention are used as medical agents to deplete specific cell populations expressing the first and second antigens recognized by the first and second antibodies according to the present invention. Therefore, the first and second antibodies according to the present invention may be used to deplete tumor cells expressing the first and second antigens recognized by the first and second antibodies, but the first and second antibodies may not deplete healthy tissue that expresses only the first antigen or healthy tissue that expresses only the second antigen. The first and second antibodies according to the present invention may also be particularly useful to deplete specific cell populations of the immune system, for example, specific subsets of lymphocytes, for example, B cells or T cells, or even subsets of B cells or T cells. For applications where cell depletion is desired, the following mutations E345K and E430G may be particularly preferred.

[0142] In another aspect of the present invention, the combination of the first antibody and the second antibody induces proliferation in cell populations expressing the first antigen and the second antigen. This describes how the first and second antibodies according to the present invention are used as a medical agent to activate a specific cell population, that is, as a medical agent to activate a specific cell population by inducing proliferation of a specific cell population expressing the first and second antigens recognized by the first and second antibodies. For applications where an agonist effect is desired, the following mutations E345R, E430Y, and E430F may be particularly preferred.

[0143] In further key aspects, the present invention is Regarding an antibody comprising the Fc region of human IgG and an antigen-binding region capable of binding to an antigen, The aforementioned Fc region is a. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and b. Replacement with K439E or S440K The antibody contains, and does not contain N-linked glycosylation at position N297.

[0144] In one embodiment, the antibody comprises an amino acid substitution, deletion, or insertion that inhibits N-linked glycosylation at position N297.

[0145] In one embodiment, the Fc region includes an amino acid substitution at position N297 or position T299, wherein the substitution at position T299 is not T299S.

[0146] In one embodiment, the Fc region includes substitutions selected from the group consisting of N297A, N297G, N297Y, N297Q, N297D, N297S, N297T, T299A, and T299G.

[0147] In one embodiment, the antibody does not contain N-linked glycosylation at any position on the antibody.

[0148] In one embodiment, the Fc region is · K439E replacement and Y436N replacement, or · K439E replacement and Q438R replacement, or · Replacement of S440K and Y436N, or • Replacement of S440K and Q438R Includes.

[0149] In one embodiment, the Fc region includes substitutions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E430Y, E345Q, E345R, and E345Y, or a combination of substitutions K248E and T437R.

[0150] In one embodiment, the Fc region includes substitutions selected from the group consisting of E345K, E430G, E345R, E430Y, E345Q, E345Y, E430S, E430T, and E430F, and / or combinations of substitution K248E and T437R.

[0151] In one embodiment, the Fc region includes substitutions selected from the group consisting of E345K, E430G, E345R, and E430Y.

[0152] In one embodiment, the Fc region includes substitutions selected from the group consisting of E430G and E345K. In one embodiment, the Fc region includes substitutions selected from the group consisting of E345R and E430Y. In one embodiment, the Fc region includes the E345K substitution. In one embodiment, the Fc region includes the E430G substitution. In one embodiment, the Fc region includes the E345R substitution. In one embodiment, the Fc region includes the E430Y substitution. In one embodiment, the Fc region includes the E345Q substitution. In one embodiment, the Fc region includes the E345Y substitution. In one embodiment, the Fc region includes the E430S substitution. In one embodiment, the Fc region includes the E430T substitution. In one embodiment, the Fc region includes the E430F substitution. In one embodiment, the Fc region includes a combination of substitutions K248E and T437R.

[0153] In one embodiment, the Fc region includes one or more substitutions selected from the group consisting of E333S, K326A, E333A, and K326W. In one embodiment, the Fc region includes substitutions of E333S and K326A.

[0154] In one embodiment, the antibody is a human IgG1, IgG2, IgG3, or IgG4 subclass.

[0155] In one embodiment, the antibody is a human IgG1, IgG2, or IgG4 subclass. In one embodiment, the antibody is a human IgG1 subclass. In one embodiment, the antibody is a human IgG2 subclass. In one embodiment, the antibody is a human IgG4 subclass.

[0156] Target and method of use The first and second antibodies according to the present invention may bind to a target expressed on the same cell. In one embodiment, the target is a target that activates, inhibits, modulates, and / or modulates a signaling pathway.

[0157] Examples of targets that may be particularly suitable as targets according to the present invention are cell surface receptors and ligands. The following protein classes: tumor necrosis receptor superfamily, GPI anchored proteins, lipid-added proteins, hydrolase (EC3.) regulator superfamily, 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, activated leukocyte immunoglobulin-like receptors, CC-motif chemokine receptors, G protein-coupled receptors, Toll-like receptors, and receptor tyrosine kinases may also be particularly suitable as antigen-binding targets for the first and / or second antibodies according to the present invention. In one embodiment of the present invention, the first antigen-binding region and the second antigen-binding region can bind to a target antigen derived 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 a target antigen derived from a different protein class.

[0158] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen derived from the protein class of a GPI anchor protein, and the second antigen-binding region can bind to a target antigen derived from the protein class of a tetraspanin.

[0159] In one embodiment of the present invention, the first antigen-binding region can bind to a target antigen derived from the protein class of the GPI anchor protein, and the second antigen-binding region can bind to a target antigen derived from the protein class of the Membrane spanning 4-domains.

[0160] In one embodiment of the present invention, a first antigen-binding domain can bind to a target antigen derived from a protein class of the tumor necrosis receptor superfamily, and a second antigen-binding domain can bind to a target antigen derived from a protein class of the tumor necrosis receptor superfamily. In one embodiment of the present invention, the first antigen and / or the second antigen are members of TNFR-SF. In one embodiment of the present invention, the first antigen and the second antigen are members of TNFR-SF.

[0161] In one embodiment of the present invention, a first antigen-binding region can bind to a target antigen derived from a protein class of the tumor necrosis receptor superfamily, and a second antigen-binding region can bind to a target antigen derived from a protein class of the immunoglobulin superfamily.

[0162] Cell surface receptors include, for example, receptors belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. Various references are available regarding receptors belonging to these receptor families and their characteristics, for example: Cooke B A., King RJ B., van der Molen H J. ed. New Comprehensive Biochemistry Vol. 18B "Hormones and their Actions Part 2" pp. 1-46 (1988) Elsevier Science Publishers BV., New York, USA; Patthy L. (1990) Cell, 61: 13-14; Ullrich A., et al. (1990) Cell, 61: 203-212; Massagul J. (1992) Cell, 69: 1067-1070; Miyajima A., et al. (1992) Annu. Rev. Immunol., 10: 295-331; Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396; Fantl W I., et al. (1993) Annu. Rev. Biochem., 62: 453-481; Smith C A., et al. (1994) Cell, 76: 959-962; Flower D R. (1999) Biochim. Biophys. Acta, 1422: 207-234; and M. Miyasaka ed., Cell Technology, supplementary volume, Handbook series, including ``Handbook for Adhesion Factors'' (1994) (Shujunsha, Tokyo, Japan).

[0163] In one embodiment of the present invention, the antibody comprises an antigen-binding domain that binds to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) or the G protein-coupled receptor (GPCR) superfamily.

[0164] In one embodiment of the present invention, a first antibody and / or a second antibody bind to cell surface receptors, such as hormone receptors and cytokine receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, and differentiation regulatory factor receptors. Examples of cytokine receptors include erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony-stimulating factor (G-CSF) receptor, macrophage colony-stimulating factor (M-CSF) receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1) receptor, interleukin-2 (IL-2) receptor, interleukin-3 (IL-3) receptor, interleukin-4 (IL-4) receptor, interleukin-5 (IL-5) receptor, interleukin-6 (IL-6) receptor, interleukin-7 (IL-7) receptor, interleukin-9 (IL-9) receptor, interleukin-10 (IL-10) receptor, interleukin-11 (IL-11) receptor, and interleukin- These include IL-12 receptors, interleukin-13 receptors, interleukin-15 receptors, interferon-α (IFN-α) receptors, interferon-β (IFN-β) receptors, interferon-γ (IFN-γ) receptors, growth hormone (GH) receptors, insulin receptors, hematopoietic stem cell growth factor (SCF) receptors, vascular epidermal growth factor (VEGF) receptors, epidermal growth factor (EGF) receptors, nerve growth factor (NGF) receptors, fibroblast growth factor (FGF) receptors, platelet-derived growth factor (PDGF) receptors, transforming growth factor-β (TGF-β) receptors, leukocyte migration inhibitor (LIF) receptors, ciliary neurotrophic factor (CNTF) receptors, oncostatin M (OSM) receptors, and Notch family receptors.

[0165] The tumor necrosis factor receptor superfamily (TNFRSF) is a group of receptors characterized by their ability to bind to tumor necrosis factor superfamily (TNFSF) ligands via extracellular cysteine-rich domains. TNF receptors form trimer complexes in the plasma membrane. TNFRSF includes the following list of 29 proteins: TNFR1 (Uniprot P19438), FAS (Uniprot P25445), DR3 (Uniprot Q93038), DR4 (Uniprot O00220), DR5 (Uniprot O14763), DR6 (Uniprot O75509), NGFR (Uniprot P08138), EDAR (Uniprot Q9UNE0), DcR1 (Uniprot O14798), DcR2 (Uniprot Q9UBN6), DcR3 (Uniprot O95407), OPG (Uniprot O00300), TROY (Uniprot Q9NS68), XEDAR (Uniprot Q9HAV5), LTbR (Uniprot P36941), HVEM (Uniprot Q92956), TWEAKR (Uniprot Q9NP84), CD120b (Uniprot P20333), OX40 (Uniprot P43489), CD40 (Uniprot P25942), CD27 (Uniprot P26842), CD30 (Uniprot Q07011), RANK (Uniprot Q9Y6Q6), TACI (Uniprot O14836), BLySR (Uniprot Q96RJ3), BCMA (Uniprot Q02223), GITR (Uniprot Q9Y5U5), RELT (Uniprot Q969Z4).

[0166] In one embodiment of the present invention, the antibody, the first antibody and / or the second antibody include an antigen-binding region that can bind to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, and PD-1.

[0167] In one embodiment of the present invention, the antibody, the first antibody and / or the second antibody include an antigen-binding region capable of binding to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, PD-1, and FAS.

[0168] In one embodiment, the antigen-binding region can bind to DR4. In one embodiment, the antigen-binding region can bind to DR5. In one embodiment, the antigen-binding region can bind to CD20. In one embodiment, the antigen-binding region can bind to CD37. In one embodiment, the antigen-binding region can bind to CD52. In one embodiment, the antigen-binding region can bind to HLA-DR. In one embodiment, the antigen-binding region can bind to CD3. In one embodiment, the antigen-binding region can bind to CD5. In one embodiment, the antigen-binding region can bind to 4-1BB. In one embodiment, the antigen-binding region can bind to PD-1. In one embodiment, the antigen-binding region can bind to FAS. In one embodiment, the antigen-binding region can bind to members of TNFR-SF.

[0169] In one embodiment of the present invention, the antibody or the first antibody and / or the second antibody is The VH region includes the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10, and the CDR3 sequence shown in SEQ ID NO:11, as well as the VL region [CD20, 11B8] including the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown in DAS, and the CDR3 sequence shown in SEQ ID NO:14; The VH region includes the CDR1 sequence shown in SEQ ID NO:43, the CDR2 sequence shown in SEQ ID NO:44, and the CDR3 sequence shown in SEQ ID NO:45, as well as the VL region [CD37] including the CDR1 sequence shown in SEQ ID NO:47, the CDR2 sequence shown in VAT, and the CDR3 sequence shown in SEQ ID NO:48; The VH region includes the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, as well as the VL region [CD52, CAMPATH-1H] including the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown in NTN, and the CDR3 sequence shown in SEQ ID NO:7; The VH region includes the CDR1 sequence shown in SEQ ID NO:118, the CDR2 sequence shown in SEQ ID NO:119, and the CDR3 sequence shown in SEQ ID NO:120, as well as the VL region [CD52, h2E8] including the CDR1 sequence shown in SEQ ID NO:122, the CDR2 sequence shown in SEQ ID NO:123, and the CDR3 sequence shown in SEQ ID NO:124; The VH region includes the CDR1 sequence shown in SEQ ID NO:104, the CDR2 sequence shown in SEQ ID NO:105, and the CDR3 sequence shown in SEQ ID NO:106, as well as the VL region [Fas, Fas-E09] including the CDR1 sequence shown in SEQ ID NO:108, the CDR2 sequence shown in YNN, and the CDR3 sequence shown in SEQ ID NO:109; The VH region includes the CDR1 sequence shown in SEQ ID NO:111, the CDR2 sequence shown in SEQ ID NO:112, and the CDR3 sequence shown in SEQ ID NO:113, as well as the VL region [4-1BB, BMS-663513] including the CDR1 sequence shown in SEQ ID NO:115, the CDR2 sequence shown in DAS, and the CDR3 sequence shown in SEQ ID NO:116; The VH region includes the CDR1 sequence shown in SEQ ID NO:90, the CDR2 sequence shown in SEQ ID NO:91, and the CDR3 sequence shown in SEQ ID NO:92, as well as the VL region [DR5, hDR5-01-G56T] including the CDR1 sequence shown in SEQ ID NO:94, the CDR2 sequence shown in FAS, and the CDR3 sequence shown in SEQ ID NO:95; The VH region includes the CDR1 sequence shown in SEQ ID NO:97, the CDR2 sequence shown in SEQ ID NO:98, and the CDR3 sequence shown in SEQ ID NO:99, as well as the VL region [DR5, hDR5-05] including the CDR1 sequence shown in SEQ ID NO:101, the CDR2 sequence shown in RTS, and the CDR3 sequence shown in SEQ ID NO:102. It includes an antigen-binding region.

[0170] In a further aspect, the present invention A composition comprising a first antibody and a second antibody, The first antibody comprises a first antigen-binding region and a first Fc region according to any embodiment disclosed herein. A composition comprising a second antibody comprising a second antigen-binding region and a second Fc region according to any aspect or embodiment disclosed herein. Regarding.

[0171] In a further aspect, the present invention A composition comprising a first antibody and a second antibody, The first antibody comprises a first antigen-binding region capable of binding to a first antigen, and a first Fc region of human IgG; The second antibody comprises a second antigen-binding region capable of binding to a second antigen, and a second Fc region of human IgG; The first Fc region described above is a. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and b. Replacement with K439E or S440K Includes; The aforementioned second Fc region is c. Replacement at position E430, or replacement at position E345, or replacement of a combination of K248E and T437R, and d. Replacement with K439E or S440K Includes; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first antibody and / or the second antibody do not contain N-linked glycosylation at position N297; A composition in which the amino acid position corresponds to human IgG1 according to the Eu numbering system. Regarding.

[0172] In one embodiment, the first and second Fc regions of the antibody present in the composition include substitutions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E430Y, E345Q, E345R, and E345Y, or a combination of substitution K248E and T437R.

[0173] In one embodiment, the first antibody and the second antibody are present in the composition in a molar ratio of about 1:50 to 50:1, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1 It exists in molar ratios of 35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 50:1, approximately 45:1, approximately 40:1, approximately 35:1, approximately 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.

[0174] In another embodiment, the first antibody and the second antibody are present in the composition in a molar ratio of about 1:50 to 50:1, for example, a molar ratio of 1:40 to 40:1, for example, a molar ratio of 1:30 to 30:1, for example, a molar ratio of 1:20 to 20:1, for example, a molar ratio of 1:10 to 10:1, for example, a molar ratio of 1:9 to 9:1, for example, a molar ratio of 1:5 to 5:1, for example, a molar ratio of 1:2 to 2:1.

[0175] In one embodiment, the first antibody and the second antibody are present in the composition in a 1:1 molar ratio.

[0176] In one embodiment, the composition further comprises a pharmaceutical carrier or excipient. In one embodiment, the composition is a pharmaceutical composition.

[0177] therapeutic use A first antibody and a second antibody, antibody, or composition according to any aspect or embodiment of the present invention may be used as a medical agent, i.e., for therapeutic purposes.

[0178] Accordingly, in one aspect, the present invention provides a first antibody and a second antibody or composition for use as a medical agent, according to any aspect or embodiment disclosed herein.

[0179] In another aspect, the present invention provides antibodies or compositions for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, or infections, according to any aspect or embodiment disclosed herein.

[0180] In another aspect, the present invention is A method for treating an individual having a disease, comprising the step of administering to the individual an effective amount of a first antibody and a second antibody or composition according to any aspect or aspect disclosed herein. Regarding this, in one embodiment, the disease is selected from the group of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0181] In one embodiment of the present invention, the method includes the step of administering a further therapeutic agent.

[0182] In one embodiment of the present invention, further therapeutic agents include, but are not limited to, chemotherapeutic agents (paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, and pemetrexed), kinase inhibitors (including, but are not limited to, sorafenib, sunitinib, or everolimus), apoptosis modulators (including, but are not limited to, recombinant human TRAIL or blinapant), RAS inhibitors, and proteasome inhibitors (including 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), nutritional supplements, 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.

[0183] In a further aspect, the present invention A method for depleting a cell population expressing a first antigen and a second antigen, comprising the step of contacting the cell population with a first antibody and a second antibody or composition according to any aspect or aspect disclosed herein. Regarding.

[0184] In one embodiment, the cell population is a tumor cell population, for example, a hematological tumor cell population or a solid tumor cell population.

[0185] In a further aspect, the present invention A method for inducing proliferation in a cell population expressing a first antigen and a second antigen, comprising the step of contacting the cell population with a first antibody and a second antibody according to the present invention or a composition according to the present invention. Regarding.

[0186] In one embodiment of the above method, the cell population is present in the blood. In one embodiment, the cell population is leukocytes, for example, a population of leukocyte cells. In one embodiment, the cell population is a subset of the population of leukocyte cells. In one embodiment, the cell population is a population of lymphocyte cells. In one embodiment, the cell population is a population of B cells. In one embodiment of the present invention, the cell population is a subset of the population of B cells. In one embodiment, the cell population is a population of T cells. In one embodiment, the cell population is a subset of the population of T cells. In one embodiment, the cell population is regulatory T cells, for example, a population of regulatory T cells. In one embodiment, the cell population is a population of NK cells. In one embodiment, the cell population is myeloid-derived suppressor cells.

[0187] Kit of Parts The following embodiments relating to the first and second antibodies should be understood to refer to antibodies that include the Fc region and antigen-binding region of an immunoglobulin.

[0188] The present invention also relates to a kit of parts for simultaneous, separate, or sequential use in a therapy comprising the first antibody and the second antibody described herein. Furthermore, such first and second antibodies can be obtained according to any method described herein.

[0189] In one aspect, the present invention relates to a kit of parts comprising an antibody or composition according to any aspect or embodiment described herein, wherein the first antibody and the second antibody or composition are contained in one or more containers such as vials.

[0190] In one embodiment, the kit of parts comprises a first antibody and a second antibody or composition, according to any aspect or embodiment described herein, for use simultaneously, separately, or sequentially in a therapy.

[0191] In another aspect, the present invention relates to the use of a first antibody and a second antibody, composition, or kit of parts in accordance with any aspect described herein for use in a diagnostic method.

[0192] In another aspect, the present invention relates to a diagnostic method comprising the step of administering a first antibody and a second antibody, composition, or kit of parts, according to any embodiment described herein, to at least a portion of the body of a human or other mammal.

[0193] In another aspect, the present invention relates to the use of a first antibody and a second antibody, composition, or kit of parts in the imaging of at least a portion of the body of a human or other mammal, according to any embodiment described herein.

[0194] In another aspect, the present invention relates to a method for imaging at least a portion of the body of a human or other mammal, comprising the step of administering a first antibody and a second antibody, composition, or kit of parts according to any embodiment described herein.

[0195] Dosage The effective dosage and administration plan of the antibody depend on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary and 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 mg / kg, for example, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg.

[0196] As described above, the antibodies of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition to be treated. Accordingly, in one embodiment, the medical agent containing the antibodies is intended to be combined with one or more further therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents. Such combination administrations may be performed simultaneously, separately, or sequentially.

[0197] In a further embodiment, the present invention provides a method for treating or preventing a disease, such as cancer, comprising the step of administering a therapeutically effective amount of a variant or pharmaceutical composition of the present invention to a subject in need of treatment or prevention of a disease, such as cancer, in combination with radiotherapy and / or surgery.

[0198] Preparation method The present invention also provides isolated nucleic acids and vectors encoding antibodies that conform to any one of the aforementioned aspects, as well as recombinant host cells capable of producing antibodies. Nucleic acid constructs, vectors, and host cells suitable for antibodies and their variants are known in the art and are described in the examples. In embodiments in which the variant antibody includes not only a heavy chain (or its Fc-containing fragment) but also a light chain, the nucleotide sequence encoding the heavy chain portion and the nucleotide sequence encoding the light chain portion may be located on the same nucleic acid or vector, or on different nucleic acids or vectors.

[0199] Accordingly, in a further aspect, the present invention relates to nucleic acids encoding antibodies or a first antibody or a second antibody according to the present invention, such as isolated nucleic acids.

[0200] Furthermore, the present invention relates to a nucleic acid, such as an isolated nucleic acid, that encodes the heavy chain of an antibody according to the present invention or a first antibody or a second antibody. The nucleic acid may be used in combination with a nucleic acid that encodes the light chain.

[0201] In a further aspect, the present invention provides an expression vector comprising nucleic acids according to the present invention, or a combination of such nucleic acids.

[0202] In a further aspect, the present invention provides a delivery vehicle comprising a nucleic acid or a combination thereof according to the present invention, optionally in a composition comprising a pharmaceutically acceptable carrier. In one embodiment, the delivery vehicle is a particle. The present invention is A method for producing an antibody in a host cell that conforms to any one of the aforementioned aspects, comprising the following steps: (a) A step of preparing a nucleotide construct encoding the antibody; (b) A step of expressing the nucleotide construct in a host cell; and (c) A step of recovering the antibody from the cell culture of the host cells. Methods including We also offer it.

[0203] In some embodiments, the antibody is a heavy-chain antibody. However, in most embodiments, the antibody also contains a light chain, and therefore the host cell further expresses a construct encoding the light chain on the same vector or on a different vector.

[0204] Host cells suitable for recombinant antibody expression are well known in the art and include CHO, HEK-293, Expi293, PER-C6, NS / 0, and Sp2 / 0 cells.

[0205] In one embodiment, the host cell is one that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97: 2426 (incorporated herein by reference) lists numerous such antibody-producing systems, e.g., Sp2 / 0, NS / 0, or transgenic mammary gland (goat), which only achieve partial removal of the C-terminal lysine.

[0206] As described above, the antibody of the present invention lacks N-linked glycosylation at position N297. In some embodiments, this can be achieved by mutating the antibody sequence such that the acceptor site around position N297 is no longer functional. In such embodiments, the host cell may be a cell capable of N-linked glycosylation of proteins, such as a eukaryotic cell, such as a mammalian cell, such as a human cell.

[0207] In another aspect, the present invention relates to a method for producing an antibody according to the present invention, comprising the step of producing the antibody in recombinant host cells in which the asparagine at position 297 of the antibody cannot be glycosylated. This may be preferred, for example, when the sequence around position N297 is not mutated. Such host cells may be prokaryotic cells, such as bacterial cells.

[0208] Therefore, in one aspect, the present invention is A method for producing an antibody in a host cell that conforms to any one of the aforementioned aspects, comprising the following steps: (a) A step of preparing a nucleotide construct encoding the antibody; (b) A step of expressing the nucleotide construct in a host cell capable of glycosylation of asparagine at position 297 of the antibody; and (c) A step of recovering the antibody from the cell culture of the host cells. Methods including To provide.

[0209] In a further aspect, the present invention A method for producing an antibody according to the present invention, comprising the steps of: producing an antibody in recombinant host cells capable of glycosylation of asparagine at position N297 of the antibody; and subsequently removing N-linked glycosylation from the produced antibody, for example, by enzymatic removal, for example, by using the deglycosylating enzyme PNGase F. Regarding.

[0210] The present invention also relates to antibodies obtained or available by the methods of the present invention described above.

[0211] (Table 1) TIFF0007880817000010.tif32156TIFF0007880817000011.tif219156TIFF0007880817000012.tif221156TIFF0007880817000013.ti f222156TIFF0007880817000014.tif222156TIFF0007880817000015.tif222156TIFF0007880817000016.tif222156TIFF00078808170 00017.tif220156TIFF0007880817000018.tif210156TIFF0007880817000019.tif212156TIFF0007880817000020.tif212156TIFF000 7880817000021.tif223156TIFF0007880817000022.tif220156TIFF0007880817000023.tif222156TIFF0007880817000024.tif140156

[0212] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. [Examples]

[0213] Example 1 Antibody expression construct To express the human antibodies and humanized antibodies used herein, the sequences of the variable heavy (VH) chain and variable light (VL) chain were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific) and cloned into a pcDNA3.3 expression vector (ThermoFisher Scientific) containing the constant region of the human IgG heavy chain (HC) (constant region human IgG1m(f)HC: SEQ ID NO 59) and / or the constant region of the human κ light chain (LC): SEQ ID NO 60. Desired mutations were introduced by gene synthesis. The CD20 antibody variant in this application has the VH and VL sequences (WO2004 / 035607; VH: SEQ ID NO 15; VL: SEQ ID NO 19) derived from the previously described CD20 antibody IgG1-CD20-11B8. The CD52 antibody variant in this application has VH and VL sequences derived from the previously described CD52 antibody CAMPATH-1H (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 8; VL: SEQ ID NO 12). 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 22; VL: SEQ ID NO 26). The Fas antibody variant in this application has VH and VL sequences derived from the previously described Fas antibody Fas-E09 (Chodorge et al., Cell Death Differ. 2012 Jul;19(7):1187-95; VH: 103; VL: 107).The DR5 antibody variant in this application has VH and VL sequences derived from the previously described DR5 antibody DR5-01-G56T (WO2017 / 093447; VH: SEQ ID NO 89; VL: SEQ ID NO 93) and VH and VL sequences derived from DR5-05 (WO2014 / 009358; VH: SEQ ID NO 96; VL: SEQ ID NO 100). The 4-1BB antibody variant in this application has VH and VL sequences derived from the previously described 4-1BB antibody BMS-663513 (US2005 / 0095244A1; VH: SEQ ID NO 110; VL: SEQ ID NO 114).

[0214] Transient expression antibody construct A plasmid DNA mixture encoding both the heavy and light chains of the antibody was transiently transfected into human Expi293F cells (Gibco, catalog number A14635) using 293fectin (Life Technologies), essentially as described by Vink et al. (Vink et al., 2014 Methods 65(1):5-10). The antibody concentration in the supernatant was measured by absorbance at 280 nm. The supernatant containing the antibody was used directly in the in vitro assay, or the antibody was purified as described below.

[0215] Antibody purification and quality assessment The antibody was purified by protein A affinity chromatography. The culture supernatant was filtered through a 0.20 μM dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), washed, and eluted with 0.02 M sodium citrate-NaOH, pH 3. Immediately after purification, the eluate was loaded onto a HiPrep Desalting column (GE Healthcare), and the antibody was buffer-exchanged in 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B. Braun or Thermo Fisher). After buffer exchange, the sample was filtered through a 0.2 μm dead-end filter. The purified protein was analyzed by numerous biochemical assays, including capillary electrophoresis and high-speed size exclusion chromatography (HP-SEC) on a sodium dodecyl sulfate-polyacrylamide gel (CE-SDS). The concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2–8°C.

[0216] Example 2: A deglycosylated antibody variant with an Fc-Fc interaction-enhancing mutation retains potent CDC activity. Glycosylation of antibody residue N297 positively impacts the potential involvement of antibodies in effector functions mediated by FcγR and complement. Complement activation may benefit from the contribution of N297-binding glycans to protein-protein Fc-Fc interactions. Therefore, deglycosylation of antibody residue N297 may suppress Fc-Fc interactions and thus inhibit antibody complex formation. Here, we investigated whether the loss of CDC efficacy in Wien 133 cells by anti-CD52 and anti-CD20 antibody variants after enzymatic deglycosylation could be restored by introducing Fc-Fc interaction-enhancing mutations.

[0217] Anti-CD52 IgG1-CAMPATH-1H antibody and anti-CD20 IgG1-11B8 antibody were introduced with different mutations: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions. To allow direct comparison between the concentrations of the individual components described in Examples 3 and 4 and the concentrations of the mixtures composed of those individual components, one antibody was mixed 1:1 with the unbound isotype control antibody IgG1-b12. Deglycosylation was performed using the Remove-iT® PNGase F kit (New England Biolabs, catalog number P0706S), which contains an amidase that cleaves between the asparagine residue in the N-linked glycoprotein and the innermost GlcNAc of the high-mannose hybrid complex oligosaccharide. First, 1 μl of Remove-iT PNGase F was added per 10 μg of each antibody variant, followed by incubation at 37°C overnight. Next, 50 μl of Chitin Magnetic Beads (New England Biolabs, catalog number E8036S) was pipetteed into a reaction vial and placed in a DynaMag™-2 magnetic separation rack (Invitrogen, catalog number 12321D). After the chitin beads were attracted to the magnet, the supernatant was pipetteed and discarded. With the vial still on the magnetic separation rack, the magnetic chitin beads were washed twice with 500 μl of PBS (Braun, catalog number 182478082). The supernatant was pipetteed and discarded. Then, the deglycosylated glycoprotein sample was added to the vial along with the magnetic chitin beads, thoroughly vortexed, and the sample was shaken at RT for 1 hour. The vial was returned to the magnetic separation rack to attract the chitin beads to the magnet. The supernatant was pipetteed and transferred to a new vial. To remove any remaining magnetic beads, the supernatant in the new vial was returned to the magnet, and the supernatant was pipetted for further use.

[0218] To confirm the glycosylation status of enzyme-treated antibody samples and controls, all samples were analyzed by mass spectrometry using an Orbitrap Q Exactive plus (Thermo Scientific). Enzymatically deglycosylated antibody samples, mock antibody samples (by omitting the PNGase F enzyme during this procedure), and untreated control samples were diluted to a concentration of 0.2 μg / mL in PBS (B. Braun, catalog number 3623140). 1 μL of 1M DTT (Sigma, catalog number D9163-5G) was added to 10 μl of each sample, followed by vortexing and brief centrifugation. After incubation at 37°C for 1 hour, all samples were vortexed, briefly centrifuged, and then acquired using the Orbitrap Q Exactive plus. Data were analyzed using Genedata Expressionist software.

[0219] Mass spectrometry analysis of enzymatically deglycosylated samples and untreated samples of wild-type IgG1-CAMPATH-1, IgG1-CAMPATH-1H-E430G-K439E (SEQ ID NO 57), and IgG1-CAMPATH-1H-E430G-S440K (SEQ ID NO 58) revealed that the treated samples were deglycosylated with high efficiency (<1% residual glycosylation; Figure 1A, B, C). Furthermore, Table 2 shows an overview of the glycosylation status of IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants that were enzymatically deglycosylated and used in Examples 2, 3, and 4, and which possess Fc-Fc interaction-enhancing mutations and autooligomerization-inhibiting mutations.

[0220] (Table 2) Glycosylation status of antibody variants tested in Examples 2, 3, and 4 after deglycosylation by enzyme. TIFF0007880817000025.tif128128

[0221] In an in vitro CDC assay on Wien133 cells using 20% ​​NHS, deglycosylated samples were tested at a range of purified antibody concentrations (final concentration range 0.004–10.0 μg / mL; 3-fold dilution). For the CDC assay, 0.1 x 10⁶ Wien133 cells (provided courtesy of Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK) co-expressing CD20 and CD52 antigens were placed in a polystyrene round-bottom 96-well plate (Greiner Bio-One catalog no. 650101) with a total volume of 80 μL in RPMI (Lonza, catalog no. BE12-115F) containing 0.2% bovine serum albumin (BSA; Roche, catalog no. 10735086001). 6 Cells were pre-incubated with purified antibody in concentration series on a shaker for 15 minutes at RT. Next, 20 μL of normal human serum (NHS; Sanquin, reference no. M0008) was added as a complement source, and the mixture was incubated in a 37°C incubator for 45 minutes (20% final NHS concentration). After stopping the reaction by placing the plate on ice, the cells were pelleted by centrifugation, and the supernatant was replaced with 30 μL of 2 μg / mL propidium iodide solution (PI; Sigma Aldrich, catalog no. P4170). The number of PI-positive cells was determined by flow cytometry using an Intellicyt iQue screener (Westburg). Percent lysis was calculated as (number of PI-positive cells / total number of cells) x 100%. Therefore, the CDC assay performed according to the above method will henceforth be called the PI CDC assay. The area under the dose-response curve for three repeated experiments was calculated using logarithmically transformed concentrations in GraphPad PRISM. The relative area under the curve (AUC) values ​​were normalized against the AUC value measured for the unbound negative control IgG1-b12 (0%) and the AUC value measured for the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0222] The wild-type antibody IgG1-CAMPATH-1H induced CDC with intermediate potency on Wien133 cells compared to a positive control mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (both containing SEQ ID NO 54) (Figure 1D). Deglycosylation of the wild-type IgG1-CAMPATH-1H antibody (indicated as "deg" in the figure) reduced the CDC potency to background levels. In contrast, (mock-treated) samples of the same antibody, processed using the same procedure without the addition of deglycosylase, showed comparable CDC potency to the untreated antibody. The introduction of Fc-Fc interaction-enhancing mutants E430G, E345K (SEQ ID NO 55), or E345R (SEQ ID NO 56) significantly increased the CDC potency of IgG1-CAMPATH-1H. The mock-treated IgG1-CAMPATH-1H-E430G antibody variant induced CDC with similar potency to the untreated IgG1-CAMPATH-1H-E430G antibody variant. Interestingly, the CDC potency of the Fc-Fc interaction-enhanced antibody variants IgG1-CAMPATH-1H-E430G and IgG1-CAMPATH-1H-E345K was only slightly impaired by deglycosylation. The CDC potency of IgG1-CAMPATH-1H-E345R was unaffected by deglycosylation. At the highest IgG concentrations tested, the deglycosylated IgG1-CAMPATH-1H variant with the Fc-Fc interaction-enhanced mutation induced CDC with comparable potency to its glycosylated counterpart (Figure 1E). In contrast, the CDC ability of IgG1-11B8-E430G was sharply reduced after deglycosylation (Figure 1). This suggests that the CDC efficacy of deglycosylated antibodies with Fc-Fc interaction-enhancing mutations may be affected by the antibody target or epitope. As shown in Figures 4G and 4H (described in Example 5), wild-type IgG1-11B8 did not induce detectable CDC on Wien133 cells, so the effect of deglycosylation on this antibody could not be evaluated.

[0223] These data suggest that deglycosylation negatively affected the CDC efficacy of the wild-type antibody IgG1-CAMPATH-1H, while it had little to no effect on the CDC efficacy of IgG1-CAMPATH-1H antibody variants with Fc-Fc interaction-enhancing mutations.

[0224] Example 3: Deglycosylation increases the selectivity of antibody variants containing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. When two antibodies containing Fc-Fc interaction-enhancing mutations also contain a further auto-oligomerization-inhibiting mutation (either K439E or S440K), a mixture of such antibodies can induce lysis of cells expressing the two targets to which these antibodies are directed. While the activity of such a codependent antibody combination was high on cells expressing both targets, the individual Campath-derived components still showed considerable activity. Therefore, undesirable activity may be induced on cells expressing one of the two targets (Figure 2A). Here, we investigated whether deglycosylation of anti-CD52 and anti-CD20 antibody variants, which have Fc-Fc interaction-enhancing mutations in addition to auto-oligomerization-inhibiting mutations, could improve the selectivity of such a codependent antibody mixture by reducing the mono-drug CDC activity of the individual antibody variants.

[0225] Anti-CD52 IgG1-CAMPATH-1H antibody and anti-CD20 IgG1-11B8 antibody were introduced with different mutations: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions; and K439E or S440K, which inhibit the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions. As a control, one antibody was mixed 1:1 with an unbound isotype control antibody IgG1-b12 to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of individual components. Deglycosylation was performed using the Remove-iT® PNGase F kit (New England Biolabs), essentially as described in Example 2. Essentially as described in Example 2, deglycosylated samples were tested in an in vitro CDC assay on Wien133 cells using 20% ​​NHS at a range of purified antibody concentrations (final concentration range 0.0023 to 10.0 μg / mL; 3-fold dilution). The percentage of cell lysis was calculated as (number of PI-positive cells / total number of cells) x 100%. The area under the dose-response curve for three repeated experiments was calculated using logarithmically transformed concentrations in GraphPad PRISM. The relative area under the curve (AUC) values ​​were normalized against the AUC value measured for the unbound negative control IgG1-b12 (0%) and the AUC value measured for the positive control mixture IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0226] On Wien133 cells expressing both CD20 and CD52, the Fc-Fc-enhanced antibody variant IgG1-CAMPATH-1H-E430G demonstrated potent CDC efficacy close to that induced by the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (Figure 2A). While the introduction of the auto-oligomerization inhibitory mutation K439E into IgG1-CAMPATH-1H-E430G only partially suppressed its CDC efficacy as a single agent, deglycosylation of this antibody variant completely suppressed its ability to induce CDC. The antibody variant IgG1-CAMPATH-1H-E430G-S440K exhibits high residual activity when used as a single agent. Deglycosylation of this antibody variant reduced its CDC efficacy to only about 4% of the level induced by the glycosylated variant. Even at the highest concentration tested (10 μg / ml), the deglycosylated IgG1-CAMPATH-1H-E430G-S440K antibody variant induced CDC at near-background levels, while the glycosylated counterpart induced CDC at levels close to those induced by glycosylated IgG1-CAMPATH-1H-E430G (Figure 2B).

[0227] A mixture of IgG1-CAMPATH-1H-E430G-K439E and IgG1-CAMPATH-1H-E430G-S440K restored CDC efficacy to the level induced by IgG1-CAMPATH-1H-E430G. Surprisingly, while single-agent activity was lost, no significant reduction in CDC efficacy was observed in the case of deglycosylated mixtures of IgG1-CAMPATH-1H-E430G-K439E or IgG1-CAMPATH-1H-E430G-S440K. Furthermore, while mixtures of both antibody variants that were only deglycosylated showed a slight reduction in CDC efficacy compared to mixtures of glycosylated variants, at the highest concentrations tested, the ability of the deglycosylated IgG1-CAMPATH-1H antibody variant mixture to induce CDC was equivalent to that of the glycosylated antibody variant mixture (Figure 2B).

[0228] The effect of deglycosylation on CDC efficacy in Wien133 cells was also evaluated using a mixture of IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing either the E430G mutation or either the K439E or S440K mutation. A potent CDC efficacy was observed in Wien133 cells for the positive control mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (Figure 2C). A mixture in which both antibody variants were deglycosylated showed a reduced ability to induce CDC to approximately 80% of the AUC measured for the glycosylated antibody variants. However, at the highest concentrations tested, cell lysis induced by the deglycosylated antibody variants was close to that of the glycosylated variants (Figure 2D). As previously shown in Figure 2A, introducing the auto-oligomerization inhibitory mutation K439E into IgG1-CAMPATH-1H-E430G only partially suppressed its CDC efficacy as a single agent, whereas deglycosylation of this antibody variant completely suppressed its ability to induce CDC (Figure 2C). The antibody variant IgG1-11B8, which contains the introduced mutations E430G and S440K, did not induce CDC as a single agent, nor did it induce a deglycosylation variant of this antibody. CDC efficacy could be restored by a mixture of the codependent IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants. Deglycosylation of either the IgG1-CAMPATH-1H-E430G-K439E or IgG1-11B8-E430G-S440K antibody variant in this mixture reduced CDC efficacy by 14% and 4%, respectively. However, in both cases, maximum cell lysis at the highest concentration tested was comparable to that of the glycosylated antibody variant mixture (Figure 2D). When both deglycosylated antibody variants were mixed in a 1:1 ratio, CDC efficacy was reduced by approximately 35% compared to the glycosylated variant mixture (Figure 2C). At the highest concentration tested for the mixture containing both deglycosylated antibody variants, maximum cell lysis decreased from 87% lysis observed for the fully glycosylated mixture to approximately 74% lysis observed for the fully deglycosylated mixture (Figure 2D).

[0229] In combination, these data indicate that deglycosylation of IgG1-CAMPATH-1H antibody variants containing the Fc-Fc interaction-enhancing mutation E430G and the auto-oligomerization-inhibiting mutation K439E or S440K reduces the single-agent CDC efficacy on Wien133 cells. CDC efficacy could be efficiently restored to a level close to that induced by a mixture of glycosylated counterparts by mixing the two deglycosylation-dependent antibody variants. While the single-agent activity of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K was completely suppressed by deglycosylation, mixtures of these antibody variants restored potent CDC efficacy, albeit at a lower level than non-selective glycosylated antibody counterparts.

[0230] Example 4: Recombinant glycan removal increases the selectivity of antibody variants containing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. In Example 3, it was explained that when an antibody variant having both an Fc-Fc interaction-enhancing mutation and an auto-oligomerization-inhibiting mutation is deglycosylated enzymatically, the single-drug CDC activity is reduced, but CDC can be (partially) restored by mixing such deglycosylated antibody variants. The CH2 domain of IgG antibodies contains a highly conserved N-glycosylation site at amino acid position N297. Here, the inventors tested whether the same effects observed for the enzyme-deglycosylated antibody variant could be observed with an unglycosylated antibody variant containing the N297Q mutation.

[0231] Unglycosyl antibodies were obtained by introducing different mutations into anti-CD52 IgG1-CAMPATH-1H antibodies and anti-CD20 IgG1-11B8 antibodies: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions; K439E or S440K, which inhibit the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions; and N297Q, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]). As a control, one antibody was also mixed 1:1 with an unbound isotype control antibody IgG1-b12 to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of the individual components. Essentially as described in Example 2, glycosyl-free samples were tested in an in vitro CDC assay on Wien133 cells using 20% ​​NHS at a range of purified antibody concentrations (final concentration range 0.002–40 μg / mL; 3-fold dilution). The percentage of cell lysis was calculated as (number of PI-positive cells / total number of cells) x 100%. The area under the dose-response curve for three repeated experiments was calculated using logarithmically transformed concentrations in GraphPad PRISM. The relative area under the curve (AUC) values ​​were normalized against the AUC value measured for the unbound negative control IgG1-b12 (0%) and the AUC value measured for the positive control mixture IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0232] A mixture of the positive control IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G demonstrated potent CDC efficacy on Wien133 cells (Figure 3A). Similar to what was observed after enzymatic deglycosylation of the antibody variant IgG1-CAMPATH-1H-E430G-K439E (Example 3), this antibody variant (SEQ ID NO 61), made unglycosyl by introducing the mutation N297Q, lost its ability to induce CDC as a single agent (Figure 3A). CDC could be partially restored to approximately 65% ​​of the level induced by the positive control mixture by mixing IgG1-CAMPATH-1H-E430G-K439E-N297Q with IgG1-11B8-E430G-S440K. At the highest concentrations tested, the latter mixture induced efficient cell lysis to approximately 97% of the cell lysis level induced by the positive control mixture (Figure 3B). CDC efficacy could also be partially restored to approximately 45% of the level induced by the positive control mixture by mixing two glycosyl antibody variants: IgG1-CAMPATH-1H-E430G-K439E-N297Q and IgG1-11B8-E430G-S440K-N297Q (SEQ ID NO 62). In contrast, IgG1-11B8-E430G-S440K-N297Q did not exhibit the ability to induce CDC (Figure 3A). A mixture of IgG1-CAMPATH-1H-E430G-K439E-N297Q and IgG1-11B8-E430G-S440K-N297Q induced cell lysis, and at the highest concentration tested, the efficiency was approximately 90% of the cell lysis level induced by the positive control mixture (Figure 3B).

[0233] In summary, these data indicate that introducing the N297Q mutation, which leads to the deglycosylation of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants via recombination, and which also possess Fc-Fc interaction-enhancing and auto-oligomerization-inhibiting mutations, weakens their ability to induce CDC on Wien133 cells, similar to the loss of CDC efficacy when enzymatically deglycosylated. CDC-induced cell lysis could be restored by mixing two codependent antibody variants, one or both of which possess Fc-Fc interaction-enhancing and auto-oligomerization-inhibiting mutations, when they were deglycosylated.

[0234] Example 5: The presence of Fc-Fc interaction-enhancing mutations promotes CDC by nonglycosyl anti-CD52 antibody variants and anti-CD20 antibody variants. In Example 2, it was shown that enzymatic deglycosylation suppresses the CDC activity of wild-type anti-CD52 IgG1-CAMPATH-1H on Wien133 cells. Here, we investigated the effects of introducing different mutations at residues N297 and T299 of anti-CD52 IgG1-CAMPATH-1H antibody variants and anti-CD20 IgG1-11B8 antibody variants with Fc-Fc interaction-enhancing mutations on CDC activity on Ramos lymphoma cells and Wien133 lymphoma cells.

[0235] Different mutations were introduced into anti-CD52 IgG1-CAMPATH-1H antibodies and anti-CD20 IgG1-11B8 antibodies: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions; and N297A, N297G, N297Q, N297D, N297Y, or T299A, which disrupt the N297 N-glycosylation consensus sequence (NX[S / T]) and produce non-glycosyl antibodies. Using the in vitro CellTiterGlo CDC assay detailed below, purified antibody variants were tested in Ramos cells (N=3) co-expressing CD20 and CD52 antigens in the presence of 10% NHS, or in Wien133 cells (N=3) in the presence of 20% NHS using the in vitro PI CDC assay described in Example 2, at a range of concentrations (final concentration range 0.0088 to 40.0 μg / mL; 3.33-fold dilution).

[0236] For the CellTtiterGlo CDC assay, 50x10 oz. of RPMI-1620 (Thermo, catalog number A1049101) containing 10% heat-inactivated FBS (Thermo, catalog number 10082147) was placed in a polystyrene flat-bottom 96-well plate with opaque walls (Corning, catalog number 3917) with a total volume of 80 μL. 3100 Ramos cells (ATCC, catalog no. CRL-1596) were pre-incubated on ice for 15 minutes with concentration-series purified antibodies. Next, 20 μL of normal human serum (diluted 1:1 with medium) (Complement Technologies, NHS Lot No. 42) was added as a complement source, and the plates were incubated in a 37°C incubator for 45 minutes (10% final NHS concentration). CellTiterGlo® reagent (Promega, catalog no. G7570) was added to all wells (50 μL / well) and thoroughly mixed using a pipette to induce cell lysis. After incubating the plates in the dark for 20 minutes to stabilize the luminescence signal, the luminescence was recorded using a microplate reader (Tecan Spark 20M). In the CellTiterGlo CDC assay, luminescence is detected by ATP-dependent luciferase-mediated luciferin conversion, which is proportional to the amount of luminescence released from viable cells remaining after incubation when the CellTiterGlo reagent is added. Maximum cytotoxicity was determined using a positive control where the remaining ATP was minimized and therefore the luminescence value was minimized as a result of lysing cells with 0.02% Triton X-100 solution (1X lysis buffer from Thermo LDH Cytotoxicity Assay Kit catalog number 88953 diluted to 1X). Minimum cytotoxicity (negative control) was determined using cells incubated with serum in the absence of IgG, where the remaining ATP was maximized and therefore the luminescence value was maximized. The luminescence values ​​were converted to percentage cytotoxicity as follows: Cytotoxicity (%)=100% * (1 - (Luminescence of test sample - Luminescence of positive control Triton X-100) / (Luminescence of negative control without antibody - Luminescence of positive control Triton X-100) .

[0237] For CellTiterGlo and PI CDC assays, the relative area under the dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%).

[0238] Wild-type IgG1-CAMPATH-1H antibody induced efficient CDC in Ramos cells. This was further enhanced by introducing E430G, E345K, or E345R mutations (Figure 4A). IgG1-CAMPATH-1H antibody variants with Fc-Fc interaction-enhancing mutations, including glycosylation site mutations N297A (E430G-N297A: SEQ ID NO 29; E345K-N297A: SEQ ID NO 35; E345R-N297A: SEQ ID NO 39), N297G (E430G-N297G: SEQ ID NO 30; E345K-N297G: SEQ ID NO 36; E345R-N297G: SEQ ID NO 40), and N297Q (E430G-N297Q: SEQ ID NO 31; E345K-N297Q: SEQ ID NO 37; E345R-N297Q: SEQ ID NO Introducing any of the following variants—41), N297D (E430G-N297D: SEQ ID NO 32), N297Y (E430G-N297Y: SEQ ID NO 33), or T299A (E430G-T299A: SEQ ID NO 34; E345K-T299A: SEQ ID NO 38)—slightly suppressed CDC activity, but all non-glycosyl variants retained potent CDC activity. At the highest antibody concentration tested (40 μg / mL), all tested IgG1-CAMPATH-1H antibody variants induced similar levels of high-efficiency CDC activity (Figure 4B).

[0239] Wild-type IgG1-CAMPATH-1H induced CDC with lower efficiency on Wien133 cells than on Ramos cells. However, introducing an aglycosyl mutation into an IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation yielded similar results on Wien133 cells compared to Ramos cells. In short, introducing an Fc-Fc interaction-enhancing mutation strongly increased the CDC activity of IgG1-CAMPATH-1H (Figure 4C). The aglycosyl mutations N297A, N297G, N297Q, N297D, N297Y, or T299A only partially suppressed CDC activity as measured by the AUC of the dose-response curve. In contrast, at the highest antibody concentrations tested, the CDC activity of the aglycosyl variant was similar to that of its glycosylated counterpart (Figure 4D). In particular, the antibody variant containing E345R showed the least suppression of CDC activity regardless of which aglycosyl mutation was introduced.

[0240] Wild-type IgG1-11B8 antibody did not induce detectable CDC in Ramos cells (Figure 4E). Efficient CDC was obtained by introducing either the E430G or E345K mutation. Similar to the enzymatic deglycosylation described in Example 2, the introduction of the nonglycosyl mutation N297A strongly suppressed the CDC activity of IgG1-11B8-E430G. In contrast, the introduction of the N297A mutation into IgG1-11B8-E345K only partially suppressed CDC activity. The maximum cell lysis induced by IgG1-11B8-E430G-N297A at an antibody concentration of 40 μg / mL was considerably lower than that induced by IgG1-11B8-E430G, but IgG1-11B8-E345K-N297A induced highly efficient CDC at the highest antibody concentration tested, comparable to the level induced by IgG1-11B8-E345K (Figure 4F).

[0241] Wild-type IgG1-11B8 did not induce CDC at detectable levels on Wien133 cells (Figure 4G). Introducing the E430G or E345K mutations resulted in partial CDC in Wien133 cells, and further introduction of the non-glycosyl mutation N297A into IgG1-CAMPATH-1H-E430G completely suppressed CDC, even at the highest antibody concentrations tested (Figure 4H). Introducing N297A into IgG1-CAMPATH-1H-E345K partially suppressed CDC activity (Figure 4G, H).

[0242] In summary, these data indicate that IgG1-CAMPATH-1H antibody variants possessing Fc-Fc interaction-enhancing mutations retain high CDC activity on Ramos cells and Wien133 cells when mutations that inhibit antibody glycosylation are introduced. While the CDC activity of IgG1-11B8-E430G was suppressed by introducing the glycosylation-inhibiting mutation N297A, IgG1-11B8-E345K-N297A showed comparable CDC activity to IgG1-11B8-E345K at the highest concentrations tested on Ramos and Wien133 cell lines.

[0243] Example 6: The nonglycosyl antibody variant with an Fc-Fc interaction mutation retains partial CDC activity against Raji and Daudi cells. In Example 5, it was shown that non-glycosyl IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants with Fc-Fc interaction-enhancing mutations retained potent CDC activity on Ramos cells and Wien133 cells. Here, we investigated whether mutations that inhibit glycosylation affect the CDC efficacy of IgG1-CD37-37-3 antibody variants and IgG1-11B8 antibody variants with Fc-Fc interaction-enhancing mutations on Raji cells and Daudi cells.

[0244] Different mutations were introduced into the anti-CD37 antibody IgG1-CD37-37-3 and the anti-CD20 antibody IgG1-11B8: E430G or E345K, which induces enhanced Fc-Fc interaction; N297A, N297G, N297Q, or T299A, which disrupt the N297 N-glycosylation consensus sequence (NX[S / T]) and produce non-glycosyl antibodies. The purified antibody variants were tested at a range of concentrations (final concentration range 0.0087 to 40.0 μg / mL; 3.33-fold dilution) in an in vitro CellTiterGlo CDC assay on Daudi (N=2) and Raji (N=3) co-expressing CD20 and CD37 antigens using 10% NHS, essentially as described in Example 5. Maximum cytotoxicity was determined using a positive control, 0.02% Triton X-100 solution (10X lysis buffer from Thermo LDH Cytotoxicity Assay Kit catalog number 88953). Minimum cytotoxicity (negative control) was determined using cells incubated with serum in the absence of IgG. Luminescence values ​​were converted to percentage cytotoxicity as follows: Cytotoxicity (%)=100% * (1 - (Luminescence of test sample - Luminescence of positive control Triton X-100) / (Luminescence of negative control without antibody - Luminescence of positive control Triton X-100) The relative area under the dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and against the AUC measured for a mixture of the positive controls IgG1-CD37-37-3-E430G and IgG1-11B8-E430G.

[0245] Wild-type IgG1-CD37-37-3 and IgG1-11B8 did not exhibit CDC activity against Daudi cells (Figure 4A). By introducing the Fc-Fc interaction-enhancing E430G mutation, these antibody variants were able to efficiently induce CDC both when used individually and in combination. Disruption of the N297 glycosylation site of antibody IgG1-CD37-37-3-E430G with N297A, N297G, N297Q, or T299A mutations reduced CDC efficacy to variable levels. At the highest antibody concentrations, the unglycosyl IgG1-CD37-37-3-E430G variant retained considerable CDC activity, inducing CDC in Daudi cells that was superior in all aspects to that of wild-type IgG1-CD37-37-3 (Figure 4B). Introducing the N297A mutation into IgG1-11B8-E430G partially reduced its CDC efficacy, and at the highest antibody concentrations, Daudi cell lysis was partially inhibited. Another nonglycosyl IgG1-11B8 variant with an Fc-Fc interaction-enhancing mutation (IgG1-11B8-E345K-N297A) also killed Daudi cells, with slightly greater efficiency than IgG1-11B8-E430G-N297A, and maximal cytotoxicity similar to IgG1-11B8-E430G.

[0246] Similar results were obtained in CDC assays on Raji cells (Figures 4C-D). Wild-type IgG1-CD37-37-3 and IgG1-11B8 showed little to no CDC activity against Raji cells, but efficient CDC was achieved by introducing the Fc-Fc interaction-enhancing E430G mutation into these variants (Figure 4C). Disrupting the N297 glycosylation site of IgG1-CD37-37-3-E430G with N297A, N297G, N297Q, or T299A mutations reduced the CDC ability of the antibody variants, but significant Raji cell lysis was maintained at the highest antibody concentrations (Figure 4D), exceeding the lysis observed for wild-type IgG1-CD37-37-3. Introducing the N297A mutation into IgG1-11B8-E430G suppressed its CDC efficacy. A non-glycosyl IgG1-11B8 variant (IgG1-11B8-E345K-N297A) with a different Fc-Fc interaction-enhancing mutation killed Raji cells more efficiently than IgG1-11B8-E430G-N297A. These data indicate that inhibiting glycosylation by modifying the N-glycosylation consensus sequence only partially inhibited the CDC efficacy of the IgG1-CD37-37-3 antibody variant and the IgG1-11B8 antibody variant with Fc-Fc interaction-enhancing mutations.

[0247] Example 7: When the N297 glycosylation site is disrupted, the selectivity of CD52 target-directed antibody variants containing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations is improved. In Example 4, the inventors demonstrated that the N297Q glycosylation site mutation in the CH2 domain of an IgG antibody improves the selectivity of antibody variants containing the E430G Fc-Fc interaction enhancing mutation and the K439E / S440K auto-oligomerization inhibiting mutation. Here, the inventors tested whether this improved selectivity could be observed when using glycosylation site mutations (N297A, N297Q) in antibody variants containing various Fc-Fc interaction enhancing mutations and auto-oligomerization inhibiting mutations.

[0248] Different mutations were introduced into the anti-CD52 IgG1-CAMPATH-1H antibody: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions; K439E or S440K, which inhibit the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions; and N297A, N297Q, or S298G-T299A, which disrupt the N297 N-glycosylation consensus sequence (NX[S / T]) to produce unglycosyl antibodies. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. The purified antibody variants were tested at a range of concentrations (0.0088 to 40.0 μg / mL final concentration at a 3.33-fold dilution for Ramos cells in the case of Ramos cells; 0.0018 to 40.0 μg / mL final concentration at a 3.33-fold dilution for Wien133 cells in the case of Wien133 cells) in the case of Wien133 cells in the case of 3.33-fold dilution for the case of CellTiterGlo and PI CDC assays) using logarithmically transformed concentrations. The area under the relative dose-response curve (AUC) was normalized for the unbound negative control IgG1-b12 (0%) and the positive control IgG1-CAMPATH-1H-E430G.

[0249] Introducing the K439E / S440K autooligomerization inhibitory mutation into the IgG1-CAMPATH-1H-E430G antibody variant, which has enhanced Fc-Fc interaction, reduced CDC efficacy on Ramos cells, but did not completely block it (Figure 6A). At the highest antibody concentrations, the K439E and S440K variants only slightly reduced CDC-mediated lysis (Figure 6B). This indicates considerable mono-drug activity. Similarly, IgG1-CAMPATH-1H antibody variants possessing Fc-Fc interaction-enhancing E345K or E345R mutations, and K439E (E345K-K439E: SEQ ID NO 50; E345R-K439E: SEQ ID NO 52) or S440K (E345K-S440K: SEQ ID NO 51; E345R-S440K: SEQ ID NO 53) auto-oligomerization inhibitory mutations, still demonstrated mono-drug activity, particularly at the highest antibody concentrations tested. However, when the N297 glycosylation site was disrupted via N297A mutations (E430G-K439E-N297A: SEQ ID NO 42; E430G-S440K-N297A: SEQ ID NO 43; E345K-K439E-N297A: SEQ ID NO 44; E345K-S440K-N297A: SEQ ID NO 45; E345R-K439E-N297A: SEQ ID NO 46; E345R-S440K-N297A: SEQ ID NO 47), the CDC efficacy of these antibody variants was almost completely inhibited at the highest antibody concentrations, and CDC-mediated Ramos cell lysis was partially or completely inhibited. Introducing the N297Q mutation reduced the monophasic activity of IgG1-CAMPATH-1H-IgG1-CAMPATH-1H-E345K-S440K (SEQ ID NO 49), whereas introducing the same mutation into E345K-K439E (SEQ ID NO 48) did not reduce its activity.

[0250] When complementary IgG1-CAMPATH-1H-K439E and -S440K antibody variants with E430G, E345K, or E345R Fc-Fc interaction-enhancing mutations were combined, nearly complete recovery of CDC efficacy (approximately 93% to 109% of the activity of the positive control IgG1-CAMPATH-1H-E430G) was observed. Disruption of the glycosylation site in these antibody variants (N297A) resulted in a slight reduction in CDC efficacy when mixed (approximately 67% to 87% compared to the positive control), and at the highest antibody concentrations, lysis levels were virtually unaffected. Efficient CDC was also observed when IgG1-CAMPATH-1H-E345K-K439E and IgG1-CAMPATH-1H-E345K-S440K antibody variants with the N297Q mutation were combined.

[0251] When IgG1-CAMPATH-1H-K439E-N297A and IgG1-CAMPATH-1H-S440K-N297A antibody variants, each possessing two different Fc-Fc interaction-enhancing mutations (E430G, E345K, or E345R), were combined, the resulting CDC was equivalent to the CDC levels induced by two of these antibody variants, each possessing the same Fc-Fc interaction-enhancing mutation. This suggests that Fc-Fc-enhancing mutations can be used without distinction in the context of non-glycosyl antibody variants.

[0252] Similar results were obtained using Wien133 cells (Figures 6C-D). The K439E and S440K autooligomerization inhibitory mutations reduced, but did not completely suppress, the CDC efficacy of IgG1-CAMPATH-1H-E430G on Wien133 cells (Figure 6C). Similarly, IgG1-CAMPATH-1H-E345R-K439E and -S440K still showed considerable monodrug activity, especially at the highest antibody concentrations (Figure 6D). Non-glycosyl variants of these antibodies via the N297A mutation substantially suppressed CDC efficacy and, at the highest antibody concentrations, strongly inhibited CDC-mediated Wien133 cell lysis (approximately 8%-28% lysis remained). IgG1-CAMPATH-1H-E345K-K439E and -S440K antibody variants with N297A or N297Q mutations also showed low CDC activity (approximately 4% to 32%) on Wien133 cells.

[0253] Complementary IgG1-CAMPATH-1H-K439E and -S440K antibody variants with E430G or E345R Fc-Fc interaction-enhancing mutations, when combined, restored CDC efficacy against Wien133 cells to almost complete levels (approximately 95%–99%). When the non-glycosyl variant (N297A) of these antibody variants was mixed, it slightly reduced CDC efficacy (approximately 70%–73%), but had virtually no effect on lysis levels at the highest antibody concentrations. Efficient CDC was also observed when IgG1-CAMPATH-1H-E345K-K439E and IgG1-CAMPATH-1H-E345K-S440K antibody variants with N297A or N297Q mutations were combined.

[0254] When IgG1-CAMPATH-1H-K439E-N297A and IgG1-CAMPATH-1H-S440K-N297A antibody variants, each possessing two different Fc-Fc interaction-enhancing mutations (E430G, E345K, or E345R), were combined, the resulting CDC was equivalent to the CDC levels induced by two of these antibody variants possessing the same Fc-Fc interaction-enhancing mutation. This suggests that Fc-Fc-enhancing mutations can be used without distinction.

[0255] These data indicate that disruption of the N297 glycosylation site reduces the monophasic activity of IgG1-CAMPATH-1H antibody variants with enhanced Fc-Fc interaction and the self-oligomerization-inhibiting K439E or S440K mutations. In contrast, when these unglycosylated antibody variants with enhanced Fc-Fc interaction and inhibited self-oligomerization were combined, their efficacy was almost completely restored in CDC assays.

[0256] Example 8: When the N297 glycosylation site is disrupted, the selectivity of CD52-targeted antibody variants and CD20-targeted antibody variants, which include Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations, is improved. In Example 7, disruption of the N297 glycosylation site in the CH2 domain of an IgG1-CAMPATH-1H antibody variant having both an Fc-Fc interaction-enhancing mutation and an auto-oligomerization-inhibiting mutation increased CDC selectivity. Here, the inventors tested whether the same principle could be extended to different antibody combinations (anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8).

[0257] Different mutations were introduced into anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibodies: E430G, E345K, or E345R, which induce enhanced Fc-Fc interactions; K439E or S440K, which inhibit the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions; and N297A, N297Q, or S298G-T299A, which disrupt the N297 N-glycosylation consensus sequence (NX[S / T]) to make it unglycosyl. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. The purified antibody variant was tested at a range of concentrations (0.01–40.0 μg / mL, 3.3-fold dilution for Wien133 cells; 0.0088–40.0 μg / mL, 3.3-fold dilution for Ramos cells) in an in vitro PI CDC assay on Wien133 cells (N=3) using 20% ​​NHS, essentially as described in Example 2, or in a CellTiterGlo CDC assay on Ramos cells (N=3) using 10% NHS, essentially as described in Example 5.

[0258] For CellTiterGlo and PI CDC assays, the relative area under the dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0259] Treatment of Wien133 cells with IgG1-CAMPATH-1H-E430G resulted in efficient CDC (Figure 7A). Introducing the K439E auto-oligomerization inhibitory mutation reduced CDC efficacy, but at antigen-saturated antibody concentrations, it had only a limited effect on mono-drug activity. Further introduction of the glycosylation-inhibiting N297A mutation reduced the mono-drug activity of this antibody variant to background levels, even at high antibody concentrations. The N297A mutation also suppressed the CDC efficacy of the IgG1-CAMPATH-1H antibody variant containing both the E345R Fc-Fc interaction-enhancing mutation and the K439E auto-oligomerization inhibitory mutation. At the highest antibody concentration, treatment of Wien133 cells with this IgG1-CAMPATH-1H-E345R-K439E-N297A antibody variant resulted in a decrease in CDC levels (approximately 28%) (Figure 7B). IgG1-CAMPATH-1H-E345K-K439E antibody variants with N297A or N297Q mutations also showed limited efficacy as monophasic agents in CDC assays, but exhibited limited activity (approximately 5% to 35%) at antigen-saturated antibody concentrations.

[0260] Compared to IgG1-CAMPATH-1H-E430G, IgG1-11B8-E430G showed limited CDC efficacy. Introducing the S440K auto-oligomerization inhibitory mutation completely suppressed mono-drug activity on Wien133 cells, thus obscuring the potential further inhibitory effects of any glycosylation-blocking mutation (N297A or N297Q). Similarly, IgG1-11B8-E345K-S440K did not mediate CDC, and in this case as well, the further inhibitory effects of any glycosylation-blocking mutation (N297A or N297Q) were obscured.

[0261] The IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants efficiently induced CDC when combined, and their efficacy was only slightly lower than that of the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G. Introducing the N297A mutation into IgG1-CAMPATH-1H-E430G-K439E slightly reduced its CDC efficacy when combined with the IgG1-11B8-E430G-S440K antibody variant, and more significantly reduced when combined with the glycosyl IgG1-11B8-E430G-S440K-N297A or IgG1-11B8-E430G-S440K-N297Q antibody variants. However, at the highest antibody concentrations, this combination had virtually no effect on Wien133 cell lysis mediated by these antibody combinations (Figure 7B). Similar levels of CDC efficacy were observed when IgG1-CAMPATH-1H-E430G-K439E-N297A was combined with IgG1-11B8-E345K-S440K-N297A or IgG1-11B8-E345K-S440K-N297Q, which have the E345K mutation instead of the E430G Fc-Fc interaction enhancing mutation.

[0262] The IgG1-CAMPATH-1H-E345K-K439E antibody variant efficiently induced CDC when combined with the IgG1-11B8-E430G-S440K or IgG1-11B8-E345K-S440K antibody variants. This indicates that when antibodies with different Fc-Fc interaction-enhancing mutations are combined, they form a codependently functioning mixture. Introducing the N297A or N297Q mutation into IgG1-CAMPATH-1H-E345K-K439E slightly reduced the CDC efficacy of this antibody variant when combined with the IgG1-11B8-E430G-S440K or IgG1-11B8-E345K-S440K antibody variants, but at the highest antibody concentrations, it did not affect Wien133 cell lysis levels. A more pronounced decrease in CDC efficacy was observed when both the IgG1-CAMPATH-1H-E345K-K439E antibody variant and IgG1-11B8-E430G-S440K or IgG1-11B8-E345K-S440K contained mutations that inhibit glycosylation (N297A or N297Q). However, the maximum level of Wien133 cell lysis remained nearly the same.

[0263] The IgG1-CAMPATH-1H-E345R-K439E variant efficiently induced CDC when combined with IgG1-11B8-E430G-S440K. The CDC efficacy of this combination was slightly reduced when IgG1-CAMPATH-1H-E345R-K439E carried the N297A mutation. The reduction in CDC efficacy was more pronounced when the IgG1-11B8-E430G-S440K antibody variant also carried a mutation that inhibited glycosylation (N297A or N297Q), although Wien133 cell lysis at the highest antibody concentrations remained unaffected.

[0264] Similar results were observed using Ramos target cells (Figures 7C-D). Introducing the K439E auto-oligomerization inhibitory mutation into IgG1-CAMPATH-1H-E430G reduced CDC efficacy (Figure 7C), but did not reduce Ramos cell lysis at the highest antibody concentration (Figure 7D). The N297A mutation reduced the mono-drug activity of this antibody variant to background levels. The N297A mutation also suppressed the mono-drug CDC efficacy of IgG1-CAMPATH-1H-E345R-K439E and IgG1-CAMPATH-1H-E345K-K439E. Introducing the N297Q mutation had only a limited effect on the CDC efficacy of IgG1-CAMPATH-1H-E345K-K439E, in contrast to the inhibitory effect observed when introduced into IgG1-CAMPATH-1H-E430G-K439E (Examples 5,4).

[0265] Compared to IgG1-CAMPATH-1H-E430G, IgG1-11B8-E430G showed limited CDC efficacy. Introducing the S440K auto-oligomerization inhibitory mutation completely suppressed single-drug activity, thus obscuring the potential further inhibitory effects of any glycosylation-blocking mutation (N297A or N297Q). Similarly, IgG1-11B8-E345K-S440K did not mediate CDC, and in this case as well, the further inhibitory effects of any glycosylation-blocking mutation (N297A, N297Q, or S298G-T299A) were obscured.

[0266] The IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants efficiently induced CDC when combined (approximately 81% of the positive control mixture). Introducing the N297A mutation into IgG1-CAMPATH-1H-E430G-K439E slightly reduced its CDC efficacy (to approximately 56%) when combined with the IgG1-11B8-E430G-S440K antibody variant, and more significantly (to 37%–52%) when combined with the glycosyl IgG1-11B8-E430G-S440K-N297A or IgG1-11B8-E345K-S440K-N297A antibody variants. However, this combination had virtually no effect on Ramos cell lysis at the highest antibody concentrations.

[0267] The IgG1-CAMPATH-1H-E345K-K439E antibody variant efficiently induced CDC when combined with IgG1-11B8-E345K-S440K. The presence of the N297A or N297Q mutation in both IgG1-CAMPATH-1H-E345K-K439E and IgG1-11B8-E345K-S440K slightly reduced this CDC efficacy, but at the highest antibody concentrations, it had a limited effect on Ramos cell lysis levels. The combination of glycosyl-free IgG1-CAMPATH-1H-E345K-K439E-N297A or -N297Q with IgG1-11B8-E430G-S440K also induced CDC. The combination of glycosyl IgG1-CAMPATH-1H-E345R-K439E-N297A and IgG1-11B8-E430G-S440K or glycosyl IgG1-11B8-E430G-S440K-N297A or -N297Q efficiently induced CDC.

[0268] These data indicate that disruption of the N297 glycosylation site inhibits the monophasic activity of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants with enhanced Fc-Fc interaction and the self-oligomerization-inhibiting K439E and S440K mutations. In contrast, a complementary combination of (K439E+S440K) unglycosyl, Fc-Fc interaction-enhanced IgG1-CAMPATH-1H and IgG1-11B8 antibody variants partially restores CDC induction ability. At antigen saturation concentrations, IgG1-CD52-CAMPATH-1H, particularly those with the E345K, K439E, and N297A mutations, exhibited a preferred minimal activity phenotype as a monophasic, while demonstrating high efficacy when used in a codependent mixture.

[0269] Example 9: When the N297 glycosylation site is disrupted, the selectivity of CD37-targeted antibody variants and CD20-targeted antibody variants, which include Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations, is improved. In Examples 7 and 8, disruption of the N297 glycosylation site in the IgG1-CAMPATH-1H antibody variant and the IgG1-11B8 antibody variant, which contain both Fc-Fc interaction-enhancing and auto-oligomerization-inhibiting mutations, improved their CDC selectivity. Here, the inventors investigated whether the N297 mutation could also limit the selectivity of different combinations of antibody variants (IgG1-CD37-37-3 and IgG1-11B8) that contain both Fc-Fc interaction-enhancing and auto-oligomerization-inhibiting mutations.

[0270] Anti-CD37 IgG1-CD37-37-3 and anti-CD20 IgG1-11B8 antibodies were introduced with different mutations: E430G or E345K, which induces enhanced Fc-Fc interactions; K439E or S440K, which inhibits the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promotes the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions; and N297A or N297Q, which disrupts the N297 N-glycosylation site to make it unglycosyl. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. Essentially as described in Example 5, purified antibody variants were tested at a range of concentrations (0.0088 to 40.0 μg / mL final concentration range; 3.33-fold dilution) in an in vitro CellTiterGlo CDC assay on Raji cells (N=3) or Daudi cells (N=2) using 10% NHS. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control mixture of IgG1-CD37-37-3-E430G and IgG1-11B8-E430G (100%).

[0271] Treatment of Raji cells with IgG1-CD37-37-3-E430G or IgG1-11B8-E430G resulted in efficient CDC (Figure 8A). Introducing the K439E mutation alone, or in combination with a glycosylation-blocking mutation (N297A or N297Q), strongly reduced its CDC-inducing efficacy. Similarly, introducing the K439E mutation into IgG1-CD37-37-3-E345K in combination with N297A or N297Q weakened the remaining CDC-inducing efficacy. For IgG1-11B8-E430G or IgG1-11B8-E345K, no single-agent CDC activity was observed after introducing the S440K mutation in combination with N297A or N297Q. When IgG1-CD37-37-3-E430G-K439E was mixed with IgG1-11B8-S440K variants containing either the E430G or E345K mutation and either the N297A or N297Q mutation, a partial recovery of CDC efficacy was observed. When IgG1-CD37-37-3-E430G-K439E was mixed with E345K-containing IgG1-11B8 variants, a stronger CDC recovery was induced, reaching approximately 75% of the level induced by the positive control (resulting in approximately 60% of the level induced by the positive control), compared to mixing with E430G-containing IgG1-11B8 variants. Mixing the unglycosyl IgG1-CD37-37-3-K439E variant containing either the E430G or E345K mutation with the unglycosyl IgG1-11B8-S440K variant containing either the E430G or E345K mutation partially restored CDC efficacy, although the recovery level was lower than that of a mixture in which one of the antibody variants was glycosylated. When two E430G-containing antibody variants containing either the N297A or N297Q mutation were mixed, CDC efficacy was approximately 45% or 50% of the level induced by the positive control, respectively. CDC recovery was even more efficient when two E345K-containing antibody variants containing either the N297A or N297Q mutation were mixed, with recovery reaching approximately 64% and 66% of the level induced by the positive control, respectively.At the highest concentration tested (40 μg / mL), all mixtures exhibited similar CDC efficacy, except for a mixture of glycosyl-free IgG1-CD37-37-3-K439E containing E430G and the IgG1-11B8-S440K variant, which induced slightly reduced CDC levels at this concentration (Figure 8B).

[0272] Similar results were obtained using Daudi target cells (Figures 8C-D). Introducing a combination of the autooligomerization-inhibiting K439E mutation and a glycosylation-blocking mutation (N297A or N297Q) into IgG1-CD37-37-3-E430G or IgG1-CD37-37-3-E345K suppressed CDC efficacy (Figure 8C). The monophasic activity of IgG1-11B8-E430G and IgG1-11B8-E345K could be suppressed by introducing the S440K mutation and a mutation that disrupts the N-linked glycosylation site (N297A or N297Q). When combined with IgG1-CD37-37-3-E430G-K439E, the CDC efficacy of the glycosyl-free IgG1-11B8-E430G-S440K-N297A and -N297Q antibody variants was partially restored, and at the highest antibody concentrations, Daudi cell lysis was close to the CDC levels induced by the positive control (Figure 8D). More efficient restoration of CDC efficacy was induced by a mixture of glycosyl-free IgG1-11B8-E345K-S440K and IgG1-CD37-37-3-E430G-K439E. Mixing glycosyl-free IgG1-11B8-E430G-S440K-N297A and -N297Q antibody variants with IgG1-CD37-37-3-E430G-K439E or CD37-37-3-E345K-K439E antibody variants, which also contain mutations that inhibit glycosylation (N297A or N297Q), restored CDC efficacy, albeit not very efficiently.

[0273] These data show that introducing Fc-Fc interaction-enhancing mutations, auto-oligomerization-inhibiting mutations, and mutations that disrupt the N297 glycosylation site suppresses the monophasic activity of the IgG1-CD37-37-3 antibody variant and the IgG1-11B8 antibody variant. In contrast, when these unglycosyl, Fc-Fc interaction-enhancing antibody variants are combined with their complementary, unglycosyl, Fc-Fc interaction-enhancing counterparts, their ability to induce CDC is significantly restored, especially at antigen-saturated antibody concentrations.

[0274] Example 10: Analysis of the effect of a mutation that inhibits glycosylation in an IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation on antibody-dependent cell phagocytosis. Fcγ receptor 2a (FcγRIIa), also known as CD32a, is expressed in B cells, macrophages, and monocytes and plays a crucial role in antibody-dependent phagocytosis (ADCP). The interaction between the Fc domain and the Fcγ receptor is strongly dependent on glycosylation. Here, we investigated whether introducing a mutation that blocks glycosylation would affect the ability of an IgG1-CAMPATH-1H antibody variant carrying an Fc-Fc interaction-enhancing mutation to activate FcγRIIa in a reporter cell assay as a surrogate for ADCP.

[0275] For ADCP reporter bioassays, IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing Fc-Fc interaction-enhancing mutations (E430G, E345K, or E345R), auto-oligomerization-inhibiting mutations (K439E or S440K), and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A, N297G, N297Q, N297D, or N297Y) were tested on Raji cells using the Bio-Glo Luciferase Assay System (Promega, catalog number G7940). This kit includes Jurkat human T cells engineered to stably express high-affinity FcγRIIa (H131 allotype) and an activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, as effector cells. In short, Raji cells (25 μL; 11,000 cells / well; Promega, catalog number G870A) were seeded into a 96-well white opaque flat-bottom plate (Corning, catalog number 3917) containing RPMI-1640 [(Promega, catalog number G708A) and 4% low-IgG serum (Promega, catalog number G711A)]. The Raji cells were pre-incubated with an antibody concentration series (25 μL; 4-fold dilution series, with final concentrations ranging from 0.15 to 40,000 ng / mL after effector cell addition) at 37°C / 5% CO2 for 15 minutes. Subsequently, thawed Jurkat FcγRIIa H131 allotype effector cells (25 μL; 50,000 cells / well; Promega, catalog number G9991) were added, and the cells were incubated at 37°C / 5% CO2 for 16-24 hours. After incubation, the cells were allowed to equilibrate to room temperature for 15 minutes. Then, 75 μL of Bio-Glo Assay Luciferase Reagent [Bio-Glo Luciferase Assay Substrate (Promega catalog number G720A)] dissolved in Bio-Glo Luciferase Assay Buffer (Promega, catalog number G719A) was added per well, and the cells were incubated in the dark at RT for 20 minutes.Luciferase production was quantified by luminescence reading using a Tecan Spark 20M luminescence plate reader (Tecan). The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%; Figure 9B).

[0276] The ability of IgG1-CAMPATH-1H-E430G to induce FcγRIIa activation was completely suppressed by introducing N-linked glycosylation site mutations N297A, N297G, N297Q, N297D, or N297Y (Figure 9A). Similarly, both IgG1-CAMPATH-1H-E345K and IgG1-CAMPATH-1H-E345R efficiently induced FcγRIIa activation, but the introduction of mutation N297A into these antibody variants completely suppressed their FcγRIIa activation ability.

[0277] A mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G efficiently induced FcγRIIa activation (Figure 9B). In contrast, the IgG1-CAMPATH-1H-E430G antibody variant with the K439E mutation did not induce FcγRIIa activation, thus obscuring the further inhibitory effect of the N297A mutation. For IgG1-11B8-E430G-S440K, a weaker FcγRIIa activation induction ability was observed compared to the positive control IgG1-CAMPATH-1H-E430G-K439E and a mixture of IgG1-11B8-E430G. Introducing the N297A mutation into the IgG1-11B8-E430G-S440K variant further suppressed its FcγRIIa activation induction ability. A mixture of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K slightly restored FcγRIIa activation, but a mixture of these antibody variants containing the non-glycosyl mutation N297A did not induce FcγRIIa activation at all.

[0278] In summary, these data indicate that introducing a non-glycosyl mutation into the IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation suppresses ADCP induction as measured by the FcγRIIa-activated reporter cell assay. A mixture of the codependent glycosylated antibody variant IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K induced partial ADCP recovery, while a mixture of the non-glycosyl variant IgG1-CAMPATH-1H-E430G-K439E-N297A and IgG1-11B8-E430G-S440K-N297A did not induce ADCP recovery.

[0279] Example 11: When an IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation also has a mutation that inhibits glycosylation, antibody-dependent cell-mediated cytotoxicity is inhibited. Fcγ receptor 3a (FcγRIIIa), also known as CD16a, is primarily expressed on natural killer cells and macrophages and plays a crucial role in antibody-dependent cell-mediated cytotoxicity (ADCC). The interaction between the Fc domain and the Fcγ receptor is strongly dependent on their glycosylation. Here, we investigated whether introducing a mutation that inhibits glycosylation would affect the ability of an IgG1-CAMPATH-1H antibody variant carrying an Fc-Fc interaction-enhancing mutation to activate FcγRIIa in a reporter cell assay surrogate for ADCC.

[0280] For the ADCC reporter bioassay, the Bio-Glo Luciferase ADCC Reporter Bioassay (Promega, catalog number G7015) against the high-affinity V158 allotype of FcγRIIIa was used to test IgG1-CAMPATH-1H antibody variants containing Fc-Fc interaction-enhancing mutations (E430G, E345K, or E345R) and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A, N297G, N297Q, N297D, or N297Y) on Raji cells. This kit contains Jurkat human T cells as effector cells, engineered to stably express the high-affinity V158 allotype of FcγRIIIa and the activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression. In short, Raji cells (25 μL; 12,500 cells / well; Promega, catalog number G870A) were seeded into a 96-well white opaque flat-bottom plate (Corning, catalog number 3917) containing RPMI-1640 [(Promega, catalog number G708A) and 10% low-IgG serum (Promega, catalog number G711A)]. The Raji cells were pre-incubated with an antibody concentration series (25 μL; 4-fold dilution series, with final concentrations ranging from 0.15 to 40,000 ng / mL after effector cell addition) at 37°C / 5% CO2 for 15 minutes. Subsequently, thawed Jurkat FcγRIIIa (V158 high affinity allotype) effector cells (25 μl; 75,000 cells / well; Promega, catalog number G701A) were added, and the cells were incubated at 37°C / 5% CO2 for 5 hours. After this incubation, the cells were allowed to equilibrate to room temperature for 15 minutes. Then, 75 μl of Bio-Glo Assay Luciferase Reagent [Bio-Glo Luciferase Assay Substrate (Promega catalog number G720A)] dissolved in Bio-Glo Luciferase Assay Buffer (Promega, catalog number G719A) was added per well, and the cells were incubated in the dark at RT for 20 minutes.Luciferase production was quantified by luminescence reading using a Tecan Spark 20M luminescence plate reader (Tecan). The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%).

[0281] The ability of IgG1-CAMPATH-1H-E430G to induce FcγRIIIa activation was completely suppressed by introducing the non-glycosyl mutations N297A, N297G, N297Q, N297D, or N297Y (Figure 10). Similarly, both IgG1-CAMPATH-1H-E345K and IgG1-CAMPATH-1H-E345R efficiently induced FcγRIIIa activation, but their FcγRIIIa activation ability was completely suppressed by introducing the N297A mutation into these antibody variants.

[0282] In summary, these data indicate that introducing a mutation that inhibits glycosylation into an IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation suppresses its ADCC-inducing ability, as measured by the FcγRIIIa-activated reporter cell assay.

[0283] Example 12: Disruption of the N297 glycosylation site in an antibody variant containing the Fc-Fc interaction-enhancing mutation K248E-T437R has a limited effect on its CDC activity. In Example 5, it was shown that disruption of the N297 N-glycosylation site induced either weak inhibition of CDC activity or no inhibition of CDC activity in antibody variants having the E430G, E345K, or E345R Fc-Fc interaction enhancing mutation. Here, the inventors tested whether this could be extended to the K248E-T437R Fc-Fc interaction enhancing mutation.

[0284] Different mutations were introduced into the anti-CD52 antibody IgG1-CAMPATH-1H: E430G or K248E-T437R, which induce enhanced Fc-Fc interaction; and N297A, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. The purified antibody variants were tested at a range of concentrations (0.0088 to 40.0 μg / mL final concentration range; 3.33-fold dilution) using a PI CDC assay on Wien133 cells (N=4) with 20% NHS, essentially as described in Example 2, or using an in vitro CellTiterGlo CDC assay on Ramos cells (N=3) with 10% NHS, essentially as described in Example 5. For CellTiterGlo and PI CDC assays, the relative area under the dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control IgG1-CAMPATH-1H-E430G.

[0285] Introducing the Fc-Fc interaction-enhancing mutation E430G or K248E-T437R (SEQ ID NO 63) into IgG1-CAMPATH-1H resulted in a potent increase in CDC activity on Wien133 cells (Figure 11A). This increase in CDC activity was also evident at antigen-saturated antibody concentrations (Figure 11B). When the N297 glycosylation site was disrupted by the N297A mutation (SEQ ID NO 64), the CDC activity of IgG1-CAMPATH-1H-K248E-T437R was slightly reduced, and CDC activity remained unaffected at the highest antibody concentrations.

[0286] Similarly, IgG1-CAMPATH-1H antibody variants with E430G or K248E-T437R Fc-Fc interaction enhancing mutations efficiently induced CDC in Ramos cells (Figure 11C-D). Inhibiting N-linked glycosylation at N297 using the N297A mutation slightly reduced the CDC efficacy of IgG1-CAMPATH-1H-K248E-T437R (Figure 11C), but CDC activity did not decrease at antigen-saturated antibody doses (Figure 11D).

[0287] In combination, these data indicate that even when the N297 glycosylation site was destroyed, the CDC activity of the IgG1-CAMPATH-1H-K248E-T437R antibody variant was largely retained.

[0288] Example 13: When the N297 glycosylation site is disrupted, the selectivity of antibody variants having the K248E-T437R Fc-Fc interaction enhancing mutation and the K439E or S440K autooligomerization inhibitory mutation is improved. In Examples 7-9, it was shown that disruption of the N297 glycosylation site improved the selectivity of antibody variants having E430G, E345K, or E345R Fc-Fc interaction enhancing mutations and K439E or S440K auto-oligomerization inhibiting mutations. Here, the inventors tested whether this could be extended to antibody variants having a combination of K248E-T437R Fc-Fc interaction enhancing mutations and auto-oligomerization inhibiting K439E or S440K mutations.

[0289] Different mutations were introduced into the negative control antibody IgG1-b12, the anti-CD52 antibody IgG1-CAMPATH-1H, and the anti-CD20 antibody IgG1-11B8: E430G or K248E-T437R which induce enhanced Fc-Fc interactions; K439E or S440K which inhibit the formation of homohexameric antibody complexes by inhibiting intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes by cross-complementary Fc-Fc interactions; and N297A which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. Essentially as described in Example 2, the purified antibody variant was tested in a PI CDC assay on Wien133 cells (N=3) using 20% ​​NHS at a range of concentrations (0.002 to 40.0 μg / mL final concentration range; 4.0-fold dilution (Figure 12A); 0.0088 to 40.0 μg / mL final concentration (Figure 12A-B); 3.33-fold dilution (Figure 12C-D)). The purified antibody variant was also tested in an in vitro CellTiterGlo CDC assay on Ramos cells (N=3) using 10% NHS, essentially as described in Example 5 (Figure 12E-F). For CellTiterGlo and PI CDC assays, the relative area under the dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-B118-E430G (100%).

[0290] The combination of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G, which have enhanced Fc-Fc interactions, efficiently induced CDC in Wien133 cells (Figure 12A). IgG1-CAMPATH-1H-K248E-T437R (SEQ ID NO 65), which has an enhanced Fc-Fc interaction and contains the auto-oligomerization inhibitory mutation K439E, showed monophasic activity when combined with the IgG1-b12-S440K (SEQ ID NO 70) negative control antibody variant, whereas IgG1-CAMPATH-1H-K248E-T437R (SEQ ID NO 66), which contains the auto-oligomerization inhibitory mutation S440K, showed monophasic activity when combined with the IgG1-b12-K439E (SEQ ID NO 69) negative control antibody variant (Figures 12A-B). Inhibiting N-linked glycosylation of the IgG1-CAMPATH-1H-K248E-T437R-K439E antibody variant by the N297A mutation (SEQ ID NO 67) efficiently inhibited its single-agent CDC activity on Wien133 cells (Figure 12C-D).

[0291] The IgG1-11B8-K248E-T437R-S440K antibody variant, in which Fc-Fc interactions were enhanced and auto-oligomerization was inhibited, already showed weak mono-drug activity on Wien133 cells (Figure 12A-B), and this remained true even after the N297 glycosylation site of this antibody variant was disrupted (SEQ ID NO 68; Figure 12C-D). When IgG1-CAMPATH-1H-K248E-T437R-N297A and IgG1-11B8-K248E-T437R-S440K-N297A were combined, CDC efficacy was partially restored, and CDC activity was greatly restored at antigen-saturated antibody concentrations.

[0292] Similar codependent restoration of CDC efficacy between glycosyl IgG1-CAMPATH-1H-K248E-T437R-N297A and IgG1-11B8-K248E-T437R-S440K-N297A was observed using Ramos cells (Figures 12C-D). In short, the IgG1-CAMPATH-1H-K248E-T437R-N297A antibody variant and the IgG1-11B8-K248E-T437R-S440K-N297A antibody variant did not exhibit CDC efficacy in Ramos cells (Figure 12C), and did not even show antigen-saturated antibody concentrations (Figure 12D). When IgG1-CAMPATH-1H-K248E-T437R-N297A and IgG1-11B8-K248E-T437R-S440K-N297A were combined, the CDC efficacy of these antibody variants was partially restored compared to the IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G positive control, and CDC activity was almost completely restored at antigen saturation concentrations.

[0293] In summary, these data indicate that disruption of the N297 glycosylation site improves the selectivity of antibody variants containing the Fc-Fc interaction-enhancing K248E-T437R and the auto-oligomerization-inhibiting K439E or S440K mutations. When the Fc-Fc interaction-enhancing K248E-T437R antibody variant was combined with complementary K439E and S440K auto-oligomerization-inhibiting mutations, its CDC activity was partially restored.

[0294] Example 14: Non-glycosyl CD37-targeted antibody variants and CD20-targeted antibody variants, which contain the Fc-Fc interaction-enhancing mutation E345R and the auto-oligomerization-inhibiting mutation, induce interdependent CDC activation. In Example 9, selective CDC was induced by glycosyl-free IgG1-CD37-37-3 antibody variants and IgG1-11B8 antibody variants containing the Fc-Fc interaction enhancing mutation E430G or E345K and an autooligomerization inhibitory mutation. Here, the inventors also tested whether CDC could be selectively induced by glycosyl-free, codependent IgG1-CD37-37-3 antibody variants and IgG1-11B8 antibody variants containing the Fc-Fc interaction enhancing mutation E345R.

[0295] Different mutations were introduced into anti-CD37 IgG1-CD37-37-3 and anti-CD20 IgG1-11B8 antibodies: E430G or E345R, which induces enhanced Fc-Fc interactions; K439E or S440K, which inhibits auto-oligomerization; and N297A, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) to produce an unglycosyl antibody. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of the mixture composed of the individual components. Where indicated, these mutations were also introduced into the IgG1-b12 unbound control antibody. In this example, except for the use of 30,000 cells / well, the purified antibody variant was tested at a range of concentrations (range 0.005 to 20.0 μg / mL final concentration; 3.33-fold dilution) in an in vitro CDC assay using 20% ​​NHS on Daudi cells (N=3) co-expressing CD20 and CD37 antigens, essentially as described in Example 2. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control mixture of IgG1-CD37-37-3-E430G and IgG1-11B8-E430G (100%).

[0296] The low single-agent CDC activity of IgG1-CD37-37-3-E430G-K439E on Daudi cells could not be further suppressed by the introduction of mutant N297A (Figure 13A). Similarly, low residual single-agent CDC activity was observed for IgG1-CD37-37-3-E345R-K439E-N297A when combined with IgG1-b12-E345R-S440K-N297A. The residual CDC activity induced by the IgG1-11B8-E430G-S440K antibody variant was lower than that induced by IgG1-CD37-37-3-E430G-K439E, and this too could not be further suppressed by the introduction of mutant N297A. IgG1-11B8-E345R-S440K-N297A also showed very low CDC activity when combined with IgG1-b12-E345R-K439E-N297A.

[0297] Compared to the positive control mixture IgG1-CD37-37-3-E430G+IgG1-11B8-E430G, introducing auto-oligomerization inhibitory mutations into these antibody variants resulted in approximately 12% lower efficiency of CDC. Introducing the N297A mutation into the positive control variant induced CDC with approximately 29% lower efficiency. When both K439E or S440K and N297A mutations were introduced into IgG1-CD37-37-3-E430G and IgG1-11B8-E430G, CDC was induced with approximately 45% lower efficiency compared to the positive control mixture. However, when E345R, instead of E430G, was introduced as an Fc-Fc interaction enhancing mutation, the CDC efficacy was reduced by only 14% compared to the positive control mixture. This result is equivalent to that of the mixture IgG1-CD37-37-3-E430G-K439E+IgG1-11B8-E430G-S440K. At the highest concentration tested (20 μg / mL), all of the above mixtures induced maximal solubility equivalent to that of the positive control mixture, whereas all single-agent CDC activity was limited to <14% solubility, and even at target saturation, a considerable window remained between the mixtures and the codependent mixtures (Figure 13B).

[0298] In conclusion, non-glycosyl variants of IgG1-CD37-37-3 and IgG1-11B8 antibodies possessing K439E or S440K autooligomerization inhibitory mutations, in addition to E430G or E345R Fc-Fc interaction enhancing mutations, were able to selectively and codependently induce CDC in Daudi cells. In particular, mixtures of antibody variants with the E345R mutation showed potent CDC selectivity. At the highest concentrations tested, all mixtures studied in this example induced efficient Daudi cell lysis.

[0299] Example 15: When the N297 glycosylation site is disrupted, the selectivity of CDC efficacy by CD52-targeted antibody variants and CD20-targeted antibody variants having the Fc-Fc interaction-enhancing mutation E430Y and the self-oligomerization-inhibiting mutation is improved. In Examples 7 and 8, it was shown that single-agent CDC efficacy on Wien133 cells could be potently reduced by introducing glycosylation-blocking mutations into CD52-targeted IgG1-CAMPATH-1H and CD20-targeted IgG1-11B8 antibody variants, which also possessed mutations that enhanced Fc-Fc interaction and mutations that inhibited autooligomerization. Codependent mixtures of such unglycosyl antibody variants were able to partially restore CDC efficacy. Here, we investigated whether unglycosyl, codependent IgG1-CAMPATH-1H and IgG1-11B8 antibody variants, possessing the Fc-Fc interaction-enhancing mutation E430Y, could selectively further optimize CDC efficacy on Wien133 cells.

[0300] Different mutations were introduced into anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibodies: E430G, E345R, or E430Y, which induce enhanced Fc-Fc interactions; K439E or S440K, which inhibit auto-oligomerization; and N297A, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of the individual components. Where indicated, the above mutations were also introduced into the IgG1-b12 unbound control antibody. In this example, except for the use of 30,000 cells / well, the purified antibody variant was tested at a range of concentrations (0.01 to 40.0 μg / mL at a 3.33-fold dilution) using a PI CDC assay on Wien133 cells (N=3) with 20% NHS, essentially as described in Example 2. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and against the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%; in the case of an IgG1-CAMPATH-1H antibody variant mixture) or the AUC measured for the positive control IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%; in the case of an IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G antibody variant mixture).

[0301] In Example 7, IgG1-CAMPATH-1H antibody variants having either E430G or E345R and K439E or S440K were observed to have residual CDC activity on Wien133 cells compared to levels induced by the positive control (E430G-K439E: approximately 33%; E345R-K439E: approximately 51%; E430G-S440K: approximately 49%; E345R-S440K: approximately 58%). In this example, both IgG1-CAMPATH-1H-E430Y-K439E (SEQ ID NO 77) and IgG1-CAMPATH-1H-E430Y-S440K (SEQ ID NO 78), when combined with complementary unbound control IgG1-b12 variants, induced residual CDC to approximately 75% and 78% of the levels induced by the IgG1-CAMPATH-1H-E430G-positive control, respectively (Figure 14A). In all cases, introducing the N297A mutation partially suppressed residual CDC activity (E430Y-K439-N297A: SEQ ID NO 79; E430Y-S440-N297A: SEQ ID NO 80).

[0302] The mixture of IgG1-CAMPATH-1H-E430Y-K439E + IgG1-CAMPATH-1H-E430Y-S440K induced the strongest CDC efficacy recovery (up to approximately 108% of the level induced by the positive control), while mixtures of such variants containing E345R (approximately 103% of the level induced by the positive control) or E430G mutation (approximately 97% of the level induced by the positive control) also efficiently restored CDC activity. The CDC efficacy of the mixture of unglycosyl IgG1-CAMPATH-1H variants was lower than that of the mixture of glycosylated variants compared to the level induced by the positive control (E430G: approximately 72%; E345R: approximately 88%; E430Y: approximately 94%), but these combinations showed high CDC selectivity because the single-drug CDC activity of the unglycosyl variants was considerably lower than that of the glycosylated antibody variants.

[0303] Similar CDC efficacy results were observed for the IgG1-CAMPATH-1H antibody variant, the IgG1-11B8 antibody variant, and mixtures thereof (Figure 14B). The introduction of mutation N297A nearly suppressed the mono-agent activity of the IgG1-CAMPATH-1H-K439E variant having either the E430G or E345R mutation (both described in Example 8), whereas the mono-agent CDC activity of IgG1-CAMPATH-1H-E430Y-K439E (in a mixture with the complementary IgG1-b12 variant) was only partially suppressed by the introduction of mutation N297A. For IgG1-11B8-E430G-S440K and IgG1-11B8-E345R-S440K, single-agent CDC activity could not be detected (Example 8), but IgG1-11B8-E430Y-S440K induced relatively high residual CDC activity (to about 39% of the level induced by the positive control mixture). The residual CDC activity of IgG1-11B8-E430Y-S440K could be partially suppressed to about 10% of the level induced by the positive control mixture by introducing mutation N297A.

[0304] A mixture of glycosylated IgG1-CAMPATH-1H and IgG1-11B8 variants containing E430G, E345R, or E430Y, along with complementary auto-oligomerization inhibitory mutations, exhibited potent CDC activity recovery. Similar to what was observed for the unglycosyl IgG1-CAMPATH-1H antibody variant mixture in Figure 14A, a mixture of unglycosyl IgG1-CAMPATH-1H and IgG1-11B8 antibody variants containing E430G, E345R, or E430Y induced weak CDC efficacy recovery. However, the unglycosyl antibody variants demonstrated high CDC selectivity, as their single-drug CDC activity was considerably lower than that of the glycosylated IgG1-CAMPATH-1H and IgG1-11B8 antibody variants.

[0305] In conclusion, introducing the N297A mutation partially suppressed the monodrug CDC activity of IgG1-CAMPATH-1H-E430Y and IgG1-11B8-E430Y antibody variants containing further auto-oligomerization inhibitory mutations on Wien133 cells. CDC efficacy could be efficiently restored by mixing these unglycosyl-codependent antibody variants. While the CDC efficacy of the mixture of glycosylated IgG1-CAMPATH-1H and IgG1-11B8 antibody variants was high, the CDC selectivity was higher because the monodrug CDC activity of the unglycosyl variants was more strongly suppressed. The IgG1-CAMPATH-1H variant with the E430Y Fc-Fc interaction enhancing mutation showed considerable residual monodrug activity, reducing the selective window, while the variant IgG1-11B8-E430Y-S440K-N297A showed minimal monodrug activity.

[0306] Example 16: The CDC efficacy of a mixed glycosyl antibody variant containing mutations that enhance Fc-Fc interaction and mutations that inhibit autooligomerization is improved by introducing further C1q binding-enhancing mutations. In Examples 7 and 8, it was shown that single-drug CDC efficacy could be potently reduced by introducing a glycosylation-blocking mutation into CD52-targeted IgG1-CAMPATH-1H antibody variant and CD20-targeted IgG1-11B8 antibody variant, which also possess the Fc-Fc interaction-enhancing mutation E345K and a self-oligomerizing mutation. Codependent mixtures of such antibody variants were able to partially restore CDC efficacy compared to the level of the antibody variant containing only the Fc-Fc interaction-enhancing mutation E430G. Here, we investigated the effect of introducing further C1q-binding-enhancing mutations on the restoration of CDC efficacy after mixing the IgG1-CAMPATH-1H variant or the IgG1-CAMPATH-1H and IgG1-11B8 variants in the antibody variant containing the Fc-Fc interaction-enhancing mutation E345K.

[0307] Different mutations were introduced into the anti-CD52 IgG1-CAMPATH-1H antibody and the anti-CD20 IgG1-11B8 antibody: E430G or E345K, which induces enhanced Fc-Fc interaction; K439E or S440K, which inhibits autooligomerization; and N297A, which disrupts the N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. To allow direct comparison between the concentrations of individual components and the concentrations of mixtures composed of these components, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody. The purified antibody variants were tested at a range of concentrations (ranging from 0.01 to 40.0 μg / mL at 3.33-fold dilutions) using a PI CDC assay on Wien133 cells (N=3) with 20% NHS, essentially as described in Example 2, except that 30,000 cells / well were used in this example. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%), the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%; in the case of a mixture of IgG1-CAMPATH-1H antibody variants), or the AUC measured for the positive control mixture IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%; in the case of a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G antibody variants).

[0308] In Example 7, single-drug CDC activity could not be detected on Wien133 cells with IgG1-CAMPATH-1H-E345K-K439E-N297A and IgG1-CAMPATH-1H-E345K-S440K-N297A. However, by introducing further mutations that enhance C1q binding (K326A-E333S) into these constructs (E345K-K439E-N297A-K326A-E333S:SEQ ID NO 83; E345K-S440K-N297A-K326A-E333S:SEQ ID NO 84), single-drug CDC activity was increased (Figure 15A). When these complementary antibody variants were mixed, a complete, codependent restoration of CDC efficacy equivalent to that of the positive control IgG1-CAMPATH-1H-E430G was obtained. Similarly, in Example 8, single-agent CDC activity was not detected for IgG1-11B8-E345K-S440K-N297A, but partial CDC activity was observed after introducing the K326A-E333S mutation into this construct (Figure 15B). The mixture of IgG1-CAMPATH-1H-E345K-K439E-N297A-K326A-E333S + IgG1-11B8-E345K-S440K-N297A-K326A-E333S restored CDC efficacy, similar to the IgG1-Campath-1H-E430G + IgG1-11B8-E430G positive control mixture.

[0309] In summary, these data indicate that introducing the C1q binding-enhancing mutations K326A and E333S reduced the selectivity for CDC induction in Wien133 cells with IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing the Fc-Fc interaction-enhancing mutation E345K and an auto-oligomerization-inhibiting mutation. However, mutations K326A and E333S enabled extremely potent CDC recovery after mixing.

[0310] Example 17: Selectivity of CDC efficacy of non-glycosyl IgG2 antibody variants and IgG4 antibody variants having Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. In previous examples, it was shown that a glycosyl IgG1 antibody variant containing mutations that enhance Fc-Fc interactions and mutations that inhibit autooligomerization can selectively and codependently induce CDC in target cells. Here, we investigated whether CDC can also be selectively induced by glycosyl IgG2 and IgG4 antibody variants containing such mutations.

[0311] Anti-CD52 IgG2-CAMPATH-1H and IgG4-CAMPATH-1H antibodies, as well as anti-CD20 IgG2-11B8 and IgG4-11B8 antibodies, were introduced with different mutations: E430G or E345R, which induces enhanced Fc-Fc interactions; K439E or S440K, which inhibits auto-oligomerization; and N297A, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. As a control, one antibody was mixed 1:1 with an unbound isotype IgG2-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. In this example, except for the use of 30,000 cells / well, the purified antibody variant was tested at a range of concentrations (0.01 to 40.0 μg / mL at a 3.33-fold dilution) using a PI CDC assay on Wien133 cells (N=3) with 20% NHS, essentially as described in Example 2. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0312] A mixture of IgG2 variants (SEQ ID NO 74) of CAMPATH-1H antibody and 11B8 antibody, both possessing the E345R (SEQ ID NO 124) mutation, induced potent CDC in Wien133 cells to approximately 111% of the level induced by the positive control (Figure 16A). While the variant IgG2-CAMPATH-1H-E345R-K439E (SEQ ID NO 125) induced residual CDC activity to approximately 34% of the level induced by the positive control, introducing the N297A mutation (SEQ ID NO 127) into this variant resulted in single-drug CDC activity of approximately 48% of the level induced by the positive control. When the S440K mutation (SEQ ID NO 126) was introduced into the IgG2-11B8-E345R variant, the single-drug CDC activity decreased to approximately 18% of the level induced by the positive control. In contrast, when the N297A mutation (SEQ ID NO 128) was introduced into IgG2-11B8-E345R-S440K, the single-drug CDC activity increased to approximately 37% of the level induced by the positive control. A potent restoration of CDC efficacy was observed when IgG2-CAMPATH-1H-E345R-K439E+IgG2-11B8-E345R-S440K was mixed (to approximately 93% of the level induced by the positive control), or when IgG2-CAMPATH-1H-E345R-K439E-N297A+IgG2-11B8-E345R-S440K-N297A was mixed (to approximately 96% of the level induced by the positive control).

[0313] A mixture of IgG4 variants (SEQ ID NO 75) of CAMPATH-1H and 11B8 antibodies, both possessing the E345R (SEQ ID NO 129) mutation, induced CDC in Wien133 cells to approximately 71% of the level induced by the positive control (Figure 16B). No significant single-agent CDC activity was detected for IgG4-CAMPATH-1H-E345R variants possessing either the K439E (SEQ ID NO 130) or K439E-N297A (SEQ ID NO 132) mutations. Similarly, IgG4-11B8-E345R variants possessing either the S440K (SEQ ID NO 131) or S440K-N297A (SEQ ID NO 133) mutations induced only very low levels of CDC activity. Mixing IgG4-CAMPATH-1H-E345R-K439E and IgG4-11B8-E345R-S440K resulted in partial recovery of CDC efficacy to approximately 50% of the level induced by the positive control. Mixing with the non-glycosyl variant of the latter antibody variant (IgG4-CAMPATH-1H-E345R-K439E-N297A + IgG4-11B8-E345R-S440K-N297A) only restored CDC efficacy to approximately 14% of the level induced by the positive control.

[0314] In conclusion, a mixture of unglycosyl IgG2 antibody variants containing the Fc-Fc interaction-enhancing mutation E345R and the auto-oligomerization-inhibiting mutation K439E or S440K was able to selectively induce CDC in Wien133 cells in a codependent manner. Individual glycosylated IgG2 antibody variants induced a considerable level of residual single-drug CDC activity, which could not be suppressed by the introduction of the N297A mutation. Although the window of CDC selectivity for IgG2 antibody variants was more limited than that of equivalent IgG1 variants, as described in Examples 23 and 24, the absence of FcγR activation by unglycosyl IgG2 variants may make unglycosyl-codependent IgG2 antibody variants usable in applications where single-drug CDC activity does not pose a potential risk. No single-drug CDC activity was observed for glycosylated IgG4 antibody variants and unglycosyl IgG4 antibody variants containing mutations that enhance Fc-Fc interaction and mutations that inhibit auto-oligomerization. However, even when IgG4 antibody variants with the above mutations were mixed in, only partial restoration of CDC efficacy could be achieved.

[0315] Example 18: Selectivity for CDC induction by a non-glycosyl IgG1-CAMPATH-1H antibody variant having Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. In previous examples, the effect of disrupting the N-glycosylation consensus sequence on CDC efficacy was evaluated for different antibody-target combinations, using IgG1 antibody variants with Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations. Here, the CDC efficacy on Wien133 cells induced by single-agent antibody variants or mixtures of such variants was evaluated for variants containing the auto-oligomerization-inhibiting mutation K439E or S440K, the N297A mutation that disrupts the N-glycosylation consensus sequence, and one of the Fc-Fc interaction-enhancing mutations E430G, E430Y, E345K, or E345R.

[0316] Different mutations were introduced into the anti-CD52 IgG1-CAMPATH-1H antibody: E430G, E430Y, E345K, or E345R, which enhance Fc-Fc interactions; K439E or S440K, which inhibit auto-oligomerization; and N297A, which disrupts the N297 N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of the mixture composed of the individual components. Where indicated, the above mutations were also introduced into the IgG1-b12 unbound control antibody. In this example, the purified antibody variant was tested at a range of concentrations (0.01 to 100.0 μg / mL final concentration at 2.5-fold dilution) using a PI CDC assay on Wien133 cells (N=3) with 20% NHS, essentially the same as described in Example 2, except that 30,000 cells / well were used. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%).

[0317] When IgG1-CAMPATH-1H-E430Y was introduced with the K439E or S440K mutation, the single-agent CDC efficacy was approximately 92% and 97% of the levels induced by the positive control IgG1-CAMPATH-1H-E430G, respectively (Figure 17A). Mixtures of these antibody variants increased CDC efficacy to approximately 122% of the levels induced by the positive control. IgG1-CAMPATH-1H variants with the K439E-N297A mutation induced varying levels of residual single-agent CDC activity, ranging from approximately 5% (E345K), 12% (E430G), and 36% (E345R) to 60% (E430Y) of the levels induced by the positive control, depending on the introduced Fc-Fc interaction-enhancing mutation. The single-drug CDC activity of IgG1-CAMPATH-1H variants containing S440K-N297A was also dependent on the induced Fc-Fc interaction enhancing mutation, but the residual CDC activity of variants containing the S440K-N297A mutation was generally higher than that of constructs containing the K439E-N297A mutation. The residual CDC activity of IgG1-CAMPATH-1H variants containing both the S440K-N297A mutation and the Fc-Fc interaction enhancing mutation was approximately 20% (E345K), 27% (E430G), and 47% (E345R) to 65% (E430Y).

[0318] Mixtures of IgG1-CAMPATH-1H antibody variants containing either the N297A mutation or an Fc-Fc interaction-enhancing mutation, in addition to the K439E or S440K mutation, efficiently restored CDC efficacy. The mixtures of IgG1-CAMPATH-1H-E430Y-K439E-N297A+IgG1-CAMPATH-1H-E430Y-S440K-N297A and IgG1-CAMPATH-1H-E345R-K439E-N297A+IgG1-CAMPATH-1H-E345R-S440K-N297A most efficiently restored CDC efficacy to approximately 95% of the level induced by the positive control. Codependent mixtures of IgG1-CAMPATH-1H variants containing either the E430G or E345K mutation restored CDC efficacy to approximately 84% and 87% of the level induced by the positive control, respectively.

[0319] At the highest concentrations tested, all mixtures induced CDC to levels comparable to the positive control (Figure 17B). IgG1-CAMPATH-1H variants with the K439E-N297A mutation and any of the E430G, E345K, E430Y, or E345R mutations induced CDC at various efficacy levels from 44% (E345K) to 82% (E430Y), while IgG1-CAMPATH-1H variants with the S440K-N297A mutation and any of the E430G, E345K, E430Y, or E345R mutations induced CDC at efficacy levels from 51% (E345K) to 82% (E430Y).

[0320] In conclusion, the non-glycosyl IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation exhibits lower residual single-agent CDC activity on Wien133 cells compared to the variant with the S440K mutation when the K439E mutation is introduced. Furthermore, the data presented in this example show that CDC efficacy was most potently restored by a mixture of codependent non-glycosyl IgG1-CAMPATH-1H antibody variants with either the E430Y or E345R Fc-Fc interaction-enhancing mutations. However, such mixtures exhibit relatively high single-agent CDC activity and are associated with low CDC selectivity. The most selective antibody variant is the one with the E430G mutation following the E345K mutation, which induced relatively low single-agent activity but relatively potent CDC recovery as a mixture.

[0321] Example 19: Analysis of the effect of introducing a mutation that disrupts glycosylation into an IgG1-CAMPATH-1H antibody variant containing both an Fc-Fc interaction-enhancing mutation and an auto-oligomerization-inhibiting mutation on FcγRIIa activation. In Example 10, it was shown that introducing a mutation that inhibits glycosylation into an IgG1-CAMPATH-1H antibody variant having an Fc-Fc interaction-enhancing mutation suppressed ADCP induction ability as measured by an FcγRIIa activation reporter cell assay. Here, the inventors investigated whether introducing the glycosylation-inhibiting mutation N297A into an IgG1-CAMPATH-1H antibody variant and an IgG1-11B8 antibody variant or a mixture thereof, having an Fc-Fc interaction-enhancing mutation E430G, E430Y, or E345R and an autooligomerization-inhibiting mutation, would affect the FcγRIIa activation ability in a reporter cell assay, which is a surrogate for antibody-dependent phagocytosis (ADCP).

[0322] For ADCP reporter bioassays, IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing Fc-Fc interaction-enhancing mutations (E430G, E430Y, or E345R), auto-oligomerization-inhibiting mutations (K439E or S440K), and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A) were tested on Raji cells using the Bio-Glo Luciferase Assay System (Promega, catalog number G7941). As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. This kit contains Jurkat human T cells engineered to stably express high-affinity FcγRIIa (H131 allotype) and an activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, as effector cells. In short, Raji cells (10 μL; 5,000 cells / well) were seeded into a 384-well white opaque flat-bottom plate (Optiplate white; Perkin-Elmer; catalog number 6007680) containing RPMI-1640 [(Promega, catalog number G708A), supplemented with 4% low-IgG serum (Promega, catalog number G711A)]. The Raji cells were pre-incubated with an antibody concentration series (10 μL; 4-fold dilution series, with final concentrations ranging from 0.2 to 40,000 ng / mL after effector cell addition) at 37°C / 5% CO2 for 15 minutes. Subsequently, thawed Jurkat FcγRIIa H131 allotype effector cells (10 μL; 30,000 cells / well; Promega, catalog number G988A) were added, and the cells were incubated at 37°C / 5% CO2 for 5.5 hours. After this incubation, the cells were allowed to equilibrate to room temperature for 15 minutes.Subsequently, 30 μL of Bio-Glo Assay Luciferase Reagent [Bio-Glo Luciferase Assay Substrate (Promega, catalog number G720A)] dissolved in Bio-Glo Luciferase Assay Buffer (Promega, catalog number G719A)] was added to each well, and incubated in the dark at RT for 15 minutes. Luciferase production was quantified by luminescence reading using an EnVision Multilabel Reader (Perkin Elmer). To suppress plate-to-plate variability in absolute luminescence intensity, raw luminescence values ​​were normalized to an internal IgG1 control antibody at a concentration of 40 μg / mL, measured three times in each plate before pooling. The area under the relative dose-response curve (AUC) using logarithmically transformed concentrations was normalized against the AUC measured for the unbound negative control IgG1-b12 (0%) and against the AUC measured for the positive control IgG1-CAMPATH-1H-E430G (100%; Figure 18A) or the AUC measured for IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%, Figure 18B).

[0323] Compared to IgG1-CAMPATH-1H-E430G, IgG1-CAMPATH-1H-E430Y (SEQ ID NO 76) exhibited enhanced FcγRIIa activation induction ability (Figure 18A). Single-agent IgG1-CAMPATH-1H antibody variants containing mutations E430Y-K439E, E430Y-K439E-N297A, E430G-K439E-N297A, or E345R-K439E-N297A did not induce near-background FcγRIIa activation or levels. Such low levels of FcγRIIa activation should be observed in relation to the high sensitivity of assays applicable to any FcγRIIa activation. IgG1-CAMPATH-1H-E430Y-S440K induced low levels of FcγRIIa activation, but introduction of mutation N297A completely suppressed FcγRIIa activation. FcγRIIa activation was not detected in non-glycosyl S440K-containing antibody variants that possessed E430G or E345R instead of E430Y.

[0324] A mixture of IgG1-CAMPATH-1H-E430Y-K439E and IgG1-CAMPATH-1H-E430Y-S440K induced FcγRIIa activation to a level comparable to that of the positive control. When one of the IgG1-CAMPATH-1H antibody variants in the mixture contained the N297A mutation, this FcγRIIa activation was strongly reduced but not completely suppressed. FcγRIIa activation was completely suppressed, or reduced to weak residual FcγRIIa activation, when the mixture contained two antibody variants, both containing the N297A mutation (either E430G or E345R).

[0325] In Example 10, the IgG1-11B8-E430G-S440K variant was shown to induce low levels of FcγRIIa activation, while FcγRIIa activation induced by IgG1-11B8 monochemicals containing the E430G-S440K-N297A, E345R-S440K-N297A, or E430Y-S440K-N297A mutations was suppressed (Figure 18B). In Example 10, it was also shown that a mixture of IgG1-CAMPATH-1H-E430G-K439E + IgG1-11B8-E430G-S440K induced low levels of FcγRIIa activation compared to a positive control. However, both variants were glycosyl, and a mixture of IgG1-CAMPATH-1H and IgG1-11B8 variants, in which Fc-Fc interactions were enhanced (E430G, E345R, or E430Y mutations) and auto-oligomerization was inhibited, did not show FcγRIIa activation.

[0326] In summary, when the N297A mutation, which disrupts the glycosylation site, was introduced into IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants with enhanced Fc-Fc interaction and inhibited self-oligomerization, the ability to induce FcγRIIa activation was efficiently suppressed in a high-sensitivity reporter assay. Similarly, when the N297A mutation was introduced into both antibody variants in a mixture, such mixtures did not induce FcγRIIa activation, except for a mixture of two IgG1-CAMPATH-1H variants containing the E430Y mutation, which induced weak residual FcγRIIa activation. This suggests that codependent mixtures of non-glycosyl, Fc-Fc interaction-enhanced antibody variants lack ADCP-inducing ability.

[0327] Example 20: Analysis of the effect of introducing a mutation that disrupts glycosylation into an IgG1-CAMPATH-1H antibody variant having mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding, on FcγRIIa activation. In Example 19, it was shown that introducing the glycosylation-disrupting mutation N297A into IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, in which Fc-Fc interaction was enhanced and auto-oligomerization was inhibited, efficiently suppressed ADCP induction ability as measured by an FcγRIIa-activated reporter cell assay. Here, we investigated whether introducing further mutations that enhance C1q binding into such IgG1-CAMPATH-1H variants and IgG1-11B8 variants or mixtures thereof would affect their ability to activate FcγRIIa in a reporter cell assay surrogate for ADCP.

[0328] For ADCP reporter bioassays, IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants having Fc-Fc interaction-enhancing mutations (E430G, E430Y, or E345R), auto-oligomerization-inhibiting mutations (K439E or S440K), mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A), and mutations enhancing C1q binding (K326A-E333S) were tested on Raji cells using the Bio-Glo Luciferase Assay System (Promega, catalog number G7941), essentially as described in Example 19. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. To suppress plate-to-plate variability in absolute luminescence intensity, raw luminescence values ​​were measured three times on each plate at a concentration of 40 μg / mL and normalized against an internal IgG1 control antibody before pooling. The area under the relative dose-response curve using logarithmically transformed concentrations (AUC values ​​in a 4-fold dilution series, with final concentrations ranging from 0.2 to 40,000 ng / mL after effector cell addition) was normalized against the AUC values ​​measured against the unbound negative control IgG1-b12 (0%) and against the AUC values ​​measured against the positive control IgG1-CAMPATH-1H-E430G (100%; Figure 19A) or against IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%, Figure 19B).

[0329] Potent FcγRIIa activation was detected by the IgG1-CAMPATH-1H-E430G antibody variant (Figure 19A). For IgG1-CAMPATH-1H variants containing the E345K-K439E-N297A or E345K-S440K-N297A mutation in addition to the C1q-binding enhancing mutation K326A-E333S, no FcγRIIa activation induction ability was detected. Similarly, a mixture of the latter two variants did not induce FcγRIIa activation at all.

[0330] The ability of the IgG1-11B8 antibody variant with the E345K-S440K-N297A-K326A-E333S mutation to induce FcγRIIa activation as a single agent was quite similar to that of the IgG1-CAMPATH-1H variant, as no single-agent FcγRIIa activation was detected (Figure 19B). Similarly, a mixture of IgG1-CAMPATH-1H-E345K-K439E-N297A-K326A-E333S and IgG1-11B8-E345K-S440K-N297A-K326A-E333S also did not induce detectable FcγRIIa activation.

[0331] In conclusion, these data indicate that the C1q-enhanced, non-glycosyl IgG1-CAMPATH-1H antibody variant and IgG1-11B8 antibody variant, possessing either the E345K or K439E or S440K mutation, lack the ability to induce FcγRIIa activation in high-sensitivity FcγRIIa reporter cell assays, whether tested as a single agent or as a mixture.

[0332] Example 21: Analysis of the effect of introducing a mutation that disrupts glycosylation into an IgG1-CAMPATH-1H antibody variant containing both an Fc-Fc interaction-enhancing mutation and a self-oligomerization-inhibiting mutation on FcγRIIIa activation. In Example 11, it was shown that the ADCC-inducing ability of an IgG1-CAMPATH-1H antibody variant with an Fc-Fc interaction-enhancing mutation could be efficiently suppressed by introducing the N297A mutation, which disrupts N-linked glycosylation, as measured by an FcγRIIIa-activating reporter cell assay. Here, the inventors investigated whether the introduction of the N297A mutation affected the ability of IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants or mixtures thereof, which have mutations that enhance Fc-Fc interaction and mutations that inhibit autooligomerization, to activate FcγRIIIa in a reporter cell assay surrogate for ADCC.

[0333] For ADCC reporter bioassays, IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing Fc-Fc interaction-enhancing mutations (E430G, E430Y, or E345R), auto-oligomerization-inhibiting mutations (K439E or S440K), and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A) were tested on Raji cells using the Bio-Glo Luciferase ADCC Reporter Bioassay (Promega, catalog number G7018) against the FcγRIIIa high-affinity V158 allotype. As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. This kit contains Jurkat human T cells engineered to stably express the high-affinity V158 allotype of FcγRIIIa and an activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, as effector cells. In this example, the assay was carried out essentially as described in Example 19, except that FcγRIIIa-V effector cells (Promega; catalog number G701A) were used. To suppress plate-to-plate variability in absolute luminescence intensity, raw luminescence values ​​were normalized before pooling to an internal IgG1 control antibody measured three times in each plate at a concentration of 40 μg / mL. The area under the relative dose-response curve using logarithmically transformed concentrations (AUC values ​​for a 4-fold dilution series, where the final concentration was 0.2 to 40,000 ng / mL after the addition of effector cells) was normalized against the AUC value (0%) measured for the unbound negative control IgG1-b12 and the AUC value (100%; Figure 20A) measured for the positive control IgG1-CAMPATH-1H-E430G or the AUC value (100%, Figure 20B) measured for IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G.

[0334] A highly sensitive ADCC reporter assay demonstrated that the IgG1-CAMPATH-1H antibody variant, either as a wild-type antibody or containing the Fc-Fc interaction-enhancing mutations E430G or E430Y, exhibited potent ability to induce FcγRIIIa activation (Figure 20A). Introducing the K439E mutation into IgG1-CAMPATH-1H-E430Y did not affect its ability to induce FcγRIIIa activation. In contrast, for the non-glycosyl variant of IgG1-CAMPATH-1H-K439E, FcγRIIIa activation was either not detected (variants containing E430G or E345R) or strongly suppressed activation induction was detected (variants containing E430Y). Similarly, the IgG1-CAMPATH-1H variant with the S440K mutation showed potent FcγRIIIa activation, whereas the glycosyl variant of the IgG1-CAMPATH-1H-S440K variant, which had enhanced Fc-Fc interactions, showed either potently reduced FcγRIIIa activation (varieties containing E430Y) or no FcγRIIIa activation at all (varieties containing E430G or E345R).

[0335] Introducing the K439E or S440K mutation into a mixture of IgG1-CAMPATH-1H-E430Y antibody variants only slightly reduced their ability to induce FcγRIIIa activation. A mixture in which one antibody variant was glycosylated and the other unglycosylated also induced FcγRIIIa activation, with the efficacy depending on which Fc-Fc interaction-enhancing mutation was introduced. Specifically, a mixture of variants containing E430Y induced the strongest FcγRIIIa activation, while variants containing E430G and E345R showed reduced efficacy in inducing FcγRIIIa activation. A mixture of two unglycosyl (containing N297A) IgG1-CAMPATH1-H-E430Y variants with either the K439E or S440K mutation strongly reduced FcγRIIIa activation. In contrast, FcγRIIIa activation could not be detected in such a mixture consisting of two nonglycosyl IgG1-CAMPATH-1H variants, each possessing either the K439E or S440K mutation in addition to the E430G or E345R mutation.

[0336] Similar results were obtained for the glycosyl IgG1-11B8 variant and for a mixture of the IgG1-CAMPATH-1H variant and the IgG1-11B8 variant (Figure 20B). The glycosyl IgG1-11B8 variant with any of the E430G, E345R, or E430Y mutations did not induce FcγRIIIa activation. Similarly, no FcγRIIIa activation was observed for a mixture of the glycosyl IgG1-CAMPATH-1H variant and the IgG1-11B8 variant with any of the E430G, E345R, or E430Y mutations in addition to the K439E or S440K mutation.

[0337] In conclusion, introducing the glycosylation-disrupting mutation N297A into IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants containing the Fc-Fc interaction-enhancing mutations E430G, E345R, or E430Y, along with the K439E or S440K mutation, strongly reduced (in the case of variants containing E430Y) or completely suppressed (in the case of variants containing E430G or E345R) their ability to induce FcγRIIIa activation when used as a single agent. Similarly, no FcγRIIIa activation was detected in a mixture of two codependent, non-glycosyl IgG1-CAMPATH-1H variants and / or IgG1-11B8 variants containing the Fc-Fc interaction-enhancing mutations, although a mixture of two IgG1-CAMPATH-1H variants containing only the E430Y mutation induced weak residual FcγRIIIa activation. It should be noted that the assay applied has high sensitivity for detecting FcγRIIIa activation. A mixture consisting of one unglycosyl antibody variant and one glycosylated antibody variant still induced FcγRIIIa activation; therefore, both antibody variants in the mixture must be unglycosyl to completely eliminate FcγRIIIa activation.

[0338] Example 22: Analysis of the effects of introducing mutations that disrupt glycosylation into IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants, which have mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding, on FcγRIIIa activation. In Example 21, it was shown that the ADCC-inducing ability of IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants having mutations that enhance Fc-Fc interaction and mutations that inhibit autooligomerization, as measured by an FcγRIIIa-activating reporter cell assay, can be efficiently suppressed by introducing the N297A mutation, which disrupts N-linked glycosylation. Here, we investigated whether the ability of such IgG1-CAMPATH-1H antibody variants and IgG1-11B8 antibody variants or mixtures thereof, further possessing the C1q-binding-enhancing mutation K326A-E333S, to activate FcγRIIIa in a reporter cell assay surrogate for ADCC is affected by introducing the N297A mutation.

[0339] For ADCC reporter bioassays, anti-CD52 IgG1-CAMPATH-1H antibody variants and anti-CD20 IgG1-11B8 antibody variants were tested on Raji cells using the Bio-Glo Luciferase ADCC Reporter Bioassay (Promega, catalog number G7018) against the FcγRIIIa high-affinity V158 allotype. These variants contained mutations that enhance Fc-Fc interaction (E430G or E345K), mutations that inhibit autooligomerization (K439E or S440K), mutations that enhance binding between C1q and the antibody Fc domain (K326A-E333S), and mutations that disrupt the N297 N-glycosylation consensus sequence (NX[S / T]; N297A). As a control, one antibody was mixed 1:1 with an unbound isotype IgG1-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of the mixtures composed of those components. This kit contains Jurkat human T cells engineered to stably express the high-affinity V158 allotype of FcγRIIIa and the activated T cell nuclear factor (NFAT) response element that drives firefly luciferase expression, as effector cells. In this example, the assay was carried out essentially as described in Example 19, except that FcγRIIIa-V effector cells (Promega; catalog number G701A) were used. To suppress plate-to-plate variability in absolute luminescence intensity, raw luminescence values ​​were normalized before pooling to an internal IgG1 control antibody measured three times in each plate at a concentration of 40 μg / mL. The area under the relative dose-response curve using logarithmically transformed concentrations (AUC values ​​for a 4-fold dilution series, where the final concentration was 0.2 to 40,000 ng / mL after the addition of effector cells) was normalized against the AUC values ​​measured for the unbound negative control IgG1-b12 (0%) and against the AUC values ​​measured for the positive control IgG1-CAMPATH-1H-E430G (100%; Figure 21A) or IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%, Figure 21B).

[0340] In a high-sensitivity ADCC reporter assay, neither the single agents IgG1-CAMPATH-1H-E345K-K439E-N297A-K326A-E333S nor IgG1-CAMPATH-1H-E345K-S440K-N297A-K326A-E333S were detected to induce FcγRIIIa activation (Figure 21A). Mixing the latter antibody variant did not restore the efficacy in inducing FcγRIIIa activation.

[0341] The efficacy of IgG1-11B8 antibody variants containing the E345K-S440K-N297A-K326A-E333S mutation in inducing FcγRIIIa activation was also studied (Figure 21B). No FcγRIIIa activation was detected with this variant, either when used as a single agent or mixed with IgG1-CAMPATH-1H-E345K-K439E-N297A-K326A-E333S.

[0342] In conclusion, nonglycosyl IgG1-11B8 antibody variants containing the Fc-Fc interaction-enhancing mutation E345K, the auto-oligomerization-inhibiting mutation (K439E or S440K), and the C1q-binding-enhancing mutation K326A-E333S did not induce FcγRIIIa activation, either as monotherapy or when mixed with complementary IgG1-CAMPATH-1H antibody variants.

[0343] Example 23: Analysis of the effects of introducing glycosylation-disrupting mutations into CAMPATH-1H and 11B8 IgG2 antibody variants and IgG4 antibody variants containing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations on FcγRIIa activation. In Example 17, it was shown that a mixture of IgG2 antibody variants containing the E345R Fc-Fc interaction enhancing mutation and the auto-oligomerization inhibiting mutation K439E or S440K could selectively induce CDC in Wien133 cells in a codependent manner. A mixture of such unglycosyl antibody variants of the IgG4 subclass could also selectively induce CDC in Wien133 cells, although CDC recovery was limited. Here, we investigated whether the FcγRIIa activation ability of CAMPATH-1H and 11B8 IgG2 antibody variants and IgG4 antibody variants or mixtures thereof, containing the Fc-Fc interaction enhancing mutation E345R and the auto-oligomerization inhibiting mutation, was affected in a reporter cell assay surrogate for ADCP by introducing the N-linked glycosylation disrupting mutation N297A.

[0344] For ADCP reporter bioassays, anti-CD52 IgG2-CAMPATH-1H antibody variants and anti-CD20 IgG2-11B8 antibody variants, as well as IgG4-CAMPATH-1H antibody variants and IgG4-11B8 antibody variants, were tested on Raji cells using the Bio-Glo Luciferase Assay System (Promega, catalog number G7940), essentially as described in Example 19, using Fc-Fc interaction enhancing mutations (E345R), auto-oligomerization inhibiting mutations (K439E or S440K), and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A). As a control, one antibody was mixed 1:1 with an unbound isotype IgG2- or IgG4-b12 control antibody to allow direct comparison of the concentrations of individual components with the concentrations of mixtures composed of those components. The area under the relative dose-response curve using logarithmically transformed concentrations (AUC values ​​for a 4-fold dilution series, where the final concentration was 0.2–40 μg / mL after the addition of effector cells) was normalized against the AUC values ​​measured for the unbound negative control IgG1-b12 (0%) and the AUC values ​​measured for the positive control mixture IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0345] Compared to the positive control mixture, the mixture of CAMPATH-1H and 11B8 IgG2 variants with the E345R mutation enhanced the ability to induce FcγRIIa activation (Figure 22A). Introducing K439E into IgG2-CAMPATH-1H sharply reduced its FcγRIIa activation ability, but no residual FcγRIIa activation was detected in the non-glycosyl variant IgG2-CAMPATH-1H-E345R-K439E-N297A. The IgG2-11B8-E345R-S440K antibody variant induced potent FcγRIIa activation to approximately 191% of the level induced by the positive control, but this activity was completely suppressed by introducing the N297A mutation. A mixture of IgG2-CAMPATH-1H-E345R-K439E and IgG2-11B8-E345R-S440K induced high levels of FcγRIIa activation (approximately 179% of the level induced by the positive control), while a mixture of these antibody variants with non-glycosyl (containing N297A) variants completely suppressed their ability to induce FcγRIIa activation.

[0346] The mixture of IgG4-CAMPATH-1H-E345R and IgG4-11B8-E345R enhanced the ability to induce FcγRIIa activation compared to the positive control mixture (Figure 22B). When used as a single agent, neither the IgG4-CAMPATH-1H-E345R antibody variant with the N297A mutation and mutations K439E or S440K, or the IgG4-11B8-E345R antibody variant with the N297A mutation but K439E or S440K, induced FcγRIIa activation. A weak recovery of FcγRIIa activation was observed only with the IgG4-CAMPATH-1H-E345R-K439E+IgG4-11B8-E345R-S440K mixture, and not with the IgG4-CAMPATH-1H-E345R-K439E-N297A+IgG4-11B8-E345R-S440K-N297A mixture.

[0347] In conclusion, introducing the N297A mutation, which disrupts the glycosylation site, into CAMPATH-1H and 11B8 antibody variants of IgG2 and IgG4 subclasses, which possess the E345R Fc-Fc interaction-enhancing mutation and the auto-oligomerization-inhibiting mutation, completely suppressed ADCP induction ability as measured by a surrogate FcγRIIa-activated reporter cell assay. This also applied to the application of monodrug antibody variants and their codependent mixtures.

[0348] Example 24: Analysis of the effects of introducing glycosylation-disrupting mutations into CAMPATH-1H and 11B8 IgG2 antibody variants and IgG4 antibody variants containing Fc-Fc interaction-enhancing mutations and auto-oligomerization-inhibiting mutations on FcγRIIIa activation. In Example 23, it was shown that introducing the glycosylation-disrupting mutation N297A into CAMPATH-1H antibody variants and 11B8 antibody variants of IgG2 and IgG4 subclasses, which have the E345R Fc-Fc interaction-enhancing mutation and the auto-oligomerization-inhibiting mutation K439E or S440K, completely suppressed ADCP induction in a surrogate FcγRIIa activation reporter cell assay. Here, we tested whether unglycosyl IgG2 antibody variants and IgG4 antibody variants of CAMPATH-1H and 11B8, or mixtures thereof, which have the Fc-Fc interaction-enhancing mutation E345R and the auto-oligomerization-inhibiting mutation, can activate FcγRIIIa in a reporter cell assay surrogate for ADCC.

[0349] For the ADCC reporter bioassay, the Bio-Glo Luciferase ADCC Reporter Bioassay (Promega, catalog number G7018) against the FcγRIIIa high affinity V158 allotype was used on Raji cells to test anti-CD52 IgG2-CAMPATH-1H antibody variants and anti-CD20 IgG2-11B8 antibody variants, as well as IgG4-CAMPATH-1H antibody variants and IgG4-11B8 antibody variants, possessing Fc-Fc interaction enhancing mutations (E345R), auto-oligomerization inhibiting mutations (K439E or S440K), and mutations disrupting the N297 N-glycosylation consensus sequence (NX[S / T]; N297A). In this example, the assay was performed essentially as described in Example 19, except that FcγRIIIa-V effector cells (Promega; catalog number G701A) were used. As a control, one antibody was mixed 1:1 with an unbound isotype IgG2- or IgG4-b12 control antibody to allow direct comparison between the concentrations of individual components and the concentrations of mixtures composed of those components. The area under the relative dose-response curve using logarithmically transformed concentrations (AUC values) for a 4-fold dilution series, resulting in a final concentration of 0.2–40 μg / mL after the addition of effector cells, was normalized against the AUC values ​​measured for the unbound negative control IgG1-b12 (0%) and the AUC values ​​measured for the positive control mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0350] The positive control mixture of IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G efficiently induced FcγRIIIa activation. However, the ability of each of the tested IgG2 and IgG4 variants, and their mixture, to induce FcγRIIIa activation was less than 5% of the level induced by the positive control mixture (Figure 23A,B). These low levels of FcγRIIIa activation prevent us from drawing conclusions about the impact of introducing the glycosylation-disrupting mutation N297A into these antibody variants.

[0351] Example 25: Induction of programmed cell death by a nonglycosyl anti-Fas antibody variant having mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding. In previous examples, it was shown that a codependent mixture of non-glycosyl antibody variants having mutations that enhance Fc-Fc interactions, mutations that inhibit autooligomerization, and / or mutations that modulate C1q binding selectively induces CDC in tumor cell lines. Here, we investigated whether a mixture of non-glycosyl anti-Fas antibody variants with such mutations can induce selective, codependent programmed cell death (PCD) in target cells.

[0352] Different mutations were introduced into the anti-Fas IgG1-Fas-E09 antibody: E345R, which enhances Fc-Fc interaction; K439E or S440K, which inhibits autooligomerization; K326A and E333S, which enhance binding between C1q and the antibody Fc domain; and N297A, which disrupts the N-glycosylation consensus sequence (NX[S / T]) and produces an unglycosyl antibody. A range of antibody variant concentrations were tested in a programmed cell death assay using B lymphoblast WIL2S-SF cells. WIL2-S SF cells were derived from WIL2-S (ATCC, CRL-8885) B lymphoblasts and adapted to grow under serum-free conditions in a culture medium (Perbio, catalog no. SH30349) prepared with HyQ-ADCF-Mab containing 50 U / mL Pen / Strep and 1 mM sodium pyruvate. WIL2S-SF cells were collected and passed through a cell strainer. Cells were pelleted by centrifugation at 300g for 5 minutes and resuspended in serum-free culture medium (HyQ ADCF-Mab + 1mM sodium pyruvate (Lonza, catalog no. BE13-115E) containing L-glutamine from HyClone.Cat SH30349). Single cell suspensions of 50,000 cells were seeded in 46 μL per well of a 96-well flat-bottom polystyrene plate (Greiner Bio-One, catalog no. 655180), and 24 μL of purified human C1q stock solution (Complement Tech, catalog no. A099, final concentration 2.5 μg / mL) was added. In addition, 50 μL of antibody dilution series samples (5-fold dilution with a final concentration range of 0.05 ng / mL to 20 μg / mL) were added. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, catalog number S6942). Cell culture viability was determined after 24 hours of incubation using the CellTiter-Glo luminescence cell viability assay (Promega, catalog number G7571), which quantifies the amount of ATP present, an indicator of metabolically active cells. 12 μL of luciferin solution reagent from the kit was added to each well. The plates were then incubated at 37°C for 1.5 hours.Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer). The relative efficacy in inducing cell death is given by the following formula: Cell death induction efficacy = [AUC(IgG1-b12)-AUC(sample)] The area under the dose-response curve (AUC) was compared using logarithmically transformed concentrations, and then normalized to the cell death-inducing efficacy (100%) obtained for the positive control IgG1-Fas-E09-E345R.

[0353] Wild-type IgG1-Fas-E09 did not induce programmed cell death (PCD) in WIL2S-SF cells, but the introduction of the E345R mutation strongly enhanced PCD induction (Figure 24A). PCD was not induced in the aglycosyl IgG1-Fas-E09 variant with both the E345R and K439E mutations, nor in the variant with the same mutation in addition to the C1q-binding-enhancing mutation K326A-E333S (SEQ ID NO 85). Similarly, the aglycosyl IgG1-Fas-E09 variant with the S440K mutation, with or without the C1q-binding-enhancing mutation K326A-E333S (SEQ ID NO 86), could not induce PCD in WIL2S-SF cells as a single agent. PCD efficacy was not observed even at the highest concentration tested (20 μg / mL; Figure 24B). A mixture of IgG1-Fas-E09-E345R-K439E-N297A and IgG1-Fas-E09-E345R-S440K-N297A antibody variants was able to partially restore the efficacy of inducing PCD to approximately 49% of the level induced by the positive control mixture. At the highest concentration tested, this mixture could only partially restore PCD. Complete restoration of PCD efficacy was induced by a codependent mixture of IgG1-Fas-E09-E345R-K439E-K326A-E333S-N297A and IgG1-Fas-E09-E345R-S440K-K326A-E333S-N297A.

[0354] In short, a mixture of non-glycosyl antibody variants containing mutations that enhance Fc-Fc interactions, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding completely restored the ability of the anti-Fas-E09 antibody to induce PCD in WIL2S-SF cells. In contrast, a mixture of antibody variants without the C1q-binding-enhancing mutation showed only partial restoration. This suggests that enhanced C1q recruitment may be particularly advantageous for the agonist applications of the interdependent antibody mixture of the present invention. While not limited by theory, this may be explained by the stabilization of the multimeric Fas complex that induces PCD signaling after binding to an antibody directed against Fas that recruits the hexavalent C1q protein, which can act as a soluble crosslinker.

[0355] Example 26: Selective DR5 agonist activity on BxPC-3 cells by a mixture of two non-competitive, glycosyl anti-DR5 antibody variants having mutations that enhance Fc-Fc interaction, mutations that inhibit self-oligomerization, and mutations that enhance C1q binding. A mixture of two anti-death receptor 5 (DR5) antibodies, IgG1-DR5-01-G56T-E430G and IgG1-DR5-05-E430G, which can independently bind to non-overlapping binding sites, acts as a DR5 agonist that induces the death of DR5-positive cancer cells (WO17093447). Here, we investigated whether a glycosyl variant of such a DR5-targeted antibody, possessing mutations that enhance Fc-Fc interaction, mutations that inhibit autooligomerization, and mutations that enhance C1q binding, can selectively induce the death of COLO205 colon cancer cells, which are relatively sensitive to DR5-mediated PCD induction, and BxPC-3 pancreatic cancer cells, which are relatively resilient to DR5-mediated PCD induction.

[0356] Different mutations were introduced into anti-DR5 IgG1-hDR5-01-G56T and IgG1-hDR5-05 antibodies: E430G, E430Y, or E345R to enhance Fc-Fc interaction; K439E or S440K to inhibit auto-oligomerization; K326A and E333S to enhance binding between C1q and the antibody Fc domain; and N297A to disrupt the N-glycosylation consensus sequence (NX[S / T]) and produce an unglycosyl antibody. A range of antibody variant concentrations were tested in killing assays using BxPC-3 or COLO205 cells. BxPC-3 cells (ATCC, catalog no. CRL-1687) and COLO205 cells (ATCC, catalog no. CCL-222) were collected by trypsin treatment and passed through a cell strainer. Cells were pelleted by centrifugation at 300g for 5 minutes and resuspended in culture medium (RPMI 1640 Medium (ATCC Modification; Life Technologies, catalog number A10491-01) + 10% DBSI (Life Technologies, catalog number 20371)). 5,000 single-cell suspensions were seeded into 46 μL of polystyrene 96-well flat-bottom plates (Greiner Bio-One, catalog number 655180) per well, and 24 μL of purified human C1q stock solution (Complement Tech, catalog number A099, final concentration 2.5 μg / mL) was added. Cells were allowed to adhere overnight at 37°C. The following day, 50 μL of antibody dilution series samples (3-fold dilution, final conc...

Claims

1. (i) A second antibody comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen. For use in combination with, A first antibody comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen, or (ii) A first antibody comprising a first Fc region of human IgG and a first antigen-binding region capable of binding to a first antigen. For use in combination with, A second antibody comprising a second Fc region of human IgG and a second antigen-binding region capable of binding to a second antigen, It is a medical drug, The first Fc region is a. E345K replacement, and b. Replacement with K439E or S440K Including; The second Fc region is c. E345K replacement, and d. Replacement with K439E or S440K Including; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first Fc region and the second Fc region contain the N297A substitution; The amino acid positions correspond to human IgG1 according to the Eu numbering system. The aforementioned medical drug.

2. The pharmaceutical agent according to claim 1, wherein the first Fc region and / or the second Fc region comprises one or more substitutions selected from the group consisting of E333S, K326A, E333A, and K326W.

3. A pharmaceutical agent according to claim 1 or 2, wherein the first Fc region and / or the second Fc region comprises E333S and K326A substitutions.

4. A medical agent according to any one of claims 1 to 3, wherein the first antibody and / or the second antibody is a human antibody, is humanized, or is a chimera.

5. A medical agent according to any one of claims 1 to 4, wherein the first antibody and / or the second antibody is a monoclonal antibody.

6. A medical agent according to any one of claims 1 to 5, wherein the first antibody and / or the second antibody is a human IgG1, IgG2, IgG3, or IgG4 subclass.

7. A medical agent according to any one of claims 1 to 6, wherein the first antibody and / or the second antibody is a human IgG1, IgG2, or IgG4 subclass.

8. A medical agent according to any one of claims 1 to 7, wherein the first antibody and / or the second antibody is a human IgG1 subclass.

9. A medical agent according to any one of claims 1 to 8, wherein both the first antigen and the second antigen are cell surface expression molecules.

10. A medical agent according to any one of claims 1 to 9, wherein the first antigen and the second antigen are co-expressed in cells or tissues that are target cells or target tissues of the disease or disorder to be treated.

11. A medical agent according to any one of claims 1 to 10, wherein the first antigen and the second antigen are not the same.

12. A medical agent according to any one of claims 1 to 11, wherein the combination of the first antibody and the second antibody depletes a population of cells that simultaneously express the first antigen and the second antigen.

13. A medical agent according to any one of claims 1 to 12, wherein a combination of a first antibody and a second antibody induces cell death in a cell population that simultaneously expresses a first antigen and a second antigen.

14. A medical agent according to any one of claims 1 to 13, wherein a combination of a first antibody and a second antibody induces proliferation in a cell population expressing a first antigen and a second antigen.

15. The medical agent according to claim 12 or 13, wherein the cell population is a tumor cell population.

16. The medical agent according to any one of claims 12, 13, and 15, wherein the cell population is a hematological tumor cell population or a solid tumor cell population.

17. A medical agent according to any one of claims 12 to 16, wherein the cell population is a population of leukocytes, lymphocytes, B cells, T cells, regulatory T cells, NK cells, myeloid-derived suppressor cells, or tumor-associated macrophage cells.

18. below: The first antibody comprises a first antigen-binding region capable of binding to a first antigen, and a first Fc region of human IgG; The second antibody comprises a second antigen-binding region capable of binding to a second antigen, and a second Fc region of human IgG; The first Fc region is a. E345K replacement, and b. Replacement with K439E or S440K Including; The second Fc region is c. E345K replacement, and d. Replacement with K439E or S440K Including; The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; The first Fc region and the second Fc region contain the N297A substitution; The first and second antibodies have amino acid positions corresponding to human IgG1 according to the Eu numbering system. A composition containing the following:

19. The first antibody and the second antibody are present in the composition in a molar ratio of approximately 1:50 to 50:1, for example, approximately 1:1, approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1 The composition according to claim 18, which exists in molar ratios of 40, approximately 1:45, approximately 1:50, approximately 50:1, approximately 45:1, approximately 40:1, approximately 35:1, approximately 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.

20. The composition according to claim 18 or 19, wherein the first antibody and the second antibody are present in the composition in a 1:1 molar ratio.

21. The composition according to any one of claims 18 to 20, further comprising a pharmaceutical carrier or excipient.

22. A pharmaceutical composition according to any one of claims 18 to 21.

23. A composition according to any one of claims 18 to 22, for use as a medical drug.

24. A pharmaceutical agent according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 23, wherein the antigen-binding region can bind to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, PD1, and FAS.

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