Combinations of antibody variants and uses thereof

Abstract

Provided herein are combinations of first and second antibodies with altered Fc effector function resulting from amino acid substitutions in the Fc region, which allow for co-dependent activation of effector functions such as CDC and / or ADCC. Also provided are combinations of first and second antibodies with agonist activity or enhanced agonist activity resulting from amino acid substitutions in the Fc region, which agonist activity is co-dependent on both the first and second antibodies.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antibodies with altered Fc effector function, i.e., increased or decreased Fc effector function, resulting from amino acid substitutions in the Fc region, and to the use of combinations of such antibodies, and to compositions comprising such antibodies. [Background technology]

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

[0003] IgG hexamerization upon target binding at the cell surface has been shown to support high-avidity C1q binding. Hexamerization is mediated by intermolecular noncovalent Fc-Fc interactions, which can be enhanced by point mutations in the CH3 domain, including E345R and E430G.

[0004] WO2013 / 004842 (Patent Document 1) discloses antibodies or polypeptides comprising variant Fc regions with one or more amino acid modifications that result in altered effector functions such as complement-dependent cytotoxicity (CDC). WO2014 / 108198 (Patent Document 2) discloses polypeptides, such as antibodies, comprising variant Fc regions with one or more amino acid modifications that result in increased complement-dependent cytotoxicity (CDC).

[0005] WO2012 / 130831 (Patent Document 3) relates to Fc region-containing polypeptides having altered effector functions as a result of one or more amino acid substitutions in the Fc region of the polypeptide. These polypeptides exhibit reduced affinity for human FcyRIIIa and / or FcyRIIa and / or FcyRI compared to polypeptides containing wild-type IgG Fc regions, and exhibit ADCC induced by the polypeptides that is at least 20% reduced compared to the ADCC induced by polypeptides containing wild-type human IgG Fc regions. WO2012 / 130831 (Patent Document 3) does not disclose antibodies with enhanced Fc-Fc interactions and / or enhanced hexamer formation ability.

[0006] As mentioned above, previous efforts to enhance Fc-Fc interactions between antibodies have the effect of enhancing effector functions such as CDC and / or ADCC, which can lead to cell death of antibody-bound target cells.

[0007] However, if the target antigen is widely expressed in the body on both healthy and pathogenic cells, the antibody can become toxic by killing normal cells. Therefore, it is necessary to make the effector function of the antibody with enhanced effector function dependent on another antibody, in combination that can be selective for pathogenic cells and thus prevent the killing of healthy cells. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2013 / 004842 [Patent Document 2] WO2014 / 108198 [Patent Document 3] WO2012 / 130831 Summary of the Invention

[0009] Object of the invention Individually acting antibodies with enhanced effector function rely solely on the antigen-binding region of the antibody to achieve specificity for target cells, which may limit suitable targets to those highly selectively expressed in diseased cells. Individually acting antibodies with mutations that reduce effector function can rescue healthy cells expressing their antigen target, but the mutations that reduce effector function may limit their efficacy. Therefore, it is clearly necessary to create antibody combinations in which each individual antibody can bind to both pathogenic and normal cells, but in which enhanced effector function is activated only, or preferentially, when both antibodies bind to the same pathogenic cell. This uncoupling of effector function activation from individual target binding allows for the creation of antibodies that bind to targets that previously could not be optimally used for antibody therapy due to undesirable toxicity to healthy cells or lack of efficacy against diseased cells, because effector function activation is selective for the cells to which the antibody combination simultaneously binds.

[0010] It is therefore an object of the present invention to provide a first antibody and a second antibody that have been modified to provide maximal activity against target cells bound by both antibodies simultaneously, where the first antibody provides no or minimal activity against target cells bound by only the first antibody, and the second antibody provides minimal or reduced activity against target cells bound by only the second antibody, compared to activity against cells bound by both antibodies simultaneously.

[0011] It is a further object of the present invention to provide a first antibody and a second antibody, both of which comprise the Fc region and antigen-binding region of human IgG, both of which have substitutions that increase Fc-Fc interactions and substitutions that inhibit self-oligomerization, and the first antibody further has substitutions that reduce effector function such as CDC and / or ADCC, such that the first antibody has reduced effector function such as CDC and / or ADCC compared to a first parent antibody that does not comprise substitutions that reduce effector function, and the combination of the first and second antibodies has enhanced effector function compared to the first and second antibodies individually.

[0012] It is another object of the present invention to provide a first antibody comprising an Fc region and an antigen-binding region of human IgG with substitutions that increase Fc-Fc interactions, self-oligomerization-inhibiting substitutions, and substitutions for reduced effector function such as CDC and / or ADCC, compared to a first parent antibody, and a second antibody comprising an Fc region and an antigen-binding region of human IgG with substitutions that increase Fc-Fc interactions, self-oligomerization-inhibiting substitutions, and substitutions for enhanced effector function such as CDC and / or ADCC, compared to a second parent antibody, wherein the activities of the first and second antibodies are made co-dependent by the complementary self-oligomerization-inhibiting substitutions.

[0013] It is a further object of the present invention to provide a first antibody comprising an Fc region and an antigen-binding region of human IgG with substitutions that increase Fc-Fc interactions, self-oligomerization-inhibiting substitutions, and substitutions for reduced effector function such as CDC and / or ADCC, relative to a first parent antibody, and a second antibody comprising an Fc region and an antigen-binding region of human IgG with substitutions that increase Fc-Fc interactions, self-oligomerization-inhibiting substitutions, and substitutions that induce agonistic activity, such as increased activation of a target receptor, when compared to a second parent antibody, wherein the activities of the first and second antibodies are made co-dependent by the complementary self-oligomerization-inhibiting substitutions.

[0014] It is a further object of the present invention to provide a first antibody comprising an Fc region of human IgG and an antigen-binding region with substitutions that increase Fc-Fc interactions, substitutions that inhibit self-oligomerization, and substitutions for reduced agonist activity, such as reduced activation of the target receptor upon binding, compared to a first parent antibody, and a second antibody comprising an Fc region of human IgG and an antigen-binding region with substitutions that increase Fc-Fc interactions, substitutions that inhibit self-oligomerization, and substitutions for reduced agonist activity, such as reduced activation of the target receptor upon binding, compared to a second parent antibody, wherein the combination of the first and second antibodies has enhanced agonist activity that is made co-dependent by the complementary self-oligomerization-inhibiting substitutions.

[0015] It is a further object of the present invention to provide a first antibody comprising an Fc region and an antigen-binding region of human IgG, which has substitutions that increase Fc-Fc interactions, self-oligomerization-inhibiting substitutions, and substitutions that reduce effector functions such as CDC and / or ADCC, relative to a first parent antibody, and a second antibody comprising an Fc region and an antigen-binding region of human IgG, which has self-oligomerization-inhibiting substitutions, substitutions that increase Fc-Fc interactions, activate signaling when the antigen-binding region of the antibody binds to a corresponding antigen, and optionally induce enhanced signaling, relative to a second parent antibody, wherein the activities of the first and second antibodies are made co-dependent by the complementary self-oligomerization-inhibiting substitutions.

[0016] Summary of the Invention As described herein, the present invention relates to a combination of a first antibody having an Fc region and an antigen-binding region, wherein the Fc region has an Fc-Fc-enhancing substitution and one or more substitutions that reduce effector function such as CDC and / or ADCC, and a second antibody comprising an Fc region and an antigen-binding region, wherein the Fc region has an Fc-Fc-enhancing substitution and, optionally, one or more substitutions that enhance Fc effector function such as CDC and / or ADCC, wherein the first and second antibodies further have complementary self-oligomerization-inhibiting substitutions that make hetero-oligomerization of the first and second antibodies co-dependent.

[0017] Without being limited by theory, it is believed that the combination of a first antibody and a second antibody of the present invention, which have complementary substitutions that make the effector or signaling functions, e.g., agonist activity, of the first and second antibodies co-dependent, can reduce the toxicity of the combination and increase the therapeutic window of the combination. Furthermore, the combination of a first antibody and a second antibody of the present invention can be used to specifically deplete cell populations expressing antigens recognized by the first and second antibodies. Thus, the combination of a first antibody and a second antibody of the present invention can be used to specifically deplete tumor cell populations expressing the first and second antigens recognized by the first and second antibodies, without depleting healthy cell populations or tissues expressing only the first or second antigens recognized by the first and second antibodies.

[0018] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, and P329R; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) if the first Fc region comprises a K322E or P329R substitution, it comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the EU numbering system (Edelman et al., Proc Natl Acad Sci U.S. A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No.91-3242); A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0019] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one or more substitutions selected from the group consisting of L234, L235, G237, G236, or one or more substitutions selected from the group consisting of K322A and E269K; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0020] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one substitution at amino acid position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0021] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing a single amino acid substitution at position P329 or a substitution of K322E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the EU numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0022] In another aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) containing the substitutions K248E and T437R; and (e) containing a K439E or S440K substitution; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0023] Substitutions at positions equivalent to E430, E345, or substitutions of S440Y or S440W are considered Fc-Fc enhancing substitutions according to the present invention.

[0024] The K439E or S440K substitution is considered a complementary self-oligomerization-inhibiting substitution according to the present invention. That is, for example, a first antibody having a K439E substitution cannot form an oligomer with another antibody having a K439E substitution, but an antibody having a K439E substitution can form an oligomer with another antibody having a S440K substitution. An antibody having an S440K substitution cannot form an oligomer with another antibody having a S440K substitution.

[0025] Substitutions of an amino acid at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, P329R, L234A, L234F, L235A, L235Q, and L235E are considered C1q binding site modulating substitutions according to the present invention and reduce effector functions such as CDC and / or ADCC.

[0026] One or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S are considered C1q binding site-modulating substitutions according to the present invention, and increase effector functions such as CDC and / or ADCC.

[0027] In one embodiment, the second Fc region comprises a G237A substitution.

[0028] The G237A substitution is considered an Fcγ receptor modulating substitution according to the present invention and reduces Fcγ receptor binding.

[0029] That is, in a first aspect of the present invention, the present inventors have discovered that by combining a first antibody having Fc-Fc-enhancing substitutions, self-oligomerization-inhibiting substitutions, and one or more substitutions that reduce effector function such as CDC and / or ADCC, and a second antibody having Fc-Fc-enhancing substitutions, self-oligomerization-inhibiting substitutions, and optionally one or more substitutions that enhance effector function such as CDC and / or ADCC, wherein the first and second antibodies have complementary oligomerization-inhibiting substitutions, such that the hetero-oligomerization of the first and second antibodies is co-dependent, it may be possible to improve the safety margin between the effector function activity in diseased cells and the effector function activity in healthy cells for the antibody combination, when compared to a combination of antibodies having Fc-Fc-enhancing substitutions and self-oligomerization-inhibiting substitutions but no C1q-modulating substitutions.

[0030] In a further aspect, the present invention provides an antibody comprising an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, The Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; An antibody is provided.

[0031] In another aspect, the present invention provides an antibody comprising an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, The Fc region (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A; (d) including K332E, E269K, L234A, L234F, L235A, L235Q, L235E, K326A, K326W, E333A, and E333S; An antibody is provided.

[0032] In one aspect, the invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region according to any embodiment or aspect described herein, and the second antibody comprises a second antigen-binding region and a second Fc region according to any embodiment or aspect described herein.

[0033] In one aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, and P329R; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) if the first Fc region comprises a K322E or P329R substitution, it comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; A composition is provided.

[0034] In one aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; A composition is provided.

[0035] In one aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing a single amino acid substitution at position P329 or a substitution of K322E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, and the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A composition is provided.

[0036] In another aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) containing the substitutions K248E and T437R; and (e) containing one K439E or S440K substitution; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A composition is provided.

[0037] In another aspect, the present invention relates to a method of depleting a cell population expressing a first antigen and a second antigen, the method comprising the step of contacting the cell population with a first and second antibody, or a composition of the first and second antibodies, or a composition as defined herein.

[0038] In another aspect, the present invention relates to a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of the first and second antibodies claimed herein or an effective amount of a composition described herein.

[0039] In another aspect, the present invention relates to a kit comprising a first container containing a first antibody as defined herein and a second container containing a second antibody as defined herein.

[0040] In another aspect, the invention relates to the first and second antibodies or compositions described herein for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0041] In another aspect, the invention relates to a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of a first and second antibody or composition described herein.

[0042] [The present invention 1001] 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 as a pharmaceutical 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, the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; The first antibody. [The present invention 1002] 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 as a pharmaceutical 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, the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing a single amino acid substitution at position P329 or a substitution of K322E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the EU numbering system; The first antibody. [The present invention 1003] 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 as a pharmaceutical 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, the first Fc region (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) containing the substitutions K248E and T437R; and (e) containing a K439E or S440K substitution; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; The first antibody. [The present invention 1004] 10. A first antibody for use as a pharmaceutical in combination with a second antibody of either of the inventions 1001 or 1002, wherein the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [The present invention 1005] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001 to 1002 and 1004 of the present invention, wherein the first and second Fc regions contain a substitution selected from the group consisting of E430G, E345K, and E345R. [The present invention 1006] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001 to 1002 and 1004 to 1005 of the present invention, wherein the first and second Fc regions comprise an E430G substitution. [The present invention 1007] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 and 1003 to 1006, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, and G237P. [The present invention 1008] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 and 1003 to 1007, wherein the first Fc region contains one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, and G237R. [The present invention 1009] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001 and 1003 to 1008 of the present invention, wherein the first Fc region comprises a G237Q substitution. [The present invention 1010] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 and 1003 to 1006, wherein the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. [The present invention 1011] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001, 1003 to 1006, and 1010 of the present invention, wherein the first Fc region comprises a G236R substitution. [The present invention 1012] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001, 1003 to 1006, and 1010 of the present invention, wherein the first Fc region comprises an E269K substitution. [The present invention 1013] A first antibody for use as a pharmaceutical in combination with a second antibody of any of 1001 and 1003 to 1006 of the present invention, wherein the first Fc region comprises a substitution of K322A. [The present invention 1014] 1007. A first antibody for use as a pharmaceutical in combination with a second antibody according to any of claims 1002 to 1006, wherein the first Fc region comprises a P329R substitution. [The present invention 1015] 1007. A first antibody for use as a pharmaceutical in combination with a second antibody according to any of claims 1002 to 1006, wherein the first Fc region comprises a K322E substitution. [The present invention 1016] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 and 1003 to 1013, wherein the second Fc region contains one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S. [The present invention 1017] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1001, 1003 to 1013, and 1016, wherein the second Fc region comprises a G237A substitution. [The present invention 1018] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1001 to 1015, wherein the second Fc region comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S. [The present invention 1019] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1001 to 1015 and 1018, wherein the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S. [The present invention 1020] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1001, 1003 to 1013, and 1016 to 1017, wherein the second Fc region comprises substitutions of G237A and E333S. [The present invention 1021] A first antibody for use as a pharmaceutical in combination with a second antibody according to any of claims 1001 to 1019 of the present invention, wherein the second Fc region comprises a substitution of K326A. [The present invention 1022] A first antibody for use as a pharmaceutical in combination with a second antibody according to any of claims 1001 to 1019, wherein the second Fc region comprises an E333S substitution. [The present invention 1023] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1001 to 1016 and 1018, wherein the second Fc region contains two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S. [The present invention 1024] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 to 1016, 1018, and 1023, wherein the second Fc region comprises substitutions of K326W and E333S. [The present invention 1025] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the present inventions 1001 to 1016, 1018, and 1023, wherein the second Fc region comprises substitutions of K326A and E333A. [The present invention 1026] Any of the above first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and / or second antibody is a human antibody, a humanized antibody, or a chimeric antibody. [The present invention 1027] Any of the above first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and / or second antibody is a monoclonal antibody. [The present invention 1028] Any of the first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and / or second antibody is of human IgG1, IgG2, IgG3, or IgG4 isotype. [The present invention 1029] Any of the above first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and / or second antibody is of the human IgG1 isotype. [The present invention 1030] Any of the above first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and second antigens are both molecules exposed on the cell surface. [The present invention 1031] A first antibody for use as a pharmaceutical in combination with a second antibody of any of the above inventions, wherein the first and second antigens co-exist in a cell or tissue that is a target cell or tissue for the disease or disorder to be treated. [The present invention 1032] Any of the above first antibodies of the present invention for use as a pharmaceutical in combination with a second antibody, wherein the first and second antigens are not identical. [The present invention 1033] Any of the foregoing first antibodies of the invention for use as a medicament in combination with a second antibody, wherein the medicament depletes a cell population expressing the first and second antigens. [The present invention 1034] A first antibody for use as a medicament in combination with a second antibody of the present invention 1033, wherein the cell population is tumor cells. [This invention 1035] A first antibody for use as a pharmaceutical in combination with a second antibody according to any of claims 1033 to 1034, wherein the cell population is blood tumor cells or solid tumor cells. [The present invention 1036] A first antibody for use as a pharmaceutical in combination with a second antibody of any of claims 1033 to 1034 of the present invention, 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. [This invention 1037] An antibody comprising an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, The Fc region is (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; The antibody. [The present invention 1038] An antibody comprising an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, The Fc region is (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A; (d) including K332E, E269K, L234A, L234F, L235A, L235Q, L235E, K326A, K326W, E333A, and E333S; The antibody. [This invention 1039] 1037. The antibody of the invention, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [The present invention 1040] The antibody of any of 1037 and 1039, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, and E345R. [The present invention 1041] The antibody of any one of 1037 and 1039 to 1041 of the present invention, wherein the Fc region comprises an E430G substitution. [The present invention 1042] Any of the antibodies of the present invention 1037 to 1041, wherein the Fc region contains one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, and G237P. [This invention 1043] The antibody of any of claims 1037 to 1042, wherein the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, and G237R. [This invention 1044] The antibody of any of claims 1037 to 1043, wherein the Fc region comprises a G237Q or G237A substitution. [This invention 1045] The antibody of any of claims 1037 to 1041, wherein the Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. [The present invention 1046] The antibody of any one of 1037 to 1041 and 1045 of the present invention, wherein the Fc region comprises a G236R substitution. [This invention 1047] The antibody of any one of 1037 to 1041 and 1045 of the present invention, wherein the Fc region comprises an E269K substitution. [This invention 1048] The antibody of any one of 1037 to 1041 of the present invention, wherein the Fc region comprises a substitution of K322A. [This invention 1049] The antibody of any one of claims 1037 to 1048 of the present invention, which is of human IgG1 isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype. [The present invention 1050] Any one of the antibodies 1037 to 1049 of the present invention, which is of human IgG1 isotype. [This invention 1051] A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region of any one of 1001 to 1015 and 1026 to 1036 of the present invention, and the second antibody comprises a second antigen-binding region and a second Fc region of any one of 1001 to 1006 and 1016 to 1036 of the present invention. [This invention 1052] A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; The amino acid positions correspond to those of human IgG1 according to the EU numbering system. The composition. [This invention 1053] A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing a single amino acid substitution at position P329 or a substitution of K322E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; The composition. [This invention 1054] A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) containing the substitutions K248E and T437R; and (e) containing one substitution of K439E or S440K; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; The composition. [This invention 1055] 1054. The composition of any of claims 1052 to 1053, wherein the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [The present invention 1056] The composition of any of claims 1052 to 1053 and 1055, wherein the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, and E345R. [This invention 1057] The composition of any of 1052 to 1053 and 1055 to 1056, wherein the first and second Fc regions comprise an E430G substitution. [This invention 1058] Any of the compositions of claims 1052 to 1057, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, and G237P. [This invention 1059] 1059. The composition of any of claims 1052 to 1058, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, and G237S. [The present invention 1060] 1059. The composition of any of claims 1052 to 1059, wherein the first Fc region comprises a G237Q substitution. [This invention 1061] The composition of any of claims 1052 to 1057, wherein the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. [This invention 1062] The composition of any of 1052 to 1057 and 1061, wherein the first Fc region comprises a G236R substitution. [This invention 1063] The composition of any of claims 1052 to 1057 and 1061, wherein the first Fc region comprises an E269K substitution. [This invention 1064] The composition of any one of 1052, 1054 to 1057, wherein the first Fc region comprises a K322A substitution. [This invention 1065] The composition of any of claims 1053 to 1057, wherein the first Fc region comprises a P329R substitution. [The present invention 1066] The composition of any of claims 1053 to 1057, wherein the first Fc region comprises a K322E substitution. [This invention 1067] The composition of any of 1052, 1054 to 1064, wherein the second Fc region comprises one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S. [The present invention 1068] Any of the compositions of 1052, 1054 to 1064, and 1067, wherein the second Fc region comprises a G237A substitution. [This invention 1069] The composition of any of claims 1052 to 1067, wherein the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S. [The present invention 1070] The composition of any of claims 1052 to 1067 and 1069, wherein the second Fc region comprises two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S. [This invention 1071] The composition of any of claims 1052 to 1067 and 1069 to 1070, wherein the second Fc region comprises the substitutions K326W and E333S. [This invention 1072] The composition of any of claims 1052 to 1067 and 1069 to 1070, wherein the second Fc region comprises substitutions of K326A and E333A. [This invention 1073] The composition of any of claims 1052 to 1067 and 1069, wherein the second Fc region comprises an E333S substitution. [This invention 1074] The composition of any of claims 1052, 1054 to 1067, wherein the second Fc region comprises G237A and E333S substitutions. [This invention 1075] The composition of any of claims 1051 to 1074, wherein the first and second antigens are both cell surface exposed molecules. [This invention 1076] The composition of any of claims 1051 to 1075, wherein the first and second antigens are not identical. [This invention 1077] The first antibody and the second antibody are present 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 1:35, about 1:40, about 1:45, or Any of compositions 1051 to 1076, wherein the hydroxybenzoates are present in the composition in a molar ratio of about 1:50, about 50:1, about 45:1, about 40:1, about 35:1, about 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. [This invention 1078] Any of the compositions of the present inventions 1051 to 1077, wherein the first antibody and the second antibody are present in the composition at 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 about 1:30 to 30:1, for example, a molar ratio of about 1:20 to 20:1, for example, a molar ratio of about 1:10 to 10:1, for example, a molar ratio of about 1:9 to 9:1, for example, a molar ratio of about 1:5 to 5:1. [This invention 1079] The composition of any of claims 1051 to 1078, wherein the first antibody and the second antibody are present in the composition in a molar ratio of 1:1. [The present invention 1080] The composition of any of claims 1051 to 1079, further comprising a pharmaceutical carrier or excipient. [This invention 1081] Any of the compositions of 1051 to 1080 of the present invention, which is a pharmaceutical composition. [This invention 1082] Any of the compositions of the present invention described above for use as a pharmaceutical. [This invention 1083] The first or second antibody of any of claims 1001 to 1036, any of claims 1037 to 1050, or any of claims 1051 to 1082, wherein the antigen-binding region is capable of binding to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, and PD1. [This invention 1084] The antigen-binding region is (a) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 196, the CDR2 sequence set forth in SEQ ID NO: 196, and the CDR3 sequence set forth in SEQ ID NO: 198, and a VL region [DR4] comprising the CDR1 sequence set forth in SEQ ID NO: 200, the CDR2 sequence set forth in AAT, and the CDR3 sequence set forth in SEQ ID NO: 201; (b) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:50, the CDR2 sequence set forth in SEQ ID NO:51, and the CDR3 sequence set forth in SEQ ID NO:52, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:54, the CDR2 sequence set forth in FAS, and the CDR3 sequence set forth in SEQ ID NO:55 [DR5-01-G56T]; (c) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:57, the CDR2 sequence set forth in SEQ ID NO:58, and the CDR3 sequence set forth in SEQ ID NO:59, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:61, the CDR2 sequence set forth in RTS, and the CDR3 sequence set forth in SEQ ID NO:62 [DR5-05]; (d) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:36, the CDR2 sequence set forth in SEQ ID NO:37, and the CDR3 sequence set forth in SEQ ID NO:38, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:40, the CDR2 sequence set forth in DAS, and the CDR3 sequence set forth in SEQ ID NO:41 [CD20, 7D8]; (e) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:13, the CDR2 sequence set forth in DAS, and the CDR3 sequence set forth in SEQ ID NO:14 [CD20, 11B8]; (f) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:43, the CDR2 sequence set forth in SEQ ID NO:44, and the CDR3 sequence set forth in SEQ ID NO:45, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:47, the CDR2 sequence set forth in VAT, and the CDR3 sequence set forth in SEQ ID NO:48 [CD37]; (g) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:6, the CDR2 sequence set forth in NTN, and the CDR3 sequence set forth in SEQ ID NO:7 [CD52, CAMPATH-1H]; (h) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 161, the CDR2 sequence set forth in SEQ ID NO: 162, and the CDR3 sequence set forth in SEQ ID NO: 163, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 165, the CDR2 sequence set forth in LVS, and the CDR3 sequence set forth in SEQ ID NO: 166 [CD52, h2E8]; (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 168, the CDR2 sequence set forth in SEQ ID NO: 169, and the CDR3 sequence set forth in SEQ ID NO: 170, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 172, the CDR2 sequence set forth in AAS, and the CDR3 sequence set forth in SEQ ID NO: 173 [HLA-DR, hul243]; (j) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 175, the CDR2 sequence set forth in SEQ ID NO: 176, and the CDR3 sequence set forth in SEQ ID NO: 177, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 179, the CDR2 sequence set forth in DNN, and the CDR3 sequence set forth in SEQ ID NO: 180 [HLA-DR, 1D09C3]; (k) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 182, the CDR2 sequence set forth in SEQ ID NO: 183, and the CDR3 sequence set forth in SEQ ID NO: 184, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 186, the CDR2 sequence set forth in DTS, and the CDR3 sequence set forth in SEQ ID NO: 187 [CD3, huCLB T3 / 4]; or (l) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 189, the CDR2 sequence set forth in SEQ ID NO: 190, and the CDR3 sequence set forth in SEQ ID NO: 191, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 193, the CDR2 sequence set forth in ATS, and the CDR3 sequence set forth in SEQ ID NO: 194 [CD5] The first or second antibody of any one of 1001 to 1036 and 1083 of the present invention, the antibody of any one of 1037 to 1050 and 1083 of the present invention, or the composition of any one of 1051 to 1083 of the present invention, comprising: [This invention 1085] A method for treating an individual having a disease, the method comprising the step of administering to the individual an effective amount of the first and second antibodies of any of 1001 to 1037 and 1083 to 1084 of the present invention, any of the antibodies of 1037 to 1050 and 1083 to 1084 of the present invention, or any of the compositions of 1051 to 1084 of the present invention. [The present invention 1086] The method of claim 1085, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. [This invention 1087] The method of any one of claims 1085 to 1086, further comprising the step of administering a further therapeutic agent. [This invention 1088] A method for depleting a cell population expressing a first antigen and a second antigen, the method comprising the step of contacting the cell population with a first and second antibody, antibody, or composition of any one of claims 1001 to 1084 of the present invention. [This invention 1089] The method of claim 1088, wherein the cell population is a tumor cell population, such as a blood tumor cell population or a solid tumor cell population. [The present invention 1090] A method for inducing proliferation in a cell population expressing a first antigen and a second antigen, the method comprising the step of contacting the cell population with any one of the first and second antibodies, antibodies, or compositions of the present inventions 1001 to 1084. [This invention 1091] The method of any one of claims 1088 to 1090, wherein the cell population is present in blood. [This invention 1092] 1091. The method of any one of claims 1088 to 1091, wherein the cell population is leukocytes. [This invention 1093] The method of any one of claims 1088 to 1092, wherein the cell population is a subset of a white blood cell population. [This invention 1094] The method of any one of claims 1088 to 1093, wherein the cell population is a lymphocyte cell population. [This invention 1095] 1094. The method of claim 1094, wherein the cell population is a B cell population, a T cell population, a NK cell population, a regulatory T cell population, or a myeloid-derived suppressor cell population. [This invention 1096] 1096. The method of any of claims 1085 to 1095, wherein the first and / or second antigen is a member of the TNFR-SF. [This invention 1097] A kit comprising a first container containing a first antibody defined in any one of 1001 to 1015, 1026 to 1036, and 1083 to 1084 of the present invention, and a second container containing a second antibody defined in any one of 1001 to 1006, 1016 to 1036, and 1083 to 1084 of the present invention. These and other aspects of the invention, particularly the various uses and therapeutic applications of the first and second antibodies, are described in further detail below. [Brief explanation of the drawings]

[0043] [Figure 1]This figure shows the selectivity of CDC activity by the mixed antibody variants, anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K, due to the introduction of the P329R mutation. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% pooled normal human serum (NHS). CDC efficacy is presented as (A) the lysis rate determined by the percentage of propidium iodide (PI)-positive cells and (B) the area under the dose-response curve (AUC), normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 2] This figure shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K due to the introduction of the K322E mutation. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as (A) lysis rate determined by the percentage of PI-positive cells and (B) AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 3A]Figure 3 shows the selectivity of CDC activity against different cell lines by introducing the K322E mutation into the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20-11B8-E430G-S440K. In vitro CDC assays were performed using 30 μg / mL of antibody in the presence of 20% NHS on Burkitt's lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C), and Ramos (D), acute lymphoblastic lymphoma (ALL) cell line REH (E), myeloma cell line U266B1 (F), and B-cell lymphoma cell line U-698-M (G). CDC activity is presented as lysis rate, determined by the percentage of PI-positive cells, normalized for each cell line to the non-binding control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%) for REH, U266B1, and Wien 133, or IgG1-11B8-E430G (100%) for Daudi, Raji, Ramos, and U-698-M. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 4]This figure shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K, each carrying a C1q binding-inhibiting mutation (G236R, K322A, E269K, K322E, or P329R). Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 5] This figure shows the selectivity of CDC activity by the mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding-enhancing mutations (E333S, K326W, or K326A / E333A). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 6A]Figure 6 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q binding-inhibiting mutations G236R (A), K322A (B), E269K (C), K322E (D), or P329R (E), plus anti-CD20 IgG1-11B8-E430G-S440K with C1q binding-enhancing mutations E333S, K326W, or K326A / E333A. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized area under the curve (AUC) of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7A] Figure 7 shows the binding of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variants (C, D) with the C1q binding-enhancing mutation E333S to the human lymphoma cell lines Wien 133 (A, C) and Raji (B, D). Antibody binding was tested by flow cytometry. Binding is expressed as the geometric mean fluorescence intensity (MFI). As negative controls for binding, samples without primary antibody or unconjugated anti-gp120 antibody IgG1-b12 were used. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A]Figure 8 shows the ADCC potential of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, C) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variant (B, D) with the C1q binding-enhancing mutation E333S. (A, B) ADCC reporter bioassays were performed by co-incubating Raji target cells with antibody dilution series and Jurkat human T cells stably expressing high-affinity FcγRIIIa (V158) and an NFAT response element driving the expression of firefly luciferase. Luciferase production was quantified by luminescence readout. (C, D) In ​​vitro europium TDA (EuTDA) ADCC assays were performed by co-incubating Wien 133 target cells with antibody dilution series and human PBMCs (E:T 100:1). Cell lysis was determined by measuring the signal of the EuTDA fluorescent chelate in the supernatant. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A] Figure 9 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-K439E with enhanced Fc-Fc interaction, carrying the C1q binding-inhibiting mutations G236R or K322A, and anti-CD20 IgG1-11B8-S440K with enhanced Fc-Fc interaction, carrying either the C1q binding-inhibiting mutation E333S or the C1q binding-enhancing mutation E430G, E345K, E345R, and E345R-E430G. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 9B]See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A] Figure 10 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E, which has a C1q binding regulatory mutation or double mutation G237A-K322A at position G236 (G236R or G236K) or G237 (G237A, G237T, G237Q, or G237R), plus anti-CD20 IgG1-11B8-E430G-S440K, with or without the C1q binding regulatory mutations E333S, E333A, K326A, K326W-E333S, G237A, or G237A-E333S. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 10B] See legend to Figure 10A. [Figure 11] Figure 1 shows the selectivity of CDC activity by mixed antibody isotype variants (IgG1, IgG2, IgG3, and hinge-stabilized IgG4) of anti-CD52 CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R and anti-CD20 11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%). [Figure 12A]Figure 12 shows the selectivity of CDC activity on (A) Daudi cells and (B) Wien 133 cells by the mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R or K322A, and anti-CD37 IgG1-CD37-37.3-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Target cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-CD37-37.3-E430G (100%). [Figure 12B] See legend to Figure 12A. [Figure 13A] Figure 13 shows the DR5-mediated cytotoxicity of (A) single antibody variants or (B) agonist mixtures of antibody variants of anti-DR5 IgG1-DR5-01-G56T-E430G-K439E with or without the C1q binding inhibitor mutation G236R + IgG1-DR5-05-E430G-S440K with or without the C1q binding enhancing mutation E333S against BxPC-3 human pancreatic cancer cells. A 3-day viability assay was performed and cell viability was determined using the CellTiter-Glo kit. [Figure 13B] See legend to Figure 13A. [Figure 14] Figure 1 shows CDC activity by antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G with C1q binding-regulating mutations G237A, G236R, A327K, K322E, or P329R. Wien 133 cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibodies IgG1-b12-S440K (0%; not shown) and IgG1-CAMPATH-1H-E430G (100%). [Figure 15A]Figure 15 shows the binding of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B, C) with the C1q binding-inhibiting mutation G236R or K322A and anti-CD20 IgG1-11B8-E430G-S440K antibody variants (D, E, F) with the C1q binding-enhancing mutation E333S to human FcRn. FcRn ELISA was performed using 5 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnhsECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 16A] Figure 16 shows the clearance rate of 500 μg of intravenously administered antibody in SCID mice. (A-C) Total human IgG in plasma samples was determined by ELISA and plotted as concentration versus time curves for (A) the IgG1-CAMPATH-1H variant, (B) the IgG1-11B8 variant, and (C) the combination of the IgG1-CAMPATH-1H variant and IgG1-11B8 variant. Each data point represents the mean + / - standard deviation of triplicate samples. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1,000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17A]Figure 17 shows the binding of immobilized IgG1-CAMPATH-1H-E430G-K439E variants with C1q binding-inhibiting mutations G236R or G237T and IgG1-11B8-E430G-S440K variants with C1q binding-enhancing mutations K326A or E333S to dimeric His-tagged, biotinylated ECDs of FcγRIIA allotype 131H (A), FcγRIIA allotype 131R (B), FcγRIIB (C), FcγRIIIA allotype 158V (D), and FcγRIIIA allotype 158F (E) tested in an ELISA assay. Binding is presented relative to the no-antibody control (background) and to binding to IgG1-11B8-E430G-S440K (100%) for a 20 μg / mL antibody sample. Detection was performed using streptavidin-polyHRP and ABTS. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 17E] See legend to Figure 17A. [Figure 18] This figure shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without C1q binding-inhibiting mutations (G236R or G237T) and anti-CD20 IgG1-11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S, and the FcγR binding-inhibiting mutation G237A. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%). [Figure 19A]Figure 19 shows the selective activity of combinations of IgG1-CAMPATH-1H and IgG1-11B8 variants in whole blood as determined by flow cytometry analysis of blood cells. Y-axis: B cell fraction (CD19 positive / CD3 negative) or T cell fraction (CD19 negative / CD3 positive) of the total lymphocyte population (CD66b negative) after overnight (o / n) incubation in the presence of effector cells. X-axis: different treatment groups. Symbols represent cells from five different healthy donors tested in two separate incubations per donor. (A) Selective activity of IgG1-CAMPATH-1H-G236R-E430G-K439E mixed with IgG1-11B8-G237A-E430G-S440K. (B) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K. (C) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G236R or G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K containing an additional C1q binding-enhancing E333S mutation. (D) Depth of B cell depletion by different B cell-targeting antibodies compared to the co-dependent antibody combination of IgG1-CAMPATH-1H-E430G-K439E with the additional mutations G236R, G237Q, or G237R mixed with IgG1-11B8-G237A-E430G-S440K. Y-axis: logarithmic scale representation of the B cell fraction determined as above. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 20-1]Figure 20 shows the selectivity of CDC activity against different cell lines with different expression levels of CD20 and CD52 by the combination of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants, which have C1q binding-inhibiting mutations in the anti-CD52 component and C1q binding-enhancing mutations in the anti-CD20 component. In vitro CDC assays were performed using the Burkitt lymphoma cell lines Daudi (A), Raji (B), and Ramos (C), the ALL cell line REH (D), and the B-cell lymphoma cell line U-698-M (E), using antibody concentrations of 0.01–40 μg / mL in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as maximal lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 20-2] See description of Figure 20-1. [Figure 20-3] See description of Figure 20-1. [Figure 20-4] See description of Figure 20-1. [Figure 20-5] See description of Figure 20-1. [Figure 20-6] See description of Figure 20-1. [Figure 20-7] See description of Figure 20-1. [Figure 20-8] See description of Figure 20-1. [Figure 20-9] See description of Figure 20-1. [Figure 20-10] See description of Figure 20-1. [Figure 21]Figure 1 shows the selectivity of CDC activity by the mixed antibody variants: anti-CD37 IgG1-CD37-37.3-E430G-K439E with or without the C1q binding-inhibiting mutation G236R, and anti-CD20 IgG1-11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. (A) Daudi cells and (B) WIL2-S cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CD37-37.3-E430G and IgG1-11B8-E430G (100%). [Figure 22] (A) Binding of antibody variants of anti-CD52 IgG1-CAMPATH-1H with Fc:Fc interaction-enhancing mutations E430G or E345R and self-oligomerization-inhibiting mutation K439E, combined with either FcγR binding-inhibiting mutations and C1q binding-modulating mutations G236R, G237A, or G237T, to the human lymphoma cell line Wien 133 is shown. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-Campath-1H (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (B) Maximum binding (Bmax) to Raji cells by IgG1-Campath-1H antibody variants with mutations E430G and K439E in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T is shown normalized to the binding of wild-type IgG1-Campath-1H. (C) Apparent Kd values ​​for binding to Raji cells of IgG1-Campath-1H antibody variants with mutations E430G and S440K in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T. [Figure 23A]Figure 23 shows the binding of antibody variants of anti-CD20 IgG1-11B8 with the Fc-Fc interaction-enhancing mutation E430G and the self-oligomerization-inhibiting mutation S440K, in combination with either the C1q binding-modulating mutations K326A or E333A (A) or E333S, G237A, or G237A-E333S (B), to the human lymphoma cell line Raji. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-11B8 (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (C, D) Maximum binding (Bmax) to Raji cells by IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (C) or E333S, G237A, or G237A-E333S (D) is shown normalized to the binding of wild-type IgG1-11B8. (E, F) Apparent Kd values ​​for binding to Raji cells of IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (E) or E333S, G237A, or G237A-E333S (F). [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 23E] See legend to Figure 23A. [Figure 23F] See legend to Figure 23A. [Figure 24A]Figure 24 shows FcRn binding of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibody variants. (A, C) Binding to human FcRn is shown for variants of the anti-CD52 antibody IgG1-CAMPATH-1H with the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation K439E, and the C1q binding-modulating mutations G237A or G237T, using an antibody concentration of 40 μg / mL at (A) pH 6.0 or (C) pH 7.4. (B, D) Binding to human FcRn by variants of the anti-CD20 antibody IgG1-11B8, which have the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation S440K, and the C1q binding-modulating mutations K326A, E333A, G237A, or G237A-E333S, using an antibody concentration of 40 μg / mL at (B) pH 6.0 or (D) pH 7.4. FcRn ELISA was performed using 2 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 25A]Figure 25 shows total human IgG (hIgG) concentrations as measured in blood samples collected from mice injected with anti-CD52 IgG1-CAMPATH-1H or anti-IgG1-11B8 antibody variants, or a mixture thereof. (A) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-CAMPATH-1H, IgG1-CAMPATH-1H-E430G-K439E-G237Q, or IgG1-CAMPATH-1H-E430G-K439E-G236R. (B) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-11B8, IgG1-11B8-E430G-S440K-G237A, or IgG1-11B8-E430G-S440K-E333S. (C) Total hIgG concentrations in blood samples collected from mice injected with a mixture of wild-type IgG1-CAMPATH-1H + IgG1-11B8 or a mixture of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations as in (A) and (B). In all figures, the dotted line represents the predicted IgG1 concentration over time for wild-type IgG1 antibodies in SCID mice. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 26A] Figure 26 shows the concentrations (in μg / ml) of C4d detected in samples incubated with IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 antibody variants carrying the mutations E430G, K439E, or S440K and G236R, G237A, G237Q, or G237R, after subtraction of the mean C4d concentration detected in negative control samples containing no antibody. Positive control samples contain antibody variants carrying the Fc-Fc interaction-enhancing mutations E345R, E430G, and S440Y (RGY). [Figure 26B]See legend to Figure 26A. [Figure 27A] Figure 27 shows C1q binding to Wien 133 cells incubated on ice with normal human serum as a complement source, as detected by flow cytometry, after opsonization with variants of antibodies IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 carrying the mutations E430G, K439E, or S440K and G236R, G237T, K326A, or E333S. Mean fluorescence intensity values ​​were normalized to the no-antibody control reaction (0%) and the maximal level (100%) of the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G, estimated by fitting a logarithmic agonist response model. (A) C1q binding of control reactions. (B–D) C1q binding of (B) IgG1-CAMPATH-E430G-K439E, (C) IgG1-CAMPATH-E430G-K439E-G236R, and (D) IgG1-CAMPATH-E430G-K439E-G237R mixed with nonbinding control IgG1-b12 or different IgG1-11B8 variants. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 27D] See legend to Figure 27A. [Figure 28A]Figure 28 shows FcγR binding by IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G antibody variants carrying self-oligomerization-inhibiting mutations K439E or S440K and C1q binding-modulating mutations. (A-E) Binding of immobilized antibody variants to dimeric His-tagged, biotinylated ECD as tested in an ELISA assay for (A) the high-affinity allotype FcγRIIA 131H, (B) the low-affinity allotype FcγRIIA 131R, (C) FcγRIIB, (D) the high-affinity allotype FcγRIIIA 158V, or (E) the low-affinity allotype FcγRIIIA 158F. (F) Binding of immobilized FcγRIa to antibody variants tested in ELISA. Binding is presented for 20 μg / mL antibody samples and normalized per experiment to the average signal observed for wild-type IgG1-CAMPATH-1H (100%) after subtraction of the signal in wells incubated without primary antibody. Detection was performed using streptavidin-polyHRP and ABTS. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 28D] See legend to Figure 28A. [Figure 28E] See legend to Figure 28A. [Figure 28F] See legend to Figure 28A. [Figure 29A]Figure 29 shows the selectivity of CDC activity by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H, anti-CD20 IgG1-11B8, and anti-CD52 IgG1-h2E8 through the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy was measured in three independent experiments and is presented as (A) the mean AUC normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and (B) the mean lysis rate determined by propidium iodide positivity at an antibody concentration of 40 μg / ml. [Figure 29B] See legend to Figure 29A. [Figure 30A] Figure 30 (A, B) shows the CDC efficacy of single-agent and combined anti-CD52 IgG1-CAMPATH-1H-E430G, anti-CD20 IgG1-11B8-E430G, and non-antigen-binding IgG1-b12-E430G antibody variants carrying the indicated self-oligomerization-inhibiting and C1q-binding-modulating mutations. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 30B] See legend to Figure 30A. [Figure 30C] See legend to Figure 30A. [Figure 30D] See legend to Figure 30A. [Figure 31]Activation of Jurkat reporter cell lines stably expressing either (A) FcγRIIa or (B) FcγRIIIa is shown, as measured by the level of luminescence (RLU), upon coculture with Raji lymphoma cells and different concentrations of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants. Luminescence values ​​were normalized per experiment to the luminescence observed for IgG1-b12 (0%) and wild-type IgG1-Campath-1H + wild-type IgG1-11B8 (100%), and then averaged across three (FcγRIIa) or two (FcγRIIIa) experimental replicates. [Figure 32] Co-dependent CDC on Wien 133 cells induced by non-equimolar mixtures of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G236R and IgG1-11B8-E430G-S440K-G237A. (B) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G237Q and IgG1-11B8-E430G-S440K-G237A. [Figure 33]Figure 1 shows the selectivity of CDC activity by mixtures of anti-CD52 IgG1-CAMPATH-1H antibody variants with either the anti-HLA-DR IgG1-HLA-DR-huL243 variant (A) or the anti-HLA-DR IgG1-HLA-DR-1D09C3 variant (B) due to the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. Oci-Ly17 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to a mixture of (A) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-huL243-E430G (100%) or (B) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-1D09C3-E430G (100%). [Figure 34] Figure 1 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. CDC efficacy is shown for variants of IgG1-CAMPATH-1H-E430G-K439E with any of the mutations L234A, L234A-L235A, L234F, L234F-L235E, L235A, L235Q, G236R, or G237Q, and for mixtures of these variants with either the non-binding control antibody IgG1-b12 or IgG1-11B8-E430G-S440K. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 35A]Figure 35 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. (A) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8, as single agents or in mixtures, harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345K, E345Q, E345R, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A. (B) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in mixtures thereof. (C) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in combination. (D) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G or K248E-T437R, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-modulating mutations G236R, G237A, or E333S, as single agents or in combination. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%). [Figure 35B] See legend to Figure 35A. [Figure 35C] See legend to Figure 35A. [Figure 35D] See legend to Figure 35A. [Figure 36A] Figure 36 shows codependent CDC on Raji lymphoma cells induced by a mixture of IgG1-CD37-37-3 and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Relative area under the curve (AUC) of cell lysis induced by dilutions of the indicated antibody variants or their mixtures, normalized to minimum lysis (0% for IgG1-b12) and maximum lysis (100% for the mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G). (B) Maximum lysis rates induced by the indicated antibody variants and their mixtures. [Figure 36B] See legend to Figure 36A. [Figure 37A] Figure 37 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Patient CLL samples were incubated with a series of antibody concentrations in the presence of 20% NHS. CDC efficacy is presented as the percentage of surviving B cells upon incubation with the antibody variants. Results are shown using CLL samples from patients 1 (A), 2 (B), and 3 (C). [Figure 37B] See legend to Figure 37A. [Figure 37C] See legend to Figure 37A. [Figure 38A]Figure 38 shows the fractions of B cells, CD4+ T cells, and CD8+ T cells detected by flow cytometry after incubation of whole blood samples with a mixture of IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-CD5-INSERM antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Percentages of (A) B cells, (B) CD4+ T cells, and (C) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-b12 antibody variants. Percentages of (D) B cells, (E) CD4+ T cells, and (F) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-CD5-INSERM, and IgG1-b12 antibody variants. Fractions were calculated as [100% × (number of cells in sample / number of cells in "no antibody sample") × (number of granulocytes in "no antibody sample" / number of granulocytes in sample)]. [Figure 38B] See legend to Figure 38A. [Figure 38C] See legend to Figure 38A. [Figure 38D] See legend to Figure 38A. [Figure 38E] See legend to Figure 38A. [Figure 38F] See legend to Figure 38A. [Figure 39]Figure 1 shows cooperative activation of programmed cell death in cancer cells by anti-DR4 and anti-DR5 antibody variants harboring mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit (G237T) or enhance (K326W-E333S) C1q binding. (A) Viability of BxPC-3 human pancreatic cancer cells after 72 hours of incubation with the indicated antibody variants. (B) Viability of COLO 205 human colon cancer cells after 72 hours of incubation with the indicated antibody variants. The percentage of viable cells was calculated using the following formula: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of sample without antibody - luminescence of staurosporine sample)] * 100. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description of the Invention In describing embodiments of the present invention, specific terminology will be used for the sake of clarity. However, it is understood that the present invention is not intended to be limited to the specific terminology so selected, and that each specific term encompasses all technical equivalents that function in a similar manner to accomplish a similar purpose.

[0045] definition The term "parent antibody" is understood to mean an antibody that is identical to an antibody according to the present invention, except that it does not have a C1q binding-modulating substitution according to the present invention. Thus, a parent antibody may have an Fc-Fc-enhancing substitution and a self-oligomerization-inhibiting substitution. The term "C1q binding-modulating substitution" is understood to mean a substitution that can inhibit C1q binding, such as a substitution of an amino acid at position G237, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, and P329R, or a substitution that can enhance C1q binding, such as one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0046] The term "polypeptide comprising an immunoglobulin Fc region and a binding region" in the context of the present invention refers to a polypeptide comprising an immunoglobulin Fc region and a binding region capable of binding to any molecule, e.g., a polypeptide, present on, for example, a cell, bacterium, or virion. The immunoglobulin Fc region is typically defined as an antibody fragment that can be generated after digestion of an antibody with papain (known to those skilled in the art) and comprises the two CH2-CH3 regions of the immunoglobulin and a connecting region, e.g., a hinge region. The constant domain of the antibody heavy chain defines the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region, together with cell surface receptors called Fc receptors and proteins of the complement system, mediates the effector functions of the antibody. The binding region can be a polypeptide sequence, e.g., a protein, a protein ligand, a receptor, an antigen-binding region, or a ligand-binding region capable of binding to a cell, bacterium, or virion. When the binding region is, for example, a receptor, the "polypeptide comprising an immunoglobulin Fc region and a binding region" may be prepared as a fusion protein of the immunoglobulin Fc region and the binding region. When the binding region is an antigen-binding region, the "polypeptide comprising an immunoglobulin Fc region and a binding region" may be an antibody such as a chimeric, humanized, or human antibody, or an antibody consisting only of a heavy chain, or an ScFv-Fc fusion. A polypeptide comprising an immunoglobulin Fc region and a binding region typically comprises a linking region, such as a hinge region and two CH2-CH3 regions of the immunoglobulin heavy chain. Therefore, a "polypeptide comprising an immunoglobulin Fc region and a binding region" may be a "polypeptide comprising at least an immunoglobulin Fc region and a binding region." The term "immunoglobulin Fc region" in the context of the present invention means the presence of joining regions, such as the hinge and CH2 and CH3 regions, depending on the subtype of immunoglobulin, e.g., human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE antibody. The polypeptide is not limited to human origin, but can be of any origin, such as mouse or cynomolgus monkey origin.The term "wild-type Fc region" as used herein means an immunoglobulin Fc region having an amino acid sequence as it occurs in nature.

[0047] The term "hinge region," as used herein, is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0048] The term "CH2 region" or "CH2 domain," as used herein, is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any other subtype described herein.

[0049] The term "CH3 region" or "CH3 domain," as used herein, is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any other subtype described herein.

[0050] The term "immunoglobulin" refers to a type of structurally related glycoprotein consisting of two pairs of polypeptide chains: one pair of low-molecular-weight light (L) chains and one pair of heavy (H) chains, all four of which are tightly interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. Heavy chains are interconnected by disulfide bonds at the so-called "hinge region." Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in sequence and / or structure-defined loop configurations), also called complementarity-determining regions (CDRs), and interspersed, more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (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); see also internet http address www.imgt.org / ).Unless otherwise indicated or contradicted by context, references herein to amino acid positions within an Fc region / Fc domain follow EU numbering (Edelman et al., Proc Natl Acad Sci U.S.A. 1969 May;63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition - 1991 NIH Publication No.91-3242).

[0051] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either of these, capable of specifically binding to an antigen. The antibody of the present invention comprises an immunoglobulin Fc domain and an antigen-binding region. Antibodies generally comprise two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least an Fc domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule comprise binding domains that interact with antigens. The constant or "Fc" region of an antibody can mediate the binding of the immunoglobulin to host tissues or elements, such as various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., C1q, the first component of the classical pathway of complement activation. The antibody may also be a multispecific antibody, e.g., a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be present on the same target or on different targets. When epitopes are present on different targets, such targets may be present on the same cell or on different cells or cell types. As noted above, unless otherwise specified or clearly contradicted by the context, the term antibody as used herein encompasses antibody fragments that contain at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be obtained by any known method, such as enzymatic cleavage, peptide synthesis, and recombinant expression. It has been shown that the antigen-binding function of an antibody can also be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies consisting of only two heavy chains, such as those naturally occurring in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche,WO2011069104), asymmetric strand-exchange engineered domains (SEED or Seed-body) and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), dual variable domain immunoglobulins (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); dual domain double-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-Diabody (ImClone / Eli Lilly), Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), Electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), dual targeting domain antibodies (GSK / Domantis), two-in-one antibodies or dual action Fabs that recognize two targets (Genentech,NovImmune, Adimab), cross-linked Mab (Karmanos Cancer Center), covalent fusion mAb (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecific (ImClone / Eli Lilly, Shen, J., et al.J Immunol Methods, 2007.318(1-2):p.65-74), TIG-body, DIG-body and PIG-body (Pharmabcine), dual affinity retargeting molecule (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybody (Zyngenia), a generic light chain approach (Crucell / Merus, US7262028) or a generic heavy chain approach (κλBody by NovImmune, WO2012023053), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment comprising an Fc region, such as scFv fusions, e.g., BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009.393(3):p.672-92), scFv fusions by Genetech / Roche, scFv fusions by Novartis, scFv fusions by Immunomedics, scFv fusions by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb by f-Star, 2(WO2008 / 003116), and double scFv fusions. It should also be understood that the term antibody, unless otherwise specified, encompasses polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies) obtained by techniques such as those used by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. Potentially, the antibodies produced can have any isotype.

[0052] The term "full-length antibody," as used herein, refers to an antibody (e.g., a parent antibody) that includes all of the heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that isotype.

[0053] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0054] The term "chimeric antibody," as used herein, refers to an antibody in which both chain types (i.e., heavy and light chains) are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (whether of non-human species origin, or synthetic or engineered from any species, including human) linked to a constant region of human origin.

[0055] The term "humanized antibody," as used herein, refers to an antibody in which both chain types have been humanized as a result of antibody engineering. Humanized chains are typically chains in which the complementarity-determining regions (CDRs) of the variable domains are foreign (originating from a species other than human or synthetic), while the remainder of the chain is of human origin. Since the assessment of humanization is based on the resulting amino acid sequence and not the methodology itself, protocols other than grafting can be used.

[0056] The terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like, as used herein, refer to a preparation of Ab molecules of monomolecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an Ab exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas, which contain B cells obtained from transgenic or transchromosomic non-human animals, e.g., transgenic mice, whose genomes have been rearranged to produce functional human antibodies and contain human heavy and light chain transgene repertoires, and fused with immortalized cells.

[0057] The term "isotype," as used herein, refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotypes thereof, e.g., IgG1m(za) and IgG1m(f)) that is encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be associated with either a kappa (κ) or lambda (λ) light chain. The term "mixed isotype," as used herein, refers to an immunoglobulin Fc region obtained by combining structural forms of one isotype with analogous regions from another isotype, thereby generating a hybrid isotype. A mixed isotype may comprise an Fc region having a sequence composed of two or more isotypes selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, thereby generating combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0058] The terms "antigen-binding region," "region that binds to an antigen," "binding region," or antigen-binding domain, as used herein, refer to the region of an antibody capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody. The VH and VL domains can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in the form of sequence- and / or structure-defined loops), also called complementarity-determining regions (CDRs), and interspersed, more conserved regions called framework regions (FRs). An antigen can be any molecule, e.g., a polypeptide, present on, for example, a cell, bacterium, or virion.

[0059] The term "target," as used herein, refers to a molecule to which the antigen-binding region of an antibody binds. Targets include any antigen against which an antibody is generated. The terms "antigen" and "target" are used interchangeably in reference to antibodies and may constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0060] The term "epitope" refers to a molecular determinant capable of specific binding to an antibody variable domain. Epitopes usually consist of surface groupings of molecules, such as amino acids, sugar side chains, or a combination thereof, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes may include amino acid residues directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues not directly involved in binding.

[0061] An "antibody" or "antibody variant" or "variant of a parent antibody" of the present invention is an antibody molecule containing one or more mutations compared to a "parent antibody." These different terms are used interchangeably and may have the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof. These different terms are used interchangeably and may have the same meaning and purpose with respect to any aspect or embodiment of the present invention. Amino acid substitutions may replace a natural amino acid with another naturally occurring amino acid or a non-naturally occurring amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes shown in one or more of the following three tables:

[0062] Classes of amino acid residues for conservative substitutions TIFF0007649139000001.tif51132

[0063] Classes of alternative conservative amino acid residue substitutions TIFF0007649139000002.tif38132

[0064] Alternative physical and functional classifications of amino acid residues TIFF0007649139000003.tif68136

[0065] In the context of the present invention, substitutions in the variants are: Original amino acid - position - substituted amino acid As shown; Amino acid residues are indicated using three-letter or one-letter codes, including the codes Xaa and X. Thus, the notation "E345R" or "Glu345Arg" means that the variant contains a substitution of arginine for glutamic acid at the amino acid position in the variant that corresponds to amino acid 345 of the parent antibody.

[0066] If the position itself is not present in the antibody, but the variant contains an amino acid insertion, e.g. Use the notation position-substituted amino acid, e.g., "448E."

[0067] Such designations are particularly relevant in the context of one or more modifications in a series of homologous polypeptides or antibodies.

[0068] Similarly, if the identity of the substituted amino acid residue or residues is not important, The original amino acid position is represented as "E345".

[0069] Modifications in which one or more of the original amino acids and / or one or more substituted amino acids may include more than one but not all amino acids, such as when glutamic acid at position 345 becomes arginine, lysine, or tryptophan; "Glu345Arg,Lys,Trp" or "E345R,K,W" or "E345R / K / W" or "E345 to R, K or W" may be used interchangeably in the context of the present invention.

[0070] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids or with other amino acids, e.g., unnatural amino acids. For example, substitution of amino acid E at position 345 encompasses each of the substitutions 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345P, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is equivalent to the designation 345X, where X represents any amino acid. Such substitutions may also be designated as E345A, E345C, etc., or E345A, C, etc., as well as E345A / C / , etc. The same applies equally to any position described herein, and any one of such substitutions is specifically encompassed herein.

[0071] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, as opposed to the recognition or activation phase of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, e.g., cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, e.g., neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and exert specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing ADCC. For example, FcR-expressing monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells are involved in the specific killing of target cells and antigen presentation to other components of the immune system, or binding to cells that present antigens. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation delivers C3 fragments onto target cells. Such C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors, such as cytokines. For example, FcγRI expression has been shown to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose and dissolve target cells.In some embodiments, antibody-driven classical complement activation produces C3 fragments on target cells.These C3 cleavage products can directly promote the phagocytosis of effector cells or indirectly promote antibody-mediated phagocytosis.

[0072] The term "Fc effector function" or "Fc-mediated effector function," as used herein, is intended to refer to a function that results from the binding of a polypeptide or antibody to its target, e.g., an antigen, on a cell membrane, where the Fc effector function is due to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) binding of an opsonizing antibody to a complement receptor mediated by the antibody, (x) opsonization, and (xi) any combination of (i)-(x).

[0073] The term "clustering-dependent function," as used herein, is intended to refer to a function that is a result of the formation of an antigen complex following oligomerization of a polypeptide or antibody bound to an antigen, optionally on a cell, cell membrane, virion, or another particle. Examples of clustering-dependent effector functions include (i) antibody oligomerization, (ii) antibody oligomer stability, (iii) antigen oligomerization, (iv) antigen oligomer stability, (v) induction of apoptosis, (vi) modulation of proliferation, e.g., reducing, inhibiting, or stimulating proliferation, and (vii) any combination of (i)-(vi).

[0074] The term "agonistic," as used herein, is understood as the stimulation or activation of a receptor on a cell membrane that results in a biological response, such as intracellular signaling. Such an agonistic effect may result in the induction of apoptosis (programmed cell death) or activation of immune cells or intracellular pathways.

[0075] Agonist activity or increased agonist activity can be determined in a viability assay, as described in Example 16, for antibodies against targets expressing an intracellular death domain, using the following steps: (i) seeding a cell line expressing the target corresponding to the antibody, e.g., DR5, into a 96-well flat-bottom plate overnight at 37°C; (ii) adding serial dilutions of a range (0.0003 to 20,000 ng / mL) of an antibody, such as an anti-DR5 antibody, and incubating at 37°C for 3 days; (iii) determining cell viability by quantifying the presence of ATP, for example, by use of the CellTiter-Glo luminescent cell viability assay; (iv) Equation: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of sample without antibody - luminescence of staurosporine sample)] * Calculate viable cells using 100.

[0076] Agonist activity or increased agonist activity may be determined in a reporter assay, as described in Examples 29, 30, 31, and 32, for antibodies against targets that activate intracellular signaling pathways, using the following steps: (i) seeding Jurkat cells stably transfected with a downstream target of an NFAT response element and a luciferase reporter gene and incubating the cells overnight at 37°C in a 96-well flat-bottom plate; (ii) adding an antibody, e.g., a range of antibodies, e.g., serial dilutions from 19.5 to 5,000 ng / mL, and incubating for 5 hours; (iii) adding a firefly luciferase substrate (5'-fluoroluciferin) to the cells and incubating for 5 to 10 minutes; (iv) determining luminescence using an Envision MultiLable Plate reader.

[0077] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of directing transcription of a nucleic acid segment ligated into it. One type of vector is a "plasmid," which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment may be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0078] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because some modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphoid cells, as well as prokaryotic cells, such as Escherichia coli (E. coli), and other eukaryotic hosts, such as plant cells and fungi.

[0079] The term "transfectoma," as used herein, includes recombinant eukaryotic host cells, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungal, e.g., yeast cells, expressing an Ab or a target antigen.

[0080] The term "preparation" refers to a preparation of antibody variants and mixtures of different antibody variants that may have increased ability to oligomerize when interacting with an antigen associated with a cell, cell membrane, virion, or other structure (e.g., an antigen expressed on the cell surface), resulting in enhanced signaling and / or activation by the antigen.

[0081] As used herein, the term "affinity" refers to the strength of binding of one molecule, e.g., an antibody, to another, e.g., a target or antigen, at a single site, e.g., the strength of monovalent binding of an individual antigen-binding site of an antibody to an antigen.

[0082] As used herein, the term "avidity" refers to the total strength of binding between two structures, for example, between multiple antigen-binding sites of an antibody that simultaneously interact with a target, or between multiple binding sites, for example, an antibody and C1q. When there is more than one binding interaction, the two structures will only dissociate when all binding sites dissociate, and therefore the dissociation rate will be slower than for individual binding sites, resulting in a greater effective total binding strength (avidity) compared to the strength with which each individual binding site binds (affinity).

[0083] As used herein, the term "oligomer" refers to a molecule (e.g., an antibody) that is composed of more than one but a limited number of monomer units, as opposed to a polymer that is composed of, at least in principle, an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to denote the number of monomer units in an oligomer; for example, a tetramer is composed of four units, and a hexamer is composed of six units.

[0084] The term "oligomerization," as used herein, refers to the process of converting monomers to a finite degree of polymerization. It has been observed herein that antibodies comprising a target-binding region according to the present invention can form oligomers, such as hexamers, after target binding, for example, on the cell surface, via non-covalent association of Fc regions. In the context of this application, "self-oligomerization" or "homo-oligomerization" refers to the process of oligomerization between antibody molecules having identical protein sequences, regardless of post-translational modifications. The term "hetero-oligomerization," as used herein, refers to the process of oligomerization between antibody molecules having different protein sequences, regardless of post-translational modifications. Different antibodies involved in hetero-oligomerization may bind to different antigens, such as different target proteins, glycoproteins, glycans, or glycolipids.

[0085] The term "self-oligomerization-inhibiting substitution" refers to a substitution in an antibody comprising an immunoglobulin Fc region and an antigen-binding region that inhibits the process of oligomerization between antibody molecules having identical protein sequences, ignoring post-translational modifications. Inhibition of self-oligomerization can result in an increase in the EC50 of the CDC activity of the polypeptide or a decrease in the maximum CDC lytic activity, as measured, for example, by the methods described in Examples 2 and 15. Examples of self-oligomerization-inhibiting substitutions are K439E and S440K.

[0086] The term "clustering," as used herein, is intended to refer to the oligomerization of antibodies, polypeptides, antigens, or other proteins through non-covalent interactions.

[0087] The term "Fc-Fc enhancement," as used herein, is intended to refer to increasing the binding strength between Fc regions of polypeptides or stabilizing the interaction between Fc regions, such that two Fc region-containing antibodies or polypeptides form oligomers after target binding.

[0088] An Fc-Fc-enhancing substitution, as used herein, refers to a substitution at position E430, E345, or S440, which corresponds to human IgG1 according to EU numbering, provided that the substitution at position S440 is S440Y or S440W. Accordingly, an Fc-Fc-enhancing substitution, as used herein, refers to the amino acid substitutions E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. In a preferred embodiment, the Fc-Fc-enhancing substitution is E430G, E345K, or E345R.

[0089] As used herein with respect to two antigens, the term "coexisting" or grammatical variations thereof refers, on the one hand, to a situation in which two antigens are co-expressed in the same cell. The antigens may already be in close proximity to one another on the cell, or the antigens may be brought into close proximity through oligomerization of binding polypeptides of the invention, e.g., antibodies. Furthermore, the term "coexisting" also refers to a situation in which two antigens are expressed in different cells, but such cells are located in close proximity to one another.

[0090] The term "co-dependence," as used herein, refers to a functional effect that is dependent on the simultaneous binding of two or more different Fc domain-containing polypeptides containing self-oligomerization-inhibiting substitutions to the same target, cell, or virion. In the context of the present invention, functional effects that may be co-dependent include clustering-dependent functions, Fc-mediated effector functions, and binding of effector molecules such as FcγR or C1, but do not necessarily include the individual binding of the Fc domain-containing polypeptides to the target antigen. As used herein, different Fc domain-containing polypeptides containing self-oligomerization-inhibiting substitutions may each individually bind to different targets, cells, or virions, but a co-dependent functional outcome depends on the simultaneous binding of two or more different components to the same target, cell, or virion. As used herein, a co-dependent functional effect is specifically recovered by two or more different Fc domain-containing polypeptides containing self-oligomerization-inhibiting substitutions by restoring non-covalent Fc-Fc interactions between the different components in the co-dependent Fc-containing polypeptide mixture.

[0091] The term "safety margin," as used herein, refers to an index of a drug's effectiveness and safety and is defined as the range between the drug's minimum therapeutic dose (effectiveness on diseased tissue) and its minimum toxic dose (effectiveness on healthy tissue).

[0092] The term "C1q binding," as used herein, refers to the direct interaction between C1q and an antibody. Direct C1q binding can be assessed, for example, by using antibodies immobilized on an artificial surface. Multivalent interactions that result in high-avidity binding of C1q to antibody oligomers can be assessed when bound to a given antigen on a cell or virion surface.

[0093] Binding of C1q to a polypeptide or antibody can be determined in an ELISA assay using the following steps. (i) Coat 96-well Microlon ELISA plates with 1 μg / mL of polypeptide or antibody in 100 μL of PBS overnight at 4°C; (ii) Incubate the plates with 100 μL / well of a serial dilution series of C1q (3-fold dilutions for a final C1q concentration range of 30–0.01 μg / mL) at 37°C for 1 hour; (iii) Incubate the plates with 100 μL / well of rabbit anti-human C1q at RT for 1 hour; (iv) Incubate the plates with 100 μL / well of swine anti-rabbit IgG-HRP at RT for 1 hour; (v) Incubate the plates with 100 μL / well of substrate, 1 mg / mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), at RT for 15 minutes; (vi) Stop the reaction by adding 100 μL of 2% oxalic acid / well. Absorbance is measured at 405 nm in a BioTek EL808 Microplate reader.

[0094] The term C1q binding substitution, as used herein, refers to a substitution in a polypeptide comprising an immunoglobulin Fc region and an antigen-binding region that enhances the direct interaction with C1q. Enhanced C1q binding results in a decreased EC50 of the interaction between C1q and a polypeptide comprising an immunoglobulin Fc region and an antigen-binding region, as measured, for example, by the method for determining C1q binding described above.

[0095] As used herein, the term "complement activation" refers to activation of the classical complement pathway, initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on a surface. C1 is a complex consisting of six recognition proteins, C1q, and the serine protease heterotetramer C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions beginning with the cleavage of C4 to C4a and C4b and C2 to C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves complement component C3 to C3b and C3a, forming C5 convertase. The C5 convertase splits C5 into C5a and C5b, the latter of which is deposited on the membrane, triggering the later events of complement activation, in which terminal complement components C5b, C6, C7, C8, and C9 are assembled into the membrane attack complex (MAC). The complement cascade leads to the generation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be assessed by using C1q availability, CDC kinetics, CDC assays (as described in WO2013 / 004842, WO2014 / 108198), or by cellular deposition of C3b and C4b, as described by Beurskens et al. in Journal of Immunology, 2012 vol. 188 no. 7, April 1, 3532-3541.

[0096] The term "complement-dependent cytotoxicity" ("CDC"), as used herein, is intended to refer to the process of antibody-mediated complement activation that results in lysis of the cell or virion as a result of holes in the membrane created by the MAC construct when the antibody is bound to a target on the cell or virion.

[0097] The term "antibody-dependent cell-mediated cytotoxicity" ("ADCC"), as used herein, is intended to refer to the mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of the bound antibody. The term "antibody-dependent cellular phagocytosis" ("ADCP"), as used herein, is intended to refer to the mechanism of removal of antibody-coated target cells or virions by internalization by phagocytes. Internalized antibody-coated target cells or virions are encapsulated in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes. ADCP can be assessed by using an in vitro cytotoxicity assay using macrophages as effector cells and videomicroscopy, as described by van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685.

[0098] The term "complement-dependent cytotoxicity" ("CDCC"), as used herein, is intended to refer to the mechanism of killing of target cells or virions by cells expressing complement receptors that recognize cleavage products of the third component of complement (C3) that is covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC can be assessed in a manner similar to that described for ADCC.

[0099] The term "plasma half-life," as used herein, refers to the time it takes for the concentration of a polypeptide in plasma to decrease to half of its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase is typically 1-3 days, during which there is an approximately 50% decrease in plasma concentration due to redistribution between plasma and tissues. Plasma half-life can be measured by methods well known in the art.

[0100] The term "plasma clearance rate," as used herein, is a quantitative measure of the rate at which a polypeptide is removed from the blood when administered to a living organism. The plasma clearance rate can be calculated as Dose / AUC (mL / day / kg), where the AUC value (area under the curve) is determined from the concentration-time curve.

[0101] The term "antibody-drug conjugate," as used herein, refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that couples the drug to, for example, the antibody. The linker is cleavable or non-cleavable in the presence of malignant cells; in this case, the antibody-drug conjugate kills the malignant cells.

[0102] The term "antibody-drug conjugate uptake," as used herein, refers to the process by which an antibody-drug conjugate binds to a target on a cell and is then internalized / encapsulated by the cell membrane, thereby being drawn into the cell. Antibody-drug conjugate uptake can be assessed as described in WO 2011 / 157741, "Antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay."

[0103] The term "apoptosis," as used herein, refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events can lead to characteristic cellular changes (morphology) and death. These changes include cytoplasmic blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis can be induced by antibody binding to certain receptors.

[0104] As used herein, the term " programmed cell death " or " PCD " refers to any form of cell death mediated by intracellular program.Different forms of PCD exist, and the commonality of various types of PCD is that they are carried out by active cellular process, which can be disrupted by interfering with intracellular signal transduction.In a specific embodiment, the occurrence of any form of PCD in cell or tissue can be examined by staining cell or tissue with conjugated Annexin V, and correlating with phosphatidylserine exposure.

[0105] The term "annexin V," as used herein, refers to a family of annexin proteins that bind to phosphatidylserine (PS) on cell surfaces.

[0106] The term "FcRn," as used herein, is intended to refer to the neonatal Fc receptor, an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk through the intestinal epithelium of neonatal rodents into the neonatal bloodstream. Further studies have revealed a similar receptor in humans. However, in humans, it is found in the placenta, where it helps facilitate the transport of maternal IgG to the developing fetus and has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at an acidic pH of 6.0–6.5 but not at neutral or higher pH. Thus, FcRn can bind IgG from the intestinal lumen (inside the intestine), which has a slightly acidic pH, ensuring efficient unidirectional transport to the basolateral side (inside the body), where the pH is neutral to basic (pH 7.0–7.5). This receptor also plays a role in IgG recycling in adults by being present in the endocytic pathway within endothelial cells. FcRn receptors in acidic endosomes bind internalized IgG by pinocytosis, recycling it to the cell surface and releasing it into the basic pH of the blood, thereby preventing lysosomal degradation, which may explain the longer half-life of IgG in the blood compared with other isotypes.

[0107] The term "Protein A," as used herein, is intended to refer to a 56-kDa MSCRAMM surface protein first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cell osmolality, and a two-component system called ArlS-ArlR. It has found utility in biochemical studies due to its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains folded into a three-helix bundle. Each domain has the ability to bind proteins from many mammalian species, most notably IgG. It binds to the Fc region of most immunoglobulin heavy chains (overlapping with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. This interaction in serum allows IgG molecules to bind to bacteria via their Fc region, rather than their Fab region alone, thereby disrupting opsonization, complement activation, and phagocytosis.

[0108] The term "protein G," as used herein, is intended to refer to an immunoglobulin-binding protein expressed by group C and group G streptococci, which is similar to, but has different specificity than, protein A. It is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) cell surface protein that has found use in antibody purification due to its binding to the Fc region.

[0109] Specific Aspects of the Invention As described herein, in a first aspect, the present invention relates to a first antibody for use as a pharmaceutical in combination with a second antibody, wherein the activities of the first and second antibodies are co-dependent, the first antibody having an Fc region with an Fc-Fc-enhancing substitution, a self-oligomerization-inhibiting substitution, and one or more substitutions that reduce effector function such as CDC and / or ADCC, and the second antibody having an Fc region with an Fc-Fc-enhancing self-oligomerization-inhibiting substitution and, optionally, one or more substitutions that enhance Fc effector function such as CDC and / or ADCC. Thus, such a combination of first and second antibodies, in which the effect of the first antibody is dependent on the presence of the second antibody and the effect of the second antibody is dependent on the presence of the first antibody, may increase the margin of safety for the use of such a combination of first and second antibodies.

[0110] As shown by the present inventors, a first antibody having an Fc region with one Fc-Fc-enhancing substitution, one or more substitutions that reduce effector function such as CDC and / or ADCC, and complementary self-oligomerization-inhibiting substitutions, when used as a single antibody, exhibits no or only very limited CDC activity. Similarly, a second antibody according to the present invention having an Fc region with one Fc-Fc-enhancing substitution, one or more substitutions that enhance Fc effector function such as CDC and / or ADCC, and complementary self-oligomerization-inhibiting substitutions, when used as a single antibody, exhibits only limited CDC activity. While the single agents exhibit no or limited ability to induce CDC, surprisingly, the combination of the first and second antibodies according to the present invention can restore the induced CDC level.

[0111] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one or more substitutions selected from the group consisting of L234, L235, G237, G236, or one or more substitutions selected from the group consisting of K322A and E269K; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0112] In another aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, P329R, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) if the first Fc region comprises a K322E or P329R substitution, it comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the EU numbering system (Edelman et al., Proc Natl Acad Sci U.S. A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No.91-3242); A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0113] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing one amino acid substitution at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0114] In one aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) comprising one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (b) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and (c) containing a single amino acid substitution at position P329 or a substitution of K322E; the second Fc region (d) one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W; and (e) containing a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and (f) comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the EU numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0115] Substitutions at positions corresponding to E430, E345, or S440Y or S440W are considered Fc-Fc-enhancing substitutions according to the present invention, and such substitutions introduce enhanced Fc-Fc interaction and oligomerization effects into a polypeptide or antibody. Enhanced oligomerization occurs when the antigen-binding region of an antibody binds to its corresponding target antigen. Enhanced oligomerization generates oligomers, such as hexamers. The generation of oligomeric structures, such as hexamers, increases the C1q binding avidity of the polypeptide, thereby enhancing Fc effector function, such as CDC.

[0116] In one embodiment, the first antibody comprises at most one substitution at a position corresponding to E430, E345, or a S440Y or S440W substitution. In one embodiment, the second antibody comprises at most one substitution at a position corresponding to E430, E345, or a S440Y or S440W substitution. Thus, in one embodiment, the Fc region comprises at most one substitution at a position corresponding to E430, E345, or a S440Y or S440W substitution. In one embodiment of the invention, the first Fc and second Fc regions comprise substitutions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. Thus, in one embodiment, a first Fc region may have an E430G substitution and a second Fc region may have an E345K substitution. In another embodiment, a first Fc region may have an E345K substitution and a second antibody may have an E430G substitution. The substitutions in the first and second Fc regions may be independently selected from a group of Fc-Fc enhancing substitutions.

[0117] In one embodiment of the invention, the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, and E345R.

[0118] In one embodiment of the invention, the first and second Fc regions comprise an E430G substitution. In one embodiment of the invention, the first and second Fc regions comprise an E345K substitution. In one embodiment of the invention, the first and second Fc regions comprise an E345R substitution. In one embodiment of the invention, the first and second Fc regions comprise an S440Y substitution.

[0119] The first and second Fc regions further comprise a K439E or S440K substitution, which are considered complementary oligomerization-inhibiting substitutions according to the present invention. That is, for example, a first antibody having a K439E substitution cannot form oligomers with another antibody having a K439E substitution, but an antibody having a K439E substitution can form oligomers with another antibody having a S440K substitution. An antibody having a S440K substitution cannot form oligomers with another antibody having a S440K substitution, but can form oligomers with an antibody having a K439E substitution. Thus, in one embodiment of the present invention, the first Fc region comprises a K439E substitution and the second Fc region comprises a S440K substitution. In one embodiment of the present invention, the first Fc region comprises a S440K substitution and the second Fc region comprises a K439E substitution. When an Fc region contains the oligomerization-inhibiting substitution S440K, the Fc region may not contain the Fc-Fc-enhancing substitutions S440Y or S440W. Thus, an Fc region having the oligomerization-inhibiting substitution S440K may have an Fc-Fc-enhancing substitution at the amino acid position corresponding to E430 or E345.

[0120] In another aspect, the present invention provides 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 as a pharmaceutical 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, wherein the first antibody comprises: the first Fc region (a) containing the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing one amino acid substitution at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region (d) containing the substitutions K248E and T437R; and (e) containing a K439E or S440K substitution; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to those of human IgG1 according to the Eu numbering system; A first antibody for use as a pharmaceutical in combination with a second antibody is provided.

[0121] An Fc-Fc enhancing substitution at a position corresponding to E430, E345, or a S440Y or S440W substitution according to any aspect or embodiment herein may be substituted with the following two substitutions: T248E and T437R.

[0122] In one embodiment of the present invention, the first Fc region comprises a substitution that reduces effector function such as CDC and / or ADCC. Accordingly, in one embodiment of the present invention, the first Fc region comprises a substitution that reduces CDC. In one embodiment of the present invention, the first Fc region comprises a substitution that reduces ADCC. In one embodiment of the present invention, the first Fc region comprises a substitution that reduces CDC and ADCC. In one embodiment of the present invention, the first Fc region comprises a single amino acid substitution at position G237. The substitution at amino acid position G237 may have the effect of reducing the ability of the antibody to induce CDC. This provides an embodiment in which the first Fc region comprises a substitution that, in addition to reducing CDC activity, can also reduce Fcγ receptor binding, thereby reducing effector function mediated by the Fcγ receptor.

[0123] In one embodiment of the invention, the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, and G237P.

[0124] The present inventors have found that substituting glycine at the position corresponding to 237 in human IgG1 with amino acids such as alanine, threonine, glutamine, or arginine, which represent various classes of naturally occurring amino acids, reduces the ability of the antibody to induce CDC. Therefore, substituting G237 with another naturally occurring amino acid is believed to reduce the ability of the antibody to induce CDC. Substitutions at amino acid position G237 can reduce the ability of antibodies with Fc-Fc enhancing substitutions and self-oligomerization inhibiting substitutions to induce CDC alone.

[0125] In one embodiment of the present invention, the first Fc region comprises one substitution selected from the group consisting of G237T, G237A, G237Q, and G237R. In one embodiment, the first Fc region comprises a G237T substitution. In one embodiment of the present invention, the first Fc region comprises a G237A substitution. In one embodiment of the present invention, the first Fc region comprises a G237S substitution. In one embodiment of the present invention, the first Fc region comprises a G237Q substitution. In one embodiment of the present invention, the first Fc region comprises a G237R substitution.

[0126] In one embodiment of the present invention, the first Fc region comprises one or more substitutions selected from the group consisting of G236R, G236K, E269K, and P329R. In one embodiment of the present invention, the first Fc region comprises substitutions L234F and L235E. This provides an embodiment in which the first Fc region comprises substitutions that not only reduce CDC activity but also reduce Fcγ receptor binding, thereby reducing Fcγ receptor-mediated effector function.

[0127] In one embodiment of the present invention, the first Fc region comprises one or more substitutions selected from the group consisting of G236R, G236K, and E269K. In one embodiment of the present invention, the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. In one embodiment of the present invention, the first Fc region comprises a G263R or G236K substitution. In one embodiment of the present invention, the first Fc region comprises a G236R substitution. In one embodiment of the present invention, the first Fc region comprises a G236K substitution. In one embodiment of the present invention, the first Fc region comprises a G269K substitution.

[0128] In one embodiment of the present invention, the first Fc region comprises a substitution selected from the group consisting of K322A and K322E, thereby providing an embodiment in which the first Fc region comprises a substitution that can reduce the ability of the antibody to induce CDC activity while retaining the ability of the antibody to bind to Fcγ receptors.

[0129] In one embodiment of the present invention, the first Fc region comprises a K322A substitution.

[0130] In one embodiment of the present invention, the first Fc region comprises a K322E substitution.

[0131] In one embodiment of the invention, the first Fc region comprises an amino acid substitution at position P329.

[0132] In one embodiment of the present invention, the first Fc region comprises one substitution selected from the group consisting of P329R, P329K, P329E, P329D, and P329A. In one embodiment of the present invention, the first Fc region comprises a P329R substitution. In one embodiment of the present invention, the first Fc region comprises a P329R substitution. In one embodiment of the present invention, the first Fc region comprises a P329K substitution. In one embodiment of the present invention, the first Fc region comprises a P329E substitution. In one embodiment of the present invention, the first Fc region comprises a P329D substitution. In one embodiment of the present invention, the first Fc region comprises a P329A substitution.

[0133] This provides an embodiment in which a first antibody comprises a first Fc region having Fc-Fc-enhancing substitutions that enhance the antibody's Fc-Fc interaction and oligomerization. Enhanced oligomerization occurs when the antibody's antigen-binding region binds to its corresponding target antigen. Enhanced oligomerization generates oligomers, such as hexamers. The generation of oligomeric structures, such as hexamers, increases the polypeptide's C1q binding avidity, thereby increasing Fc effector function, such as CDC. However, by introducing substitutions that inhibit self-oligomerization and substitutions that reduce effector function, such as CDC and / or ADCC, antibodies with reduced effector function, such as CDC and / or ADCC, can be generated, which may allow for improved modulation of the toxicity profile of an antibody combined with a second antibody. A first antibody is understood to have reduced effector function, such as CDC and / or ADCC, when compared to a parent antibody that has the same Fc-Fc enhancing substitutions and self-oligomerization inhibiting substitutions, but does not have the substitutions that reduce effector function.

[0134] In one embodiment of the invention, the second Fc region comprises substitutions that increase effector function, such as CDC and / or ADCC. Accordingly, in one embodiment, the second Fc region comprises substitutions that increase CDC. In one embodiment, the second Fc region comprises substitutions that increase ADCC. In one embodiment, the second Fc region comprises substitutions that increase CDC and ADCC. A second antibody is said to have increased effector function, such as CDC and / or ADCC, when compared to a parent antibody that has the same Fc-Fc-enhancing substitutions and self-oligomerization-inhibiting substitutions, but does not have the substitutions that enhance effector function.

[0135] In one embodiment of the invention, the second Fc region comprises one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S.

[0136] In one embodiment of the invention, the second Fc region comprises a substitution of G237A. In one embodiment of the invention, the second Fc region comprises a substitution that reduces Fcγ receptor binding, such as G237A.

[0137] In one embodiment of the invention, the second Fc region comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0138] In one embodiment of the invention, the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S.

[0139] In one embodiment of the present invention, the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S. In one embodiment of the present invention, the second Fc region comprises a K326A substitution. In one embodiment of the present invention, the second Fc region comprises a K326W substitution. In one embodiment of the present invention, the second Fc region comprises an E333A substitution. In one embodiment of the present invention, the second Fc region comprises an E333S substitution.

[0140] In one embodiment of the invention, the second Fc region comprises two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0141] In one embodiment of the invention, the second Fc region comprises the substitutions K326W and E333S. In one embodiment of the invention, the second Fc region comprises the substitutions K326A and E333A.

[0142] In one embodiment of the invention, the second Fc region comprises the substitutions G237A and E333S.

[0143] In one embodiment of the invention, the second Fc region comprises a K326A substitution.

[0144] In one embodiment of the invention, the second Fc region comprises an E333S substitution.

[0145] This provides an embodiment in which the second antibody comprises a second Fc region having Fc-Fc-enhancing substitutions that enhance the antibody's Fc-Fc interaction and oligomerization. Enhanced oligomerization occurs when the antibody's antigen-binding region binds to its corresponding target antigen. Enhanced oligomerization generates oligomers, such as hexamers. The generation of oligomeric structures, such as hexamers, has the effect of increasing Fc effector function, such as CDC, by increasing the C1q binding avidity of the polypeptide. However, by introducing substitutions that increase effector function, such as CDC and / or ADCC, an antibody with increased oligomerization and effector function, such as CDC and / or ADCC, can be generated, which may enable improved efficacy of the antibody in combination with the first antibody. A second antibody is understood to have reduced effector function, such as CDC and / or ADCC, when compared to a parent antibody that has the same Fc-Fc enhancing substitutions and self-oligomerization inhibiting substitutions, but does not have substitutions that increase effector function.

[0146] The following table provides a non-limiting list of embodiments describing combinations of first and second polypeptides comprising specific substitutions, thus, for example, embodiment 1 in the table below is a combination of a first antibody comprising substitutions at positions corresponding to E430G, K439E, and G236R, respectively, of human IgG, with a second antibody comprising E430G and S440K substitutions, respectively, of human IgG. All first and second antibodies of embodiments 1-177 may optionally comprise further substitutions as described herein.

[0147] TIFF0007649139000004.tif115129TIFF0007649139000005.tif214129TIFF0007649139000006.tif214129TIFF0007649139000007.tif21412 9TIFF0007649139000008.tif214129TIFF0007649139000009.tif214129TIFF0007649139000010.tif214129TIFF0007649139000011.tif66129

[0148] The following table provides a preferred list of embodiments describing combinations of first and second antibodies comprising specific substitutions, thus, for example, embodiment 1 in the table below is a combination of a first antibody comprising substitutions at positions corresponding to E430G, K439E, and G236R, respectively, of human IgG, with a second antibody comprising E430G and S440K substitutions, respectively, of human IgG. As described herein, all of the first and second antibodies of embodiments 1 to 36 below may optionally comprise further substitutions.

[0149] TIFF0007649139000012.tif87128TIFF0007649139000013.tif206120TIFF0007649139000014.tif42128

[0150] In one embodiment, the first Fc region comprises E430G, K439E, and G236R substitutions, and the second Fc region comprises E430G and S440K substitutions. In one embodiment, the first Fc region comprises E430G, K439E, and G237Q substitutions, and the second Fc region comprises E430G and S440K substitutions. In one embodiment, the first Fc region comprises E430G and K439E substitutions, and the second Fc region comprises E430G, S440K, and G236R substitutions. In one embodiment, the first Fc region comprises E430G and K439E substitutions, and the second Fc region comprises E430G, S440K, and G237Q substitutions. In one embodiment, the first Fc region comprises substitutions of E430G, K439E, and G236R, and the second Fc region comprises substitutions of E430G, S440K, and G237A. In one embodiment, the first Fc region comprises substitutions of E430G, K439E, and G236R, and the second Fc region comprises substitutions of E430G, S440K, and E333S. In one embodiment, the first Fc region comprises substitutions of E430G, K439E, and G237A, and the second Fc region comprises substitutions of E430G, S440K, and G236R. In one embodiment, the first Fc region comprises E430G, K439E, and E333S substitutions, and the second Fc region comprises E430G, S440K, and G236R substitutions. In one embodiment, the first Fc region comprises E430G, K439E, and G237Q substitutions, and the second Fc region comprises E430G, S440K, and G237A substitutions. In one embodiment, the first Fc region comprises E430G, K439E, and G237Q substitutions, and the second Fc region comprises E430G, S440K, and E333S substitutions. In one embodiment, the first Fc region comprises E430G, K439E, and G237A substitutions, and the second Fc region comprises E430G, S440K, and G237Q substitutions. In one embodiment, the first Fc region comprises E430G, K439E, and E333S substitutions, and the second Fc region comprises E430G, S440K, and G237Q substitutions.In one embodiment, the first Fc region comprises substitutions of E345K, K439E, and G236R, and the second Fc region comprises substitutions of E345K, S440K, and E333S. In one embodiment, the first Fc region comprises substitutions of E345R, K439E, and G236R, and the second Fc region comprises substitutions of E345K, S440K, and E333S. In one embodiment, the first Fc region comprises substitutions of E345R, K439E, and G237Q, and the second Fc region comprises substitutions of E345K and S440K.

[0151] In one embodiment of the present invention, the first and / or second antibody is a human antibody, a humanized antibody, or a chimeric antibody. In one embodiment of the present invention, the first and second antibodies are human antibodies, a humanized antibody, or a chimeric antibody. In one embodiment of the present invention, the first and second antibodies are human antibodies. In one embodiment of the present invention, the first and second antibodies are humanized antibodies. In one embodiment of the present invention, the first antibody is a human antibody and the second antibody is a humanized antibody. In one embodiment of the present invention, the first antibody is a humanized antibody and the second antibody is a human antibody.

[0152] In one embodiment of the present invention, the first and / or second antibody is a monoclonal antibody. In one embodiment of the present invention, the first and second antibodies are monoclonal antibodies. In one embodiment of the present invention, the first and second antibodies 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.

[0153] The embodiments described herein with respect to antibodies refer to antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin; it is understood that the antibody may also be a multispecific antibody, such as a bispecific antibody, having 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.

[0154] In one embodiment of the present invention, the first and / or second antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment of the present invention, the first and second antibodies are an IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment of the present invention, the first and second antibodies are a human IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment of the present invention, the first and second antibodies are an IgG1, IgG2, or IgG4 isotype. In one embodiment of the present invention, the first and second antibodies are a human IgG1, IgG2, or IgG4 isotype. In one embodiment of the present invention, the first and / or second antibody is an IgG1 isotype. In one embodiment of the present invention, the first and / or second antibody is a human IgG1 isotype. In one embodiment of the present invention, the first and second antibody is an IgG1 isotype. In one embodiment, the first and second antibodies are of the IgG2 isotype. In one embodiment of the present invention, the first and second antibodies are of the IgG4 isotype. In one embodiment of the present invention, the first antibody is of the IgG1 isotype and the second antibody is of the IgG2 isotype. In one embodiment of the present invention, the first antibody is of the IgG2 isotype and the second antibody is of the IgG1 isotype.

[0155] In one embodiment of the invention, the first antibody comprises a heavy chain of the IgG1 isotype. In one embodiment of the invention, the second antibody comprises a heavy chain of the IgG1 isotype. In one embodiment of the invention, the first antibody comprises a heavy chain of the IgG2 isotype. In one embodiment of the invention, the second antibody comprises a heavy chain of the IgG2 isotype. In one embodiment of the invention, the first antibody comprises a heavy chain of the IgG3 isotype. In one embodiment of the invention, the second antibody comprises a heavy chain of the IgG3 isotype. In one embodiment of the invention, the first antibody comprises a heavy chain of the IgG4 isotype. In one embodiment of the invention, the second antibody comprises a heavy chain of the IgG4 isotype.

[0156] In one embodiment of the invention, the first antibody comprises a heavy chain of the IgG1 isotype and the second antibody comprises a heavy chain of the IgG1 isotype.

[0157] In preferred embodiments, the first antibody comprises a first Fc region of the human IgG1 isotype and / or the second antibody comprises a second Fc region of the human IgG1 isotype.

[0158] In one embodiment of the invention, the antibody, or the first and / or second antibody, comprises a first and / or second constant region comprising a sequence selected from Table 1.

[0159] In one embodiment of the present invention, the antibody, or the first and / or second antibody, comprises a first and / or second constant region comprising a sequence selected from the group consisting of SEQ ID NOs: 63-122, 135-138, 140-145.

[0160] In one embodiment of the invention, the first and / or second antibody comprises a first and / or second heavy chain constant region comprising a sequence selected from the group consisting of SEQ ID NOs: 63-122, 135-138, 140-145, wherein the first and second heavy chain sequences are independently selected from the group.

[0161] In one embodiment of the invention, the antibody, or the first and / or second antibody, comprises a first and / or second constant region comprising a sequence selected from the group consisting of SEQ ID NOs: 63-122, 135-138, and 140-145, wherein at most five additional substitutions have been introduced, such as at most four additional substitutions, for example, at most three additional substitutions, such as at most two additional substitutions, for example, at most one additional substitution.

[0162] In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:84. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:87. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:101. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:107. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:105. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:103. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:85. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:104. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:82. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:66. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:68. In one embodiment of the invention, the antibody, e.g., the first or second antibody, comprises a constant region comprising the sequence set forth in SEQ ID NO:73.

[0163] In one embodiment of the present invention, the first and second antigens are both cell surface-exposed molecules. In one embodiment, the first and second antigens are expressed in the same cell. In one embodiment, the first and second antigens are expressed in the same tissue.

[0164] In one embodiment of the invention, the first and second antigens co-exist in a cell or tissue that is a target cell or tissue for the disease or disorder to be treated. In one embodiment of the invention, the first and second antigens are not identical.

[0165] In one embodiment of the invention, the first and second antibodies deplete cell populations expressing the first and second antigens.

[0166] In one embodiment of the invention, the cell population is tumor cells.

[0167] In one embodiment of the invention, the cell population is hematological tumor cells or solid tumor cells.

[0168] In one embodiment of the invention, the cell population is a white blood cell population, such as a white blood cell population.

[0169] In one embodiment of the invention, the cell population is lymphocytes, such as a lymphocyte cell population.

[0170] In one embodiment of the invention, the cell population is B cells, such as a B cell population. In one embodiment of the invention, the cell population is a subset of a B cell population.

[0171] In one embodiment of the invention, the cell population is a T cell, such as a T cell population. In one embodiment of the invention, the cell population is a subset of a T cell population. In one embodiment of the invention, the cell population is regulatory T cells.

[0172] In one embodiment of the invention, the cell population is NK cells. In one embodiment of the invention, the cell population is myeloid-derived suppressor cells. In one embodiment of the invention, the cell population is tumor-associated macrophages.

[0173] The present specification describes an embodiment in which the first and second antibodies according to the present invention are used as pharmaceuticals for depleting specific cell populations expressing the first and second antigens recognized by the first and second antibodies. Thus, the first and second antibodies according to the present invention can be used to deplete tumor cells expressing the first and second antigens recognized by the first and second antibodies, without depleting healthy tissues expressing only the first or second antigen. The first and second antibodies according to the present invention can also be particularly useful in depleting specific cell populations of the immune system, such as specific subsets of lymphocytes, for example, B cells or T cells, or even subsets of B cells or T cells.

[0174] In one embodiment of the invention, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. In one embodiment of the invention, the antibody is a natural antibody.

[0175] A naturally occurring antibody, e.g., having a human Fc domain, may also have mutations other than those of the present invention, such as those affecting glycosylation or enabling the antibody to be a bispecific antibody. The term "natural antibody" refers to an antibody that does not contain genetically introduced mutations. Thus, antibodies containing naturally occurring modifications, e.g., different allotypes, should be understood as "natural antibodies" within the meaning of the present invention and, therefore, may be understood as parent antibodies. A natural antibody can serve as a template for at least three substitutions in a first antibody or at least two substitutions in a second antibody according to the present invention to provide the first and second antibodies of the present invention. An example of a parent antibody containing substitutions other than those of the present invention is a bispecific antibody, such as that described in WO2011 / 131746 (Genmab), which utilizes reducing conditions to promote the exchange of half of the molecules of two antibodies containing IgG4-like CH3 regions, thus forming a bispecific antibody without the simultaneous formation of aggregates. Other examples of parent antibodies include, but are not limited to, bispecific antibodies such as heterodimeric bispecific antibodies: Triomab (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); Knobs-into-holes (Genentech); Electrostatic steering (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies, or Fc-containing antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof.

[0176] In one aspect of the present invention, the antibody comprises an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, wherein the Fc region comprises: one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and including the substitution K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and It comprises a single substitution of the amino acid at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E.

[0177] In another aspect, the antibody comprises an Fc region of human IgG and an antigen-binding region capable of binding to an antigen, wherein the Fc region comprises: containing the substitutions K248E and T437R, and containing a K439E or S440K substitution, and It comprises a single substitution of an amino acid at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, K332E, E269K, L234A, L234F, L235A, L235Q, L235E, K326A, K326W, E333A, and E333S.

[0178] In one embodiment of the invention, the Fc region comprises a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

[0179] In one embodiment of the invention, the Fc region comprises a substitution selected from the group consisting of E430G, E345K, and E345R.

[0180] In one embodiment of the invention, the Fc region comprises an E430G substitution.

[0181] In one embodiment of the invention, the Fc region comprises at most one substitution of an amino acid at a position selected from the group consisting of E430, E345, and S440, provided that the substitution at S440 is S440Y or S440W.

[0182] In one embodiment of the invention, the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, G237P.

[0183] In one embodiment of the invention, the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R, In one embodiment of the invention, the Fc region comprises a G237Q substitution.

[0184] In one embodiment of the invention, the Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K.

[0185] In one embodiment of the invention, the Fc region comprises a G236R substitution.

[0186] In one embodiment of the invention, the Fc region comprises an E269K substitution.

[0187] In one embodiment of the invention, the Fc region comprises a K322A substitution.

[0188] Targets and Methods of Use The first and / or second antibodies according to the invention can bind to targets expressed in the same cell. In one embodiment, the target is a target that activates, inhibits, modulates and / or regulates a signal transduction pathway.

[0189] Examples of targets that may be particularly suitable as targets according to the present invention are cell surface receptors and ligands.

[0190] The following protein classes may also be particularly suitable as antigen binding targets for the first and / or second antibodies of the present invention: tumor necrosis receptor superfamily, GPI-anchored proteins, lipidated proteins, hydrolase (EC 3.) and 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, transmembrane 4 domains, activated leukocyte immunoglobulin-like receptors, CC motif chemokine receptors, G protein-coupled receptors, Toll-like receptors, and receptor tyrosine kinases. In one embodiment of the present invention, the first and second antigen-binding regions are capable of binding to target antigens from the same protein class. In one embodiment of the present invention, the first and second antigen-binding regions are capable of binding to target antigens from different protein classes. In one embodiment of the present invention, the first antigen-binding region is capable of binding to a target antigen from the GPI-anchored protein class, and the second antigen-binding region is capable of binding to a target antigen from the tetraspanin protein class. In one embodiment of the present invention, the first antigen-binding region is capable of binding to a target antigen from the tetraspanin protein class, and the second antigen-binding region is capable of binding to a target antigen from the GPI-anchored protein protein class. In one embodiment of the present invention, the first antigen-binding region is capable of binding to a target antigen from the GPI-anchored protein protein class, and the second antigen-binding region is capable of binding to a target antigen from the four-transmembrane protein class. In one embodiment of the present invention, the first antigen-binding region is capable of binding to a target antigen from the four-transmembrane protein class, and the second antigen-binding region is capable of binding to a target antigen from the GPI-anchored protein protein class.

[0191] In one embodiment of the invention, the first antigen-binding region is capable of binding to a target antigen from the tumor necrosis receptor superfamily of proteins, and the second antigen-binding region is capable of binding to a target antigen from the tumor necrosis receptor superfamily of proteins.

[0192] In one embodiment of the present invention, the first antigen-binding region is capable of binding to a target antigen from the tumor necrosis receptor superfamily of proteins, and the second antigen-binding region is capable of binding to a target antigen from the immunoglobulin superfamily of proteins.

[0193] Cell surface receptors include 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 regarding the receptors belonging to these receptor families and their characteristics are available, for example Cooke B A., King RJ B., van der Molen H J. eds. New Comprehensive Biochemistry Vol. 18B "Hormones and their Actions Part II" 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. Re(v) 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, "Handbook for Adhesion Factors" (1994) (Shujunsha, Tokyo, Japan).

[0194] In one embodiment of the invention, the antibody comprises an antigen-binding region that binds to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) or the G protein-coupled receptor (GPCR) superfamily.

[0195] In one embodiment of the present invention, the first antibody and / or the second antibody bind to a cell surface receptor, and cell surface receptors include, for example, hormone receptors and cytokine receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation regulator receptors, etc. 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-12 (IL-13) receptor. These include interleukin-12 (IL-12) receptor, interleukin-13 (IL-13) receptor, interleukin-15 (IL-15) receptor, interferon-α (IFNα) receptor, interferon-β (IFNβ) receptor, interferon-γ (IFNγ) receptor, growth hormone (GH) receptor, insulin receptor, hematopoietic stem cell growth factor (SCF) receptor, vascular epithelial growth factor (VEGF) receptor, epidermal growth factor (EGF) receptor, nerve growth factor (NGF) receptor, fibroblast growth factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-β (TGFβ) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M (OSM) receptor, and Notch family receptors.

[0196] The tumor necrosis factor receptor superfamily (TNFRSF) is a group of receptors characterized by their ability to bind to ligands of the tumor necrosis factor superfamily (TNFSF) through their extracellular cysteine-rich domains. TNF receptors form trimeric complexes in the cell membrane. TNFRSF includes a 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), and 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).

[0197] In one embodiment of the present invention, the antibody, or the first and / or second antibody, comprises an antigen-binding region capable of binding to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, and CD5. In one embodiment, the antigen-binding region is capable of binding to DR4. In one embodiment, the antigen-binding region is capable of binding to DR5. In one embodiment, the antigen-binding region is capable of binding to CD20. In one embodiment, the antigen-binding region is capable of binding to CD37. In one embodiment, the antigen-binding region is capable of binding to CD52. In one embodiment, the antigen-binding region is capable of binding to HLA-DR. In one embodiment, the antigen-binding region is capable of binding to CD3. In one embodiment, the antigen-binding region is capable of binding to CD5.

[0198] In one embodiment of the invention, the antibody, or the first and / or second antibody, a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 196, the CDR2 sequence set forth in SEQ ID NO: 196, and the CDR3 sequence set forth in SEQ ID NO: 198, and a VL region [DR4] comprising the CDR1 sequence set forth in SEQ ID NO: 200, the CDR2 sequence set forth in AAT, and the CDR3 sequence set forth in SEQ ID NO: 201; a VH region comprising the CDR1 sequence shown in SEQ ID NO:50, the CDR2 sequence shown in SEQ ID NO:51, and the CDR3 sequence shown in SEQ ID NO:52, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:54, the CDR2 sequence shown in FAS, and the CDR3 sequence shown in SEQ ID NO:55 [DR5-01-G56T]; a VH region comprising the CDR1 sequence shown in SEQ ID NO:57, the CDR2 sequence shown in SEQ ID NO:58, and the CDR3 sequence shown in SEQ ID NO:59, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:61, the CDR2 sequence shown in RTS, and the CDR3 sequence shown in SEQ ID NO:62 [DR5-05]; a VH region comprising the CDR1 sequence shown in SEQ ID NO:36, the CDR2 sequence shown in SEQ ID NO:37, and the CDR3 sequence shown in SEQ ID NO:38, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:40, the CDR2 sequence shown in DAS, and the CDR3 sequence shown in SEQ ID NO:41 [CD20, 7D8]; a VH region comprising 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 a VL region comprising 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 [CD20, 11B8]; a VH region comprising 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 a VL region comprising 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 [CD37]; a VH region comprising 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 a VL region comprising 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 [CD52, CAMPATH-1H]; a VH region comprising the CDR1 sequence shown in SEQ ID NO: 161, the CDR2 sequence shown in SEQ ID NO: 162, and the CDR3 sequence shown in SEQ ID NO: 163, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 165, the CDR2 sequence shown in LVS, and the CDR3 sequence shown in SEQ ID NO: 166 [CD52, h2E8]; a VH region comprising the CDR1 sequence shown in SEQ ID NO: 168, the CDR2 sequence shown in SEQ ID NO: 169, and the CDR3 sequence shown in SEQ ID NO: 170, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 172, the CDR2 sequence shown in AAS, and the CDR3 sequence shown in SEQ ID NO: 173 [HLA-DR, hul243]; a VH region comprising the CDR1 sequence shown in SEQ ID NO: 175, the CDR2 sequence shown in SEQ ID NO: 176, and the CDR3 sequence shown in SEQ ID NO: 177, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 179, the CDR2 sequence shown in DNN, and the CDR3 sequence shown in SEQ ID NO: 180 [HLA-DR, 1D09C3]; a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 182, the CDR2 sequence set forth in SEQ ID NO: 183, and the CDR3 sequence set forth in SEQ ID NO: 184, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 186, the CDR2 sequence set forth in DTS, and the CDR3 sequence set forth in SEQ ID NO: 187 [CD3, huCLB T3 / 4]; or A VH region comprising the CDR1 sequence shown in SEQ ID NO: 189, the CDR2 sequence shown in SEQ ID NO: 190, and the CDR3 sequence shown in SEQ ID NO: 191, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 193, the CDR2 sequence shown in ATS, and the CDR3 sequence shown in SEQ ID NO: 194 [CD5] The antigen-binding region comprises:

[0199] In one aspect, the invention relates to a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region according to any embodiment disclosed herein, and the second antibody comprises a second antigen-binding region and a second Fc region according to any aspect or embodiment disclosed herein.

[0200] In another aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and including the substitution K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and comprising one substitution of an amino acid at position G237 or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, K322E, P329R, L234A, L234F, L235A, L235Q, and L235E; the second Fc region one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and and (d) a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and if the first Fc region comprises a K322E or P329R substitution, it further comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; It relates to a composition.

[0201] In one aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and including the substitution K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and comprising one substitution of an amino acid at position G237, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, E269K, L234A, L234F, L235A, L235Q, and L235E; the second Fc region one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and including the substitution K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; A composition is provided.

[0202] In one aspect, the present invention provides a composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG, and 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 one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and including the substitution K439E or S440K, provided that the substitution is not S440K when the substitution in (a) is S440Y or S440W; and containing a single amino acid substitution at position P329, or a substitution of K322E; the second Fc region one amino acid substitution at a position selected from the group consisting of E430, E345, and S440, with the proviso that the substitution at S440 is S440Y or S440W; and and (d) a substitution of K439E or S440K, provided that the substitution is not S440K when the substitution in (d) is S440Y or S440W; and comprising one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; the first Fc region has a K439E substitution and the second Fc region has a S440K substitution, or the first Fc region has a S440K substitution and the second Fc region has a K439E substitution, the amino acid positions corresponding to those of human IgG1 according to the EU numbering system; A composition is provided.

[0203] In one embodiment of the invention, the composition comprising the first and second Fc regions comprises a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

[0204] In one embodiment of the invention, the composition comprising the first and second Fc regions comprises a substitution selected from the group consisting of E430G, E345K, and E345R.

[0205] In one embodiment of the invention, the composition comprising the first and second Fc regions comprises an E430G substitution.

[0206] In one embodiment of the invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, G237S, G237N, G237D, G237E, G237K, G237V, G237M, G237I, G237L, G237H, G237F, G237Y, G237W, and G237P.

[0207] In one embodiment of the present invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, and G237S.

[0208] In one embodiment of the invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, and G237R.

[0209] The composition of any one of claims 43 to 50, wherein the first Fc region comprises a G237Q substitution.

[0210] The composition of any one of claims 43 to 50, wherein the first Fc region comprises a G237T substitution.

[0211] In one embodiment of the present invention, the composition comprising a first Fc region comprises one or more substitutions selected from the group consisting of G236R and G236K.

[0212] In one embodiment of the present invention, a composition comprising a first Fc region comprises one or more substitutions selected from the group consisting of G236R, G236K, and E269K. In one embodiment of the present invention, a composition comprising a first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. In one embodiment of the present invention, a composition comprising a first Fc region comprises a G263R or G236K substitution.

[0213] In one embodiment of the present invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of K322E and K322A.

[0214] In one embodiment of the invention, the composition comprising a first Fc region comprises a P329R substitution.

[0215] In one embodiment of the invention, the composition comprising a first Fc region comprises a G236R substitution.

[0216] In one embodiment of the invention, the composition comprising the first Fc region comprises one substitution of the amino acid at position P329.

[0217] In one embodiment of the invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of P329R, P329A, P329T, P329Q, P329R, P329S, P329N, P329D, P329E, P329K, P329V, P329M, P329I, P329L, P329H, P329F, P329Y, P329W, and P329P.

[0218] In one embodiment of the invention, the composition comprising a first Fc region comprises one substitution selected from the group consisting of P329R, P329K, P329E, P329D, and P329A.

[0219] In one embodiment of the invention, the composition comprising a first Fc region comprises a P329R substitution.

[0220] In one embodiment of the invention, the composition comprising a first Fc region comprises a K322A substitution.

[0221] In one embodiment of the invention, the composition comprising a first Fc region comprises an E269K substitution.

[0222] In one embodiment of the invention, the composition comprising a second Fc region comprises one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S.

[0223] In one embodiment of the invention, the composition comprising a second Fc region comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0224] In one embodiment of the invention, the composition comprising a second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S.

[0225] In one embodiment of the invention, the composition comprising a second Fc region comprises a G237A substitution.

[0226] In one embodiment of the invention, the composition comprising a second Fc region comprises a K326A substitution.

[0227] In one embodiment of the invention, the composition comprising a second Fc region comprises an E333S substitution.

[0228] In one embodiment of the invention, the composition comprising a second Fc region comprises two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

[0229] In one embodiment of the invention, the composition comprising a second Fc region comprises the substitutions K326W and E333S.

[0230] In one embodiment of the invention, the composition comprising a second Fc region comprises the following substitutions: K326A and E333A.

[0231] In one embodiment of the invention, the composition comprising a second Fc region comprises the substitutions G237A and E333S.

[0232] In one embodiment of the present invention, a composition comprising a first antibody and a second antibody is prepared by dissolving the first antibody and the second antibody in a molar ratio of 1:50 to 50:1 in the composition, for example, a molar ratio of 1:1, a molar ratio of 1:2, a molar ratio of 1:3, a molar ratio of 1:4, a molar ratio of 1:5, a molar ratio of 1:6, a molar ratio of 1:7, a molar ratio of 1:8, a molar ratio of 1:9, a molar ratio of 1:10, a molar ratio of 1:15, a molar ratio of 1:20, a molar ratio of 1:25, a molar ratio of 1:30, a molar ratio of 1:35, or the like. They are present in molar ratios of 1:40, 1:45, 1:50, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1.

[0233] In one embodiment of the present invention, the first antibody and the second antibody are present in the composition at a molar ratio of about 1:50 to 50:1, for example, a molar ratio of about 1:40 to 40:1, for example, a molar ratio of about 1:30 to 30:1, for example, a molar ratio of about 1:20 to 20:1, for example, a molar ratio of about 1:10 to 10:1, for example, a molar ratio of about 1:9 to 9:1, for example, a molar ratio of about 1:5 to 5:1.

[0234] In one embodiment of the invention, the first and second antibody and / or additional polypeptide are present in the composition in an equimolar ratio.

[0235] In one embodiment of the invention, the composition according to any aspect or embodiment is a pharmaceutical composition.

[0236] In one aspect, the invention relates to a method of depleting a cell population expressing a first antigen and a second antigen, comprising contacting the cell population with a first and a second antibody or composition according to any aspect or embodiment disclosed herein.

[0237] In one embodiment of the invention, the cell population is a tumor cell population, such as a blood tumor cell population or a solid tumor cell population.

[0238] In one embodiment of the invention, the cell population is present in blood.

[0239] In one embodiment of the invention, the cell population is a white blood cell population, such as a white blood cell population.

[0240] In one embodiment of the invention, the cell population is a subset of a white blood cell population.

[0241] In one embodiment of the invention, the cell population is a lymphocyte cell population.

[0242] In one embodiment of the invention, the cell population is a B cell population. In one embodiment of the invention, the cell population is a subset of a B cell population.

[0243] In one embodiment of the invention, the cell population is a T cell population. In one embodiment of the invention, the cell population is a subset of a T cell population. In one embodiment of the invention, the cell population is regulatory T cells, such as a regulatory T cell population.

[0244] In one embodiment of the invention, the cell population is a NK cell population.

[0245] In one embodiment of the invention, the cell population is myeloid-derived suppressor cells.

[0246] therapeutic use The first and second antibodies, bispecific antibodies, or compositions according to any aspect or embodiment of the invention may be used as medicaments, i.e. for therapeutic applications.

[0247] In one aspect, the invention provides a first and second antibody or composition according to any aspect or embodiment disclosed herein for use as a medicament.

[0248] In another aspect, the invention provides an antibody or composition according to any aspect or embodiment disclosed herein for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0249] In another aspect, the invention relates to a method of treating an individual having a disease, comprising administering to the individual an effective amount of a first and a second antibody or composition according to any aspect or embodiment disclosed herein.

[0250] In an aspect, the present invention relates to a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of a first and a second antibody according to any aspect or embodiment disclosed herein, or an effective amount of a composition according to any aspect or embodiment disclosed herein.

[0251] In one aspect of the invention, the disease is selected from the group of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0252] In one aspect of the invention, the method comprises administering an additional therapeutic agent.

[0253] In one embodiment of the invention, the method according to any aspect or embodiment disclosed herein relates to further administering an additional therapeutic agent.

[0254] In one aspect of the invention, the additional therapeutic agent is a chemotherapeutic agent (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), a kinase inhibitor (including but not limited to sorafenib, sunitinib, or everolimus), an apoptosis modulator (including but not limited to recombinant human TRAIL or birinapant), a RAS inhibitor, a proteasome inhibitor (including but not limited to and one or more anti-cancer agents selected from the group consisting of: anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR antibodies and antibody mimetics; and antibody-drug conjugates.

[0255] kit-of-parts It will be understood that the embodiments described below with respect to the first and second antibodies refer to antibodies comprising an immunoglobulin Fc region and an antigen-binding region.

[0256] The present invention also relates to a kit of parts for simultaneous, separate or sequential use in therapy, comprising a first and a second antibody as described herein, wherein said first and second antibodies can be obtained according to any of the methods described herein.

[0257] In one aspect, the invention relates to a kit-of-parts comprising an antibody or composition according to any aspect or embodiment described herein, wherein the first and second antibody or composition are present in one or more containers, e.g., vials.

[0258] In one embodiment of the invention, the kit of parts comprises a first and a second antibody or composition according to any aspect or embodiment described herein for simultaneous, separate or sequential use in therapy.

[0259] In another aspect, the present invention relates to the use of a first and second antibody, composition, or kit of parts according to any of the embodiments described herein for use in a diagnostic method.

[0260] In another aspect, the present invention relates to a diagnostic method comprising administering a first and a second antibody, composition, or kit-of-parts according to any embodiment described herein to at least a part of the body of a human or other mammal.

[0261] In another aspect, the invention relates to the use of the first and second antibodies, compositions, or kits of parts according to any of the embodiments described herein in imaging at least a portion of the body of a human or other mammal.

[0262] In another aspect, the invention relates to a method for imaging at least a portion of a human or other mammalian body, comprising administering a first and a second antibody, composition, or kit-of-parts according to any embodiment described herein.

[0263] Further Uses It will be understood that the embodiments described below with respect to a first and second antibody or antibodies refer to a first and second antibody or antibodies, each comprising an immunoglobulin Fc region and an antigen-binding region.

[0264] In a further aspect, the present invention relates to the first and second antibodies of the invention as described above for use as medicaments, in particular for use as medicaments for the treatment of diseases or disorders, including but not limited to cancer, autoimmune diseases, inflammatory diseases, infectious diseases, bacterial, viral, or fungal infections.

[0265] In another aspect, the present invention relates to the first and second antibodies, bispecific antibodies, compositions, and kits of parts described herein for the treatment of disease, e.g., cancer.

[0266] In another aspect, the invention relates to a method for the treatment of a human disease comprising the administration of a first and second antibody, composition, or kit of parts described herein.

[0267] In another aspect, the invention relates to a method for the treatment of cancer in a human comprising the administration of a first and a second antibody, composition, or kit of parts.

[0268] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of alleviating, ameliorating, arresting, or eradicating (curing) the symptoms or disease state.

[0269] An "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the dosage and for the period of time required. A therapeutically effective amount of an antibody may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0270] Dosage It will be understood that the embodiments described below with respect to antibodies refer to antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin; the antibody may also be a multispecific antibody, such as a bispecific antibody having 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.

[0271] The effective dosage and dosage regimen of the antibody depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range of a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg / kg, e.g., about 0.1 to 50 mg / kg, e.g., about 0.1 to 20 mg / kg, e.g., about 0.1 to 10 mg / kg, e.g., about 0.5, e.g., about 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0272] The antibody of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is intended for use in combination with one or more additional therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or angiogenesis inhibitors. Such combined administration may be simultaneous, separate, or sequential.

[0273] In a further aspect, the present invention provides a method for treating or preventing a disease, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a variant or pharmaceutical composition of the present invention in combination with radiation therapy and / or surgery.

[0274] Preparation method It will be understood that the embodiments described below with respect to antibodies refer to antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin; the antibody may also be a multispecific antibody having a first Fc region and a first antigen-binding region of an immunoglobulin and a second antibody having a second Fc region and a second antigen-binding region of an immunoglobulin.

[0275] The present invention also provides isolated nucleic acids and vectors encoding antibodies according to any one of the above aspects, as well as vectors and expression systems encoding the antibodies. Suitable nucleic acid constructs, vectors, and expression systems for antibodies and variants thereof are known in the art and are described in the Examples. In embodiments in which the variant antibody comprises not only a heavy chain (or an Fc-containing fragment thereof) but also a light chain, the nucleotide sequences encoding the heavy and light chain portions can be present in the same nucleic acid or vector or in different nucleic acids or vectors.

[0276] The present invention also provides a method for producing an antibody according to any one of the above aspects in a host cell, wherein the polypeptide or antibody comprises at least an Fc region of a heavy chain, the method comprising the steps of: a) providing a nucleotide construct encoding a variant Fc region; b) expressing the nucleotide construct in a host cell; and c) recovering the antibody variant from the cell culture of host cells Also provided is a method comprising:

[0277] In some aspects, the antibody is a heavy chain antibody, however, in most aspects, the antibody also comprises a light chain, and therefore the host cell additionally expresses a light chain-encoding construct on either the same or a different vector.

[0278] Host cells suitable for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293, PER-C6, NS / 0, and Sp2 / 0 cells. In one embodiment, the host cell is a cell in which Asn-linked glycosylation of proteins can be performed, e.g., a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In a further embodiment, the host cell is a non-human cell genetically modified to produce glycoproteins with human-like or human-type glycosylation. Examples of such cells are genetically modified methylotrophic yeast (Pichia pastoris) (Hamilton et al., Science 301(2003)1244-1246; Potgieter et al., J.Biotechnology 139(2009)318-325) and genetically modified duckweed (Lemna minor) (Cox et al., Nature Biotechnology 12(2006)1591-1597).

[0279] In one embodiment, the host cell is a host cell that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists several such antibody production systems, such as Sp2 / 0, NS / 0, or the mammary gland of transgenic animals (goats), in which only partial C-terminal lysine removal is obtained. In one embodiment, the host cell is a host cell with an altered glycosylation mechanism. Such cells have been reported in the art and can be used as host cells for expressing the variants of the present invention, thereby producing antibodies with altered glycosylation. See, e.g., Shields, R. Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP 1176195; WO 03 / 035835; and WO 99 / 54342. Additional methods for generating engineered glycoforms are known in the art and include, but are not limited to, Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation modification technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0280] The present invention also relates to antibodies obtained or obtainable by the methods of the invention described above.

[0281] In a further aspect, the present invention relates to a host cell capable of producing an antibody of the present invention. In one embodiment, the host cell is transformed or transfected with a nucleotide construct of the present invention.

[0282] This invention is further illustrated by the following examples which should not be construed as further limiting.

[0283] Table 1: Sequence Listing TIFF0007649139000015.tif207149TIFF0007649139000016.tif216149TIFF0007649139000017.tif214149TIFF0007649139000018.tif212149TI FF0007649139000019.tif216149TIFF0007649139000020.tif217149TIFF0007649139000021.tif213149TIFF0007649139000022.tif211149TIFF0 007649139000023.tif211149TIFF0007649139000024.tif207149TIFF0007649139000025.tif220149TIFF0007649139000026.tif217149TIFF0007649139000027.tif216149TIFF0007649139000028.tif216149TIFF0007649139000029.tif216149TIFF0007649139000030.tif205149TIFF000764 9139000031.tif220149TIFF0007649139000032.tif222149TIFF0007649139000033.tif216149TIFF0007649139000034.tif216149TIFF0007649139000035.tif216149TIFF0007649139000036.tif210149TIFF0007649139000037.tif222149TIFF0007649139000038.tif216149TIFF00076491390 00039.tif216149TIFF0007649139000040.tif216149TIFF0007649139000041.tif210149TIFF0007649139000042.tif217149TIFF00076491390000 43.tif216149TIFF0007649139000044.tif216149TIFF0007649139000045.tif210149TIFF0007649139000046.tif215149TIFF0007649139000047.tif217149TIFF0007649139000048.tif216149TIFF0007649139000049.tif212149TIFF0007649139000050.tif213149TIFF0007649 139000051.tif212149TIFF0007649139000052.tif216149TIFF0007649139000053.tif216149TIFF0007649139000054.tif171149.

[0284] Table defining the substitutions tested in Examples 5-23 TIFF0007649139000055.tif198163

[0285] Table defining self-oligomerization inhibitor substitutions TIFF0007649139000056.tif17128

[0286] Table defining FcγR binding-inhibiting C1q binding-inhibiting substitutions TIFF0007649139000057.tif77163 [Example]

[0287] Example 1 Antibody expression constructs For expression of the human and humanized antibodies used herein, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific) and cloned into pcDNA3.3 expression vectors (ThermoFisher Scientific) containing the constant region of the human IgG heavy chain (HC) (constant region human IgG1m(f)HC: SEQ ID NO 22; constant region human IgG2 HC: SEQ ID NO 31; constant region human IgG3 HC: SEQ ID NO 32; or constant region human IgG4 HC: SEQ ID NO 33) and / or the constant region of the human kappa light chain (LC) (SEQ ID NO 34). Desired mutations were introduced by gene synthesis. The CD20 antibody variants in this application have VH and VL sequences derived from the previously described CD20 antibody IgG1-CD20-11B8 (WO2004 / 035607; VH:SEQ ID NO 8; VL:SEQ ID NO 12). The CD52 antibody variants in this application have 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) and the previously described CD52 antibody h2E8 (US2014 / 0127236; VH:SEQ ID NO 160; VL:SEQ ID NO 164). The CD37 antibody variants in this application have VH and VL sequences derived from the previously described CD37 antibody IgG1-CD37-37.3 (WO2011 / 112978; VH: SEQ ID NO 42; VL: SEQ ID NO 46). The DR4 antibody variants in this application have VH and VL sequences derived from the previously described DR4 antibody DR4-chCTB007 (US2009 / 0136503A1; VH: SEQ ID NO 200; VL: SEQ ID NO 204).The DR5 antibody variants in this application have VH and VL sequences derived from the previously described DR5 antibody DR5-01-G56T (WO 2017 / 093447; VH: SEQ ID NO 49; VL: SEQ ID NO 53) and VH and VL sequences derived from DR5-05 (WO 2014 / 009358; VH: SEQ ID NO 56; VL: SEQ ID NO 60). The HLA-DR antibody variants in this application have VH and VL sequences derived from the previously described HLA-DR antibody HLA-DR-hul243 (US8722047B2; VH:SEQ ID NO 168; VL:SEQ ID NO 172) and the previously described HLA-DR antibody HLA-DR-1D09C3 (US7521047B2; VH:SEQ ID NO 176; VL:SEQ ID NO 180). The CD3 antibody variants in this application have VH and VL sequences derived from the previously described CD3 antibody huCLB-T3 / 4 (Parren et al., Res. Immunol 1991 Nov-Dec;142(9):749-63; VH:SEQ ID NO 184; VL:SEQ ID NO 188). The CD5 antibody variants in this application have VH and VL sequences derived from the previously described CD5 antibody CD5-INSERM (WO2010145895; VH: SEQ ID NO 192; VL: SEQ ID NO 196). The human IgG1 antibody b12, an HIV gp120-specific antibody, was used as a negative control in some experiments (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-23; VH: SEQ ID NO 15; VL: SEQ ID NO 19).

[0288] Transiently expressed antibody constructs Plasmid DNA mixtures encoding both the antibody heavy and light chains were transiently transfected into Expi293F cells (Gibco, Cat No. A14635) using 293fectin (Life Technologies) essentially as described by Vink et al. (Vink et al., 2014 Methods 65(1):5-10). Antibody concentrations in the supernatants were measured by absorbance at 280 nm. Antibody-containing supernatants were either used directly for in vitro assays or purified as described below.

[0289] Antibody purification and quality assessment Antibodies were purified by Protein A affinity chromatography. Culture supernatants were filtered through a 0.20 μm dead-end filter and loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), washed, and eluted with 0.02 M sodium citrate-NaOH (pH 3). Immediately after purification, the eluate was loaded onto a HiPrep desalting column (GE Healthcare), and the antibodies were buffer-exchanged into 12.6 mM NaH2PO4, 140 mM NaCl (pH 7.4) buffer (B. Braun or Thermo Fisher). After buffer exchange, the samples were sterile-filtered through a 0.2 μm dead-end filter. Purified proteins were analyzed by multiple bioanalytical assays, including sodium dodecyl sulfate-polyacrylamide gel capillary electrophoresis (CE-SDS) and high-performance size-exclusion chromatography (HP-SEC). Concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2–8°C.

[0290] Example 2: Selectivity of CDC activity by mixed antibody variants obtained by introducing the P329R mutation into anti-CD52 IgG1-CAMPATH-1H-K439E and anti-CD20 IgG1-11B8-S440K, which have the Fc-Fc interaction-enhancing mutation E430G. The effect of the P329R mutation on in vitro CDC efficacy was tested using a mixture of variants of the anti-CD20 antibody IgG1-11B8 and the anti-CD52 antibody IgG1-CAMPATH-1H. Different concentrations of purified antibodies (final concentrations ranging from 0.001 to 60.0 μg / mL) were tested in an in vitro CDC assay on Wien 133 cells using 20% ​​NHS. Different mutations were introduced into the IgG1-11B8 and IgG1-CAMPATH-1H antibodies: E430G (which induces enhanced Fc-Fc interactions); P329R (which inhibits direct C1q binding to the antibody); and either the K439E or S440K mutations (which inhibit the formation of homohexameric antibody complexes through the inhibition of intermolecular Fc-Fc interactions and promote the formation of heterohexameric antibody complexes through cross-complementary Fc-Fc interactions). As a control, single antibodies were also mixed 1:1 with the non-binding isotype control antibody IgG1-b12 or IgG1-b12-E430G to allow direct comparison of the concentrations of the individual components and the mixtures comprising them. For the CDC assay, 0.1 × 10 antibodies were mixed in RPMI (Lonza, Cat No. BE12-115F) containing 0.2% bovine serum albumin (BSA; Roche, Cat No. 10735086001). 6Wien 133 cells (kindly provided by Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK) were preincubated with a concentration series of purified antibodies in a total volume of 80 μL in a polystyrene round-bottom 96-well plate (Greiner Bio-One Cat # 650101) on a shaker for 15 min at RT. Next, 20 μL of normal human serum (NHS; Sanquin, Reference No. M0008) was added as a complement source, and the cells were incubated for 45 min in a 37°C incubator (20% NHS final concentration; 3-fold dilutions for final antibody concentrations of 0.001–10.0 μg / mL). The reaction was stopped by placing the plate on ice, after which the cells were pelleted by centrifugation, and the supernatant was replaced with 20 μL of 2 μg / mL propidium iodide solution (PI; Sigma-Aldrich, Cat No. P4170). The number of PI-positive cells was determined by flow cytometry on an Intellicyt iQue screener (Westburg), and percent lysis was calculated as (number of PI-positive cells / total number of cells) × 100%. Data were analyzed using best-fit nonlinear dose-response fitting with log-transformed concentrations in GraphPad PRISM, and the area under the dose-response curve for three experimental replicates was calculated. Relative area under the curve (AUC) values ​​represent normalization to minimum lysis (0% for IgG1-b12) and maximum lysis (100% for the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G).

[0291] A 1:1 mixture of anti-CD52 IgG1-CAMPATH-1H-E430G and anti-CD20 IgG1-11B8-E430G (both containing SEQ ID NO 26) induced efficient cytolysis of Wiens 133 cells (Figure 1 shows the selectivity of CDC activity by the mixed antibody variants anti-CD52 IgG1-CAMPATH-1H-E430G-K439E and anti-CD20 IgG1-11B8-E430G-S440K due to the introduction of the P329R mutation. Wiens 133 cells were incubated with a concentration series of antibody in the presence of 20% pooled normal human serum (NHS). CDC efficacy is presented as (A) the lysis rate determined by the percentage of propidium iodide (PI)-positive cells and (B) the area under the dose-response curve (AUC), normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%).

[0292] Figure 2 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with the introduction of the K322E mutation. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as (A) lysis rate determined by the percentage of PI-positive cells and (B) AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0293] Figure 3 shows the selectivity of CDC activity against different cell lines by introducing the K322E mutation into the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20-11B8-E430G-S440K. In vitro CDC assays were performed using 30 μg / mL of antibody in the presence of 20% NHS on Burkitt's lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C), and Ramos (D), acute lymphoblastic lymphoma (ALL) cell line REH (E), myeloma cell line U266B1 (F), and B-cell lymphoma cell line U-698-M (G). CDC activity is presented as lysis rate, determined by the percentage of PI-positive cells, normalized for each cell line to the non-binding control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%) for REH, U266B1, and Wien 133, or IgG1-11B8-E430G (100%) for Daudi, Raji, Ramos, and U-698-M.

[0294] Figure 4 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K, each carrying a C1q binding-inhibiting mutation (G236R, K322A, E269K, K322E, or P329R). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0295] Figure 5 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding-enhancing mutations (E333S, K326W, or K326A / E333A). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0296] Figure 6 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q binding-inhibiting mutations G236R (A), K322A (B), E269K (C), K322E (D), or P329R (E), plus anti-CD20 IgG1-11B8-E430G-S440K with C1q binding-enhancing mutations E333S, K326W, or K326A / E333A. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized area under the curve (AUC) of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0297] Figure 7 shows the binding of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variants (C, D) with the C1q binding-enhancing mutation E333S to the human lymphoma cell lines Wien 133 (A, C) and Raji (B, D). Antibody binding was tested by flow cytometry. Binding is expressed as the geometric mean fluorescence intensity (MFI). As negative controls for binding, samples without primary antibody or unconjugated anti-gp120 antibody IgG1-b12 were used.

[0298] Figure 8 shows the ADCC potential of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, C) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variant (B, D) with the C1q binding-enhancing mutation E333S. (A, B) ADCC reporter bioassays were performed by co-incubating Raji target cells with antibody dilution series and Jurkat human T cells stably expressing high-affinity FcγRIIIa (V158) and an NFAT response element driving the expression of firefly luciferase. Luciferase production was quantified by luminescence readout. (C, D) In ​​vitro europium TDA (EuTDA) ADCC assays were performed by co-incubating Wien 133 target cells with antibody dilution series and human PBMCs (E:T 100:1). Cell lysis was determined by measuring the signal of the EuTDA fluorescent chelate in the supernatant.

[0299] Figure 9 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-K439E with enhanced Fc-Fc interaction, carrying the C1q binding-inhibiting mutations G236R or K322A, and anti-CD20 IgG1-11B8-S440K with enhanced Fc-Fc interaction, carrying either the C1q binding-inhibiting mutation E333S or the C1q binding-enhancing mutation E430G, E345K, E345R, and E345R-E430G. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0300] Figure 10 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E, which has a C1q binding regulatory mutation or double mutation G237A-K322A at position G236 (G236R or G236K) or G237 (G237A, G237T, G237Q, or G237R), plus anti-CD20 IgG1-11B8-E430G-S440K, with or without the C1q binding regulatory mutations E333S, E333A, K326A, K326W-E333S, G237A, or G237A-E333S. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0301] Figure 11 shows the selectivity of CDC activity by mixed antibody isotype variants (IgG1, IgG2, IgG3, and hinge-stabilized IgG4) of anti-CD52 CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R and anti-CD20 11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%).

[0302] Figure 12 shows the selectivity of CDC activity on (A) Daudi cells and (B) Wien 133 cells by the mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R or K322A, and anti-CD37 IgG1-CD37-37.3-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Target cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-CD37-37.3-E430G (100%).

[0303] Figure 13 shows the DR5-mediated cytotoxicity of (A) single antibody variants or (B) agonist mixtures of antibody variants of anti-DR5 IgG1-DR5-01-G56T-E430G-K439E with or without the C1q binding inhibitor mutation G236R + IgG1-DR5-05-E430G-S440K with or without the C1q binding enhancing mutation E333S against BxPC-3 human pancreatic cancer cells. A 3-day viability assay was performed and cell viability was determined using the CellTiter-Glo kit.

[0304] Figure 14 shows CDC activity by antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G with C1q binding-modulating mutations G237A, G236R, A327K, K322E, or P329R. Wien 133 cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibodies IgG1-b12-S440K (0%; not shown) and IgG1-CAMPATH-1H-E430G (100%).

[0305] Figure 15 shows the binding of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B, C) with the C1q binding-inhibiting mutation G236R or K322A and anti-CD20 IgG1-11B8-E430G-S440K antibody variants (D, E, F) with the C1q binding-enhancing mutation E333S to human FcRn. FcRn ELISA was performed using 5 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnhsECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm.

[0306] Figure 16 shows the clearance rate of 500 μg of intravenously administered antibody in SCID mice. (A-C) Total human IgG in plasma samples was determined by ELISA and plotted as concentration versus time curves for (A) the IgG1-CAMPATH-1H variant, (B) the IgG1-11B8 variant, and (C) the combination of the IgG1-CAMPATH-1H variant and IgG1-11B8 variant. Each data point represents the mean + / - standard deviation of triplicate samples. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1,000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve.

[0307] Figure 17 shows the binding of immobilized IgG1-CAMPATH-1H-E430G-K439E variants with C1q binding-inhibiting mutations G236R or G237T and IgG1-11B8-E430G-S440K variants with C1q binding-enhancing mutations K326A or E333S to dimeric His-tagged, biotinylated ECDs of FcγRIIA allotype 131H (A), FcγRIIA allotype 131R (B), FcγRIIB (C), FcγRIIIA allotype 158V (D), and FcγRIIIA allotype 158F (E) tested in an ELISA assay. Binding is presented relative to the no-antibody control (background) and to binding to IgG1-11B8-E430G-S440K (100%) for a 20 μg / mL antibody sample. Detection was performed using streptavidin-polyHRP and ABTS.

[0308] Figure 18 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without C1q binding-inhibiting mutations (G236R or G237T) + anti-CD20 IgG1-11B8-E430G-S440K with the FcγR binding-inhibiting mutation G237A with or without the C1q binding-enhancing mutation E333S. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%).

[0309] Figure 19 shows the selective activity of combinations of IgG1-CAMPATH-1H and IgG1-11B8 variants in whole blood as determined by flow cytometry analysis of blood cells. Y-axis: B cell fraction (CD19 positive / CD3 negative) or T cell fraction (CD19 negative / CD3 positive) of the total lymphocyte population (CD66b negative) after overnight (o / n) incubation in the presence of effector cells. X-axis: different treatment groups. Symbols represent cells from five different healthy donors tested in two separate incubations per donor. (A) Selective activity of IgG1-CAMPATH-1H-G236R-E430G-K439E mixed with IgG1-11B8-G237A-E430G-S440K. (B) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K. (C) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G236R or G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K containing an additional C1q binding-enhancing E333S mutation. (D) Depth of B cell depletion by different B cell-targeting antibodies compared to the co-dependent antibody combination of IgG1-CAMPATH-1H-E430G-K439E with the additional mutations G236R, G237Q, or G237R mixed with IgG1-11B8-G237A-E430G-S440K. Y-axis: logarithmic scale representation of the B cell fraction determined as above.

[0310] Figure 20 shows the selectivity of CDC activity against different cell lines with different expression levels of CD20 and CD52 by the combination of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants, which have C1q binding-inhibiting mutations in the anti-CD52 component and C1q binding-enhancing mutations in the anti-CD20 component. In vitro CDC assays were performed using the Burkitt lymphoma cell lines Daudi (A), Raji (B), and Ramos (C), the ALL cell line REH (D), and the B-cell lymphoma cell line U-698-M (E), using antibody concentrations of 0.01–40 μg / mL in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as maximal lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0311] Figure 21 shows the selectivity of CDC activity by the mixed antibody variants: anti-CD37 IgG1-CD37-37.3-E430G-K439E with or without the C1q binding-inhibiting mutation G236R, and anti-CD20 IgG1-11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. (A) Daudi cells and (B) WIL2-S cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CD37-37.3-E430G and IgG1-11B8-E430G (100%).

[0312] Figure 22 shows (A) binding of antibody variants of anti-CD52 IgG1-CAMPATH-1H with the Fc:Fc interaction-enhancing mutations E430G or E345R and the self-oligomerization-inhibiting mutation K439E, combined with either an FcγR binding-inhibiting mutation and a C1q binding-modulating mutation G236R, G237A, or G237T, to the human lymphoma cell line Wien 133. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-Campath-1H (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (B) Maximum binding (Bmax) to Raji cells by IgG1-Campath-1H antibody variants with mutations E430G and K439E in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T is shown normalized to the binding of wild-type IgG1-Campath-1H. (C) Apparent Kd values ​​for binding to Raji cells of IgG1-Campath-1H antibody variants with mutations E430G and S440K in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T.

[0313] Figure 23 shows the binding of antibody variants of anti-CD20 IgG1-11B8 with the Fc-Fc interaction-enhancing mutation E430G and the self-oligomerization-inhibiting mutation S440K, in combination with either the C1q binding-modulating mutations K326A or E333A (A) or E333S, G237A, or G237A-E333S (B), to the human lymphoma cell line Raji. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-11B8 (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (C, D) Maximum binding (Bmax) to Raji cells by IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (C) or E333S, G237A, or G237A-E333S (D) is shown normalized to the binding of wild-type IgG1-11B8. (E, F) Apparent Kd values ​​for binding to Raji cells of IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (E) or E333S, G237A, or G237A-E333S (F).

[0314] Figure 24 shows FcRn binding of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibody variants. (A, C) Binding to human FcRn is shown for variants of the anti-CD52 antibody IgG1-CAMPATH-1H with the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation K439E, and the C1q binding-modulating mutations G237A or G237T, using an antibody concentration of 40 μg / mL at (A) pH 6.0 or (C) pH 7.4. (B, D) Binding to human FcRn by variants of the anti-CD20 antibody IgG1-11B8, which have the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation S440K, and the C1q binding-modulating mutations K326A, E333A, G237A, or G237A-E333S, using an antibody concentration of 40 μg / mL at (B) pH 6.0 or (D) pH 7.4. FcRn ELISA was performed using 2 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm.

[0315] Figure 25 shows total human IgG (hIgG) concentrations as measured in blood samples collected from mice injected with anti-CD52 IgG1-CAMPATH-1H or anti-IgG1-11B8 antibody variants, or a mixture thereof. (A) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-CAMPATH-1H, IgG1-CAMPATH-1H-E430G-K439E-G237Q, or IgG1-CAMPATH-1H-E430G-K439E-G236R. (B) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-11B8, IgG1-11B8-E430G-S440K-G237A, or IgG1-11B8-E430G-S440K-E333S. (C) Total hIgG concentrations in blood samples collected from mice injected with a mixture of wild-type IgG1-CAMPATH-1H + IgG1-11B8 or a mixture of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations as in (A) and (B). In all figures, the dotted line represents the predicted IgG1 concentration over time for wild-type IgG1 antibodies in SCID mice. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve.

[0316] Figure 26 shows the concentrations (in μg / ml) of C4d detected in samples incubated with IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 antibody variants carrying the mutations E430G, K439E, or S440K and G236R, G237A, G237Q, or G237R, after subtraction of the mean C4d concentration detected in negative control samples containing no antibody. Positive control samples contain antibody variants carrying the Fc-Fc interaction-enhancing mutations E345R, E430G, and S440Y (RGY).

[0317] Figure 27 shows C1q binding to Wien 133 cells incubated on ice with normal human serum as a complement source, as detected by flow cytometry, after opsonization with variants of antibodies IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 carrying the mutations E430G, K439E, or S440K and G236R, G237T, K326A, or E333S. Mean fluorescence intensity values ​​were normalized to the no-antibody control reaction (0%) and the maximal level (100%) of the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G, estimated by fitting a logarithmic agonist response model. (A) C1q binding of control reactions. (B–D) C1q binding of (B) IgG1-CAMPATH-E430G-K439E, (C) IgG1-CAMPATH-E430G-K439E-G236R, and (D) IgG1-CAMPATH-E430G-K439E-G237R mixed with nonbinding control IgG1-b12 or different IgG1-11B8 variants.

[0318] Figure 28 shows FcγR binding by IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G antibody variants carrying self-oligomerization-inhibiting mutations K439E or S440K and C1q binding-modulating mutations. (A-E) Binding of immobilized antibody variants to dimeric His-tagged, biotinylated ECD as tested in an ELISA assay for (A) the high-affinity allotype FcγRIIA 131H, (B) the low-affinity allotype FcγRIIA 131R, (C) FcγRIIB, (D) the high-affinity allotype FcγRIIIA 158V, or (E) the low-affinity allotype FcγRIIIA 158F. (F) Binding of immobilized FcγRIa to antibody variants tested in ELISA. Binding is presented for 20 μg / mL antibody samples and normalized per experiment to the average signal observed for wild-type IgG1-CAMPATH-1H (100%) after subtraction of the signal in wells incubated without primary antibody. Detection was performed using streptavidin-polyHRP and ABTS.

[0319] Figure 29 shows the selectivity of CDC activity by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H, anti-CD20 IgG1-11B8, and anti-CD52 IgG1-h2E8 through the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy was measured in three independent experiments and is presented as (A) the mean AUC normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and (B) the mean lysis rate determined by propidium iodide positivity at an antibody concentration of 40 μg / ml.

[0320] Figure 30 (A, B) shows the CDC efficacy of single-agent and combined anti-CD52 IgG1-CAMPATH-1H-E430G, anti-CD20 IgG1-11B8-E430G, and non-antigen-binding IgG1-b12-E430G antibody variants carrying the indicated self-oligomerization-inhibiting and C1q-binding-modulating mutations. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0321] Figure 31 shows the activation of Jurkat reporter cell lines stably expressing either (A) FcγRIIa or (B) FcγRIIIa, as measured by the level of luminescence (RLU), upon co-culture with Raji lymphoma cells and different concentrations of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants. Luminescence values ​​were normalized per experiment to the luminescence value observed for IgG1-b12 (0%) and wild-type IgG1-Campath-1H + wild-type IgG1-11B8 (100%), and then averaged across three (FcγRIIa) or two (FcγRIIIa) experimental replicates.

[0322] Figure 32 shows co-dependent CDC on Wien 133 cells induced by non-equimolar mixtures of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G236R and IgG1-11B8-E430G-S440K-G237A. (B) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G237Q and IgG1-11B8-E430G-S440K-G237A.

[0323] Figure 33 shows the selectivity of CDC activity by mixtures of anti-CD52 IgG1-CAMPATH-1H antibody variants with either the anti-HLA-DR IgG1-HLA-DR-huL243 variant (A) or the anti-HLA-DR IgG1-HLA-DR-1D09C3 variant (B) due to the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. Oci-Ly17 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to a mixture of (A) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-huL243-E430G (100%) or (B) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-1D09C3-E430G (100%).

[0324] Figure 34 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. CDC efficacy is shown for variants of IgG1-CAMPATH-1H-E430G-K439E with any of the mutations L234A, L234A-L235A, L234F, L234F-L235E, L235A, L235Q, G236R, or G237Q, and for mixtures of these variants with either the non-binding control antibody IgG1-b12 or IgG1-11B8-E430G-S440K. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0325] Figure 35 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. (A) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8, as single agents or in mixtures, harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345K, E345Q, E345R, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A. (B) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in mixtures thereof. (C) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in combination. (D) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G or K248E-T437R, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-modulating mutations G236R, G237A, or E333S, as single agents or in combination. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0326] Figure 36 shows codependent CDC on Raji lymphoma cells induced by a mixture of IgG1-CD37-37-3 and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Relative area under the curve (AUC) of cell lysis induced by dilutions of the indicated antibody variants or their mixtures, normalized to minimum lysis (0% for IgG1-b12) and maximum lysis (100% for the mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G). (B) Maximum lysis rates induced by the indicated antibody variants and their mixtures.

[0327] Figure 37 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Patient CLL samples were incubated with a series of antibody concentrations in the presence of 20% NHS. CDC efficacy is presented as the percentage of surviving B cells upon incubation with the antibody variants. Results are shown using CLL samples from patients 1 (A), 2 (B), and 3 (C).

[0328] Figure 38 shows the fractions of B cells, CD4+ T cells, and CD8+ T cells detected by flow cytometry after incubation of whole blood samples with a mixture of IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-CD5-INSERM antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Percentages of (A) B cells, (B) CD4+ T cells, and (C) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-b12 antibody variants. Percentages of (D) B cells, (E) CD4+ T cells, and (F) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-CD5-INSERM, and IgG1-b12 antibody variants. Fractions were calculated as [100% × (number of cells in sample / number of cells in "no antibody sample") × (number of granulocytes in "no antibody sample" / number of granulocytes in sample)].

[0329] Figure 39 shows the cooperative activation of programmed cell death in cancer cells by anti-DR4 and anti-DR5 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit (G237T) or enhance (K326W-E333S) C1q binding. (A) Viability of BxPC-3 human pancreatic cancer cells after 72 hours of incubation with the indicated antibody variants. (B) Viability of COLO 205 human colon cancer cells after 72 hours of incubation with the indicated antibody variants. The percentage of viable cells was calculated using the following formula: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of sample without antibody - luminescence of staurosporine sample)] * 100.

[0330] Figure 1A, set at 100%, shows the selectivity of CDC activity by the mixed antibody variants anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with the introduction of the P329R mutation. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% pooled normal human serum (NHS). CDC efficacy is presented as (A) the lysis rate determined by the percentage of propidium iodide (PI)-positive cells and (B) the area under the dose-response curve (AUC), normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0331] Figure 2 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with the introduction of the K322E mutation. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as (A) lysis rate determined by the percentage of PI-positive cells and (B) AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0332] Figure 3 shows the selectivity of CDC activity against different cell lines by introducing the K322E mutation into the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20-11B8-E430G-S440K. In vitro CDC assays were performed using 30 μg / mL of antibody in the presence of 20% NHS on Burkitt's lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C), and Ramos (D), acute lymphoblastic lymphoma (ALL) cell line REH (E), myeloma cell line U266B1 (F), and B-cell lymphoma cell line U-698-M (G). CDC activity is presented as lysis rate, determined by the percentage of PI-positive cells, normalized for each cell line to the non-binding control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%) for REH, U266B1, and Wien 133, or IgG1-11B8-E430G (100%) for Daudi, Raji, Ramos, and U-698-M.

[0333] Figure 4 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K, each carrying a C1q binding-inhibiting mutation (G236R, K322A, E269K, K322E, or P329R). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0334] Figure 5 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding-enhancing mutations (E333S, K326W, or K326A / E333A). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0335] Figure 6 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q binding-inhibiting mutations G236R (A), K322A (B), E269K (C), K322E (D), or P329R (E), plus anti-CD20 IgG1-11B8-E430G-S440K with C1q binding-enhancing mutations E333S, K326W, or K326A / E333A. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized area under the curve (AUC) of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0336] Figure 7 shows the binding of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variants (C, D) with the C1q binding-enhancing mutation E333S to the human lymphoma cell lines Wien 133 (A, C) and Raji (B, D). Antibody binding was tested by flow cytometry. Binding is expressed as the geometric mean fluorescence intensity (MFI). As negative controls for binding, samples without primary antibody or unconjugated anti-gp120 antibody IgG1-b12 were used.

[0337] Figure 8 shows the ADCC potential of the anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, C) with the C1q binding-inhibiting mutations G236R or K322A and the anti-CD20 IgG1-11B8-E430G-S440K antibody variant (B, D) with the C1q binding-enhancing mutation E333S. (A, B) ADCC reporter bioassays were performed by co-incubating Raji target cells with antibody dilution series and Jurkat human T cells stably expressing high-affinity FcγRIIIa (V158) and an NFAT response element driving the expression of firefly luciferase. Luciferase production was quantified by luminescence readout. (C, D) In ​​vitro europium TDA (EuTDA) ADCC assays were performed by co-incubating Wien 133 target cells with antibody dilution series and human PBMCs (E:T 100:1). Cell lysis was determined by measuring the signal of the EuTDA fluorescent chelate in the supernatant.

[0338] Figure 9 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-K439E with enhanced Fc-Fc interaction, carrying the C1q binding-inhibiting mutations G236R or K322A, and anti-CD20 IgG1-11B8-S440K with enhanced Fc-Fc interaction, carrying either the C1q binding-inhibiting mutation E333S or the C1q binding-enhancing mutation E430G, E345K, E345R, and E345R-E430G. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0339] Figure 10 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E, which has a C1q binding regulatory mutation or double mutation G237A-K322A at position G236 (G236R or G236K) or G237 (G237A, G237T, G237Q, or G237R), plus anti-CD20 IgG1-11B8-E430G-S440K, with or without the C1q binding regulatory mutations E333S, E333A, K326A, K326W-E333S, G237A, or G237A-E333S. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and maximum lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0340] Figure 11 shows the selectivity of CDC activity by mixed antibody isotype variants (IgG1, IgG2, IgG3, and hinge-stabilized IgG4) of anti-CD52 CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R and anti-CD20 11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%).

[0341] Figure 12 shows the selectivity of CDC activity on (A) Daudi cells and (B) Wien 133 cells by the mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without the C1q binding-inhibiting mutation G236R or K322A, and anti-CD37 IgG1-CD37-37.3-E430G-S440K with or without the C1q binding-enhancing mutation E333S. Target cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-CD37-37.3-E430G (100%).

[0342] Figure 13 shows the DR5-mediated cytotoxicity of (A) single antibody variants or (B) agonist mixtures of antibody variants of anti-DR5 IgG1-DR5-01-G56T-E430G-K439E with or without the C1q binding inhibitor mutation G236R + IgG1-DR5-05-E430G-S440K with or without the C1q binding enhancing mutation E333S against BxPC-3 human pancreatic cancer cells. A 3-day viability assay was performed and cell viability was determined using the CellTiter-Glo kit.

[0343] Figure 14 shows CDC activity by antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G with C1q binding-modulating mutations G237A, G236R, A327K, K322E, or P329R. Wien 133 cells were incubated with a concentration series of antibody in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibodies IgG1-b12-S440K (0%; not shown) and IgG1-CAMPATH-1H-E430G (100%).

[0344] Figure 15 shows the binding of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E antibody variants (A, B, C) with the C1q binding-inhibiting mutation G236R or K322A and anti-CD20 IgG1-11B8-E430G-S440K antibody variants (D, E, F) with the C1q binding-enhancing mutation E333S to human FcRn. FcRn ELISA was performed using 5 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnhsECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm.

[0345] Figure 16 shows the clearance rate of 500 μg of intravenously administered antibody in SCID mice. (A-C) Total human IgG in plasma samples was determined by ELISA and plotted as concentration versus time curves for (A) the IgG1-CAMPATH-1H variant, (B) the IgG1-11B8 variant, and (C) the combination of the IgG1-CAMPATH-1H variant and IgG1-11B8 variant. Each data point represents the mean + / - standard deviation of triplicate samples. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1,000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve.

[0346] Figure 17 shows the binding of immobilized IgG1-CAMPATH-1H-E430G-K439E variants with C1q binding-inhibiting mutations G236R or G237T and IgG1-11B8-E430G-S440K variants with C1q binding-enhancing mutations K326A or E333S to dimeric His-tagged, biotinylated ECDs of FcγRIIA allotype 131H (A), FcγRIIA allotype 131R (B), FcγRIIB (C), FcγRIIIA allotype 158V (D), and FcγRIIIA allotype 158F (E) tested in an ELISA assay. Binding is presented relative to the no-antibody control (background) and to binding to IgG1-11B8-E430G-S440K (100%) for a 20 μg / mL antibody sample. Detection was performed using streptavidin-polyHRP and ABTS.

[0347] Figure 18 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without C1q binding-inhibiting mutations (G236R or G237T) + anti-CD20 IgG1-11B8-E430G-S440K with the FcγR binding-inhibiting mutation G237A with or without the C1q binding-enhancing mutation E333S. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G (100%).

[0348] Figure 19 shows the selective activity of combinations of IgG1-CAMPATH-1H and IgG1-11B8 variants in whole blood as determined by flow cytometry analysis of blood cells. Y-axis: B cell fraction (CD19 positive / CD3 negative) or T cell fraction (CD19 negative / CD3 positive) of the total lymphocyte population (CD66b negative) after overnight (o / n) incubation in the presence of effector cells. X-axis: different treatment groups. Symbols represent cells from five different healthy donors tested in two separate incubations per donor. (A) Selective activity of IgG1-CAMPATH-1H-G236R-E430G-K439E mixed with IgG1-11B8-G237A-E430G-S440K. (B) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K. (C) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variants containing an additional G236R or G237 mutation mixed with IgG1-11B8-G237A-E430G-S440K containing an additional C1q binding-enhancing E333S mutation. (D) Depth of B cell depletion by different B cell-targeting antibodies compared to the co-dependent antibody combination of IgG1-CAMPATH-1H-E430G-K439E with the additional mutations G236R, G237Q, or G237R mixed with IgG1-11B8-G237A-E430G-S440K. Y-axis: logarithmic scale representation of the B cell fraction determined as above.

[0349] Figure 20 shows the selectivity of CDC activity against different cell lines with different expression levels of CD20 and CD52 by the combination of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants, which have C1q binding-inhibiting mutations in the anti-CD52 component and C1q binding-enhancing mutations in the anti-CD20 component. In vitro CDC assays were performed using the Burkitt lymphoma cell lines Daudi (A), Raji (B), and Ramos (C), the ALL cell line REH (D), and the B-cell lymphoma cell line U-698-M (E), using antibody concentrations of 0.01–40 μg / mL in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as maximal lysis. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0350] Figure 21 shows the selectivity of CDC activity by the mixed antibody variants: anti-CD37 IgG1-CD37-37.3-E430G-K439E with or without the C1q binding-inhibiting mutation G236R, and anti-CD20 IgG1-11B8-E430G-S440K with or without the C1q binding-enhancing mutation E333S. (A) Daudi cells and (B) WIL2-S cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as lysis at 40 μg / mL of IgG. Normalization was performed against the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CD37-37.3-E430G and IgG1-11B8-E430G (100%).

[0351] Figure 22 shows (A) binding of antibody variants of anti-CD52 IgG1-CAMPATH-1H with the Fc:Fc interaction-enhancing mutations E430G or E345R and the self-oligomerization-inhibiting mutation K439E, combined with either an FcγR binding-inhibiting mutation and a C1q binding-modulating mutation G236R, G237A, or G237T, to the human lymphoma cell line Wien 133. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-Campath-1H (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (B) Maximum binding (Bmax) to Raji cells by IgG1-Campath-1H antibody variants with mutations E430G and K439E in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T is shown normalized to the binding of wild-type IgG1-Campath-1H. (C) Apparent Kd values ​​for binding to Raji cells of IgG1-Campath-1H antibody variants with mutations E430G and S440K in combination with either the C1q binding regulatory mutations G236R, G237A, or G237T.

[0352] Figure 23 shows the binding of antibody variants of anti-CD20 IgG1-11B8 with the Fc-Fc interaction-enhancing mutation E430G and the self-oligomerization-inhibiting mutation S440K, in combination with either the C1q binding-modulating mutations K326A or E333A (A) or E333S, G237A, or G237A-E333S (B), to the human lymphoma cell line Raji. Antibody binding was tested by flow cytometry and is presented normalized to the Bmax value of wild-type IgG1-11B8 (100%). The non-binding anti-gp120 antibody IgG1-b12 was used as a negative control for binding. (C, D) Maximum binding (Bmax) to Raji cells by IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (C) or E333S, G237A, or G237A-E333S (D) is shown normalized to the binding of wild-type IgG1-11B8. (E, F) Apparent Kd values ​​for binding to Raji cells of IgG1-11B8 antibody variants with mutations E430G and S440K in combination with either the C1q binding modulation mutations K326A or E333A (E) or E333S, G237A, or G237A-E333S (F).

[0353] Figure 24 shows FcRn binding of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibody variants. (A, C) Binding to human FcRn is shown for variants of the anti-CD52 antibody IgG1-CAMPATH-1H with the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation K439E, and the C1q binding-modulating mutations G237A or G237T, using an antibody concentration of 40 μg / mL at (A) pH 6.0 or (C) pH 7.4. (B, D) Binding to human FcRn by variants of the anti-CD20 antibody IgG1-11B8, which have the Fc-Fc interaction-enhancing mutation E430G, the self-oligomerization-inhibiting mutation S440K, and the C1q binding-modulating mutations K326A, E333A, G237A, or G237A-E333S, using an antibody concentration of 40 μg / mL at (B) pH 6.0 or (D) pH 7.4. FcRn ELISA was performed using 2 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and an antibody dilution series. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. Absorbance was measured at 405 nm.

[0354] Figure 25 shows total human IgG (hIgG) concentrations as measured in blood samples collected from mice injected with anti-CD52 IgG1-CAMPATH-1H or anti-IgG1-11B8 antibody variants, or a mixture thereof. (A) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-CAMPATH-1H, IgG1-CAMPATH-1H-E430G-K439E-G237Q, or IgG1-CAMPATH-1H-E430G-K439E-G236R. (B) Total hIgG concentrations in blood samples collected from mice injected with wild-type IgG1-11B8, IgG1-11B8-E430G-S440K-G237A, or IgG1-11B8-E430G-S440K-E333S. (C) Total hIgG concentrations in blood samples collected from mice injected with a mixture of wild-type IgG1-CAMPATH-1H + IgG1-11B8 or a mixture of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations as in (A) and (B). In all figures, the dotted line represents the predicted IgG1 concentration over time for wild-type IgG1 antibodies in SCID mice. (D) Clearance up to 21 days after administration of the antibody was determined according to the formula D*1000 / AUC, where D is the injected dose and AUC is the area under the concentration-time curve.

[0355] Figure 26 shows the concentrations (in μg / ml) of C4d detected in samples incubated with IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 antibody variants carrying the mutations E430G, K439E, or S440K and G236R, G237A, G237Q, or G237R, after subtraction of the mean C4d concentration detected in negative control samples containing no antibody. Positive control samples contain antibody variants carrying the Fc-Fc interaction-enhancing mutations E345R, E430G, and S440Y (RGY).

[0356] Figure 27 shows C1q binding to Wien 133 cells incubated on ice with normal human serum as a complement source, as detected by flow cytometry, after opsonization with variants of antibodies IgG1-CAMPATH-1H, IgG1-11B8, and IgG1-b12 carrying the mutations E430G, K439E, or S440K and G236R, G237T, K326A, or E333S. Mean fluorescence intensity values ​​were normalized to the no-antibody control reaction (0%) and the maximal level (100%) of the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G, estimated by fitting a logarithmic agonist response model. (A) C1q binding of control reactions. (B–D) C1q binding of (B) IgG1-CAMPATH-E430G-K439E, (C) IgG1-CAMPATH-E430G-K439E-G236R, and (D) IgG1-CAMPATH-E430G-K439E-G237R mixed with nonbinding control IgG1-b12 or different IgG1-11B8 variants.

[0357] Figure 28 shows FcγR binding by IgG1-CAMPATH-1H-E430G and IgG1-11B8-E430G antibody variants carrying self-oligomerization-inhibiting mutations K439E or S440K and C1q binding-modulating mutations. (A-E) Binding of immobilized antibody variants to dimeric His-tagged, biotinylated ECD as tested in an ELISA assay for (A) the high-affinity allotype FcγRIIA 131H, (B) the low-affinity allotype FcγRIIA 131R, (C) FcγRIIB, (D) the high-affinity allotype FcγRIIIA 158V, or (E) the low-affinity allotype FcγRIIIA 158F. (F) Binding of immobilized FcγRIa to antibody variants tested in ELISA. Binding is presented for 20 μg / mL antibody samples and normalized per experiment to the average signal observed for wild-type IgG1-CAMPATH-1H (100%) after subtraction of the signal in wells incubated without primary antibody. Detection was performed using streptavidin-polyHRP and ABTS.

[0358] Figure 29 shows the selectivity of CDC activity by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H, anti-CD20 IgG1-11B8, and anti-CD52 IgG1-h2E8 through the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy was measured in three independent experiments and is presented as (A) the mean AUC normalized to the nonbinding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%), and (B) the mean lysis rate determined by propidium iodide positivity at an antibody concentration of 40 μg / ml.

[0359] Figure 30 (A, B) shows the CDC efficacy of single-agent and combined anti-CD52 IgG1-CAMPATH-1H-E430G, anti-CD20 IgG1-11B8-E430G, and non-antigen-binding IgG1-b12-E430G antibody variants carrying the indicated self-oligomerization-inhibiting and C1q-binding-modulating mutations. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0360] Figure 31 shows the activation of Jurkat reporter cell lines stably expressing either (A) FcγRIIa or (B) FcγRIIIa, as measured by the level of luminescence (RLU), upon co-culture with Raji lymphoma cells and different concentrations of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants. Luminescence values ​​were normalized per experiment to the luminescence value observed for IgG1-b12 (0%) and wild-type IgG1-Campath-1H + wild-type IgG1-11B8 (100%), and then averaged across three (FcγRIIa) or two (FcγRIIIa) experimental replicates.

[0361] Figure 32 shows co-dependent CDC on Wien 133 cells induced by non-equimolar mixtures of IgG1-CAMPATH-1H and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G236R and IgG1-11B8-E430G-S440K-G237A. (B) Co-dependent CDC induced by mixtures containing equimolar and non-equimolar ratios of IgG1-CAMPATH-1H-E430G-K439E-G237Q and IgG1-11B8-E430G-S440K-G237A.

[0362] Figure 33 shows the selectivity of CDC activity by mixtures of anti-CD52 IgG1-CAMPATH-1H antibody variants with either the anti-HLA-DR IgG1-HLA-DR-huL243 variant (A) or the anti-HLA-DR IgG1-HLA-DR-1D09C3 variant (B) due to the introduction of mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. Oci-Ly17 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to a mixture of (A) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-huL243-E430G (100%) or (B) IgG1-CAMPATH-1H-E430G + IgG1-HLA-DR-1D09C3-E430G (100%).

[0363] Figure 34 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. CDC efficacy is shown for variants of IgG1-CAMPATH-1H-E430G-K439E with any of the mutations L234A, L234A-L235A, L234F, L234F-L235E, L235A, L235Q, G236R, or G237Q, and for mixtures of these variants with either the non-binding control antibody IgG1-b12 or IgG1-11B8-E430G-S440K. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0364] Figure 35 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit C1q binding. (A) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8, as single agents or in mixtures, harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345K, E345Q, E345R, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A. (B) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in mixtures thereof. (C) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V, or E345Y, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-inhibiting mutations G236R or G237A, as single agents or in combination. (D) CDC efficacy of antibody variants of IgG1-CAMPATH-1H and IgG1-11B8 harboring matched Fc-Fc interaction-enhancing mutations E430G or K248E-T437R, self-oligomerization-inhibiting mutations K439E or S440K, and C1q binding-modulating mutations G236R, G237A, or E333S, as single agents or in combination. CDC efficacy is presented as AUC normalized to the non-binding control antibody IgG1-b12 (0%) and to the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0365] Figure 36 shows codependent CDC on Raji lymphoma cells induced by a mixture of IgG1-CD37-37-3 and IgG1-11B8 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. (A) Relative area under the curve (AUC) of cell lysis induced by dilutions of the indicated antibody variants or their mixtures, normalized to minimum lysis (0% for IgG1-b12) and maximum lysis (100% for the mixture of IgG1-CD37-37-3-E430G + IgG1-11B8-E430G). (B) Maximum lysis rates induced by the indicated antibody variants and their mixtures.

[0366] Figure 37 shows the selective co-dependent CDC activity of mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introducing mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Patient CLL samples were incubated with a series of antibody concentrations in the presence of 20% NHS. CDC efficacy is presented as the percentage of surviving B cells upon incubation with the antibody variants. Results are shown using CLL samples from patients 1 (A), 2 (B), and 3 (C).

[0367] Figure 38 shows the fractions of B cells, CD4+ T cells, and CD8+ T cells detected by flow cytometry after incubation of whole blood samples with a mixture of IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-CD5-INSERM antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and modulate C1q binding. Percentages of (A) B cells, (B) CD4+ T cells, and (C) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-b12 antibody variants. Percentages of (D) B cells, (E) CD4+ T cells, and (F) CD8+ T cells detected in whole blood samples from four donors after incubation with the indicated IgG1-CAMPATH-1H, IgG1-CD5-INSERM, and IgG1-b12 antibody variants. Fractions were calculated as [100% × (number of cells in sample / number of cells in "no antibody sample") × (number of granulocytes in "no antibody sample" / number of granulocytes in sample)].

[0368] Figure 39 shows the cooperative activation of programmed cell death in cancer cells by anti-DR4 and anti-DR5 antibody variants carrying mutations that enhance Fc-Fc interactions, inhibit self-oligomerization, and inhibit (G237T) or enhance (K326W-E333S) C1q binding. (A) Viability of BxPC-3 human pancreatic cancer cells after 72 hours of incubation with the indicated antibody variants. (B) Viability of COLO 205 human colon cancer cells after 72 hours of incubation with the indicated antibody variants. The percentage of viable cells was calculated using the following formula: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of sample without antibody - luminescence of staurosporine sample)] * 100.

[0369] B) was found to be more efficient than the individual single antibodies. (Figure 1 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with the introduction of the P329R mutation. Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% pooled normal human serum (NHS). CDC efficacy is presented as (A) the lysis rate determined by the percentage of propidium iodide (PI)-positive cells and (B) the area under the dose-response curve (AUC), normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0370] Figure 2 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with the introduction of the K322E mutation. Vienna 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as (A) lysis rate determined by the percentage of PI-positive cells and (B) AUC normalized to the non-binding control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0371] Figure 3 shows the selectivity of CDC activity against different cell lines by introducing the K322E mutation into the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20-11B8-E430G-S440K. In vitro CDC assays were performed using 30 μg / mL of antibody in the presence of 20% NHS on Burkitt's lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C), and Ramos (D), acute lymphoblastic lymphoma (ALL) cell line REH (E), myeloma cell line U266B1 (F), and B-cell lymphoma cell line U-698-M (G). CDC activity is presented as lysis rate, determined by the percentage of PI-positive cells, normalized for each cell line to the non-binding control antibody IgG1-b12 (0%) and IgG1-CAMPATH-1H-E430G (100%) for REH, U266B1, and Wien 133, or IgG1-11B8-E430G (100%) for Daudi, Raji, Ramos, and U-698-M.

[0372] Figure 4 shows the selectivity of CDC activity by the mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K, each carrying a C1q binding-inhibiting mutation (G236R, K322A, E269K, K322E, or P329R). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0373] Figure 5 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding-enhancing mutations (E333S, K326W, or K326A / E333A). Wien 133 cells were incubated with a concentration series of antibodies in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. Normalization was performed against the non-binding control antibody mix IgG1-b12-K439E + IgG1-b12-S440K (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0374] Figure 6 shows the selectivity of CDC activity by mixed antibody variants: anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q binding-inhibiting mutations G236R (A), K322A (B), E269K (C), K322E (D), or P329R (E), plus anti-CD20 IgG1-11B8-E430G-S440K with C1q binding-enhancing mutations E333S, K326W, o...

Claims

1. A medicament comprising a first antibody comprising a first Fc region of human IgG1 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 IgG1 and a second antigen-binding region capable of binding to a second antigen, comprising: The first Fc region is (a) E430G, K439E, and G236R, (b) E345K, K439E, and G236R, (c) E345R, K439E, and G236R, or (d) E430G, K439E, and G237A including the substitution: The second Fc region containing one substitution selected from the group consisting of E430G, E345K, and E345R; and Contains the S440K substitution, The amino acid positions correspond to those of human IgG1 according to the Eu numbering system. The said medicine.

2. The pharmaceutical of claim 1, wherein the first Fc region comprises a G236R substitution.

3. The pharmaceutical of claim 1 or 2, wherein the second Fc region comprises a G237A substitution.

4. The pharmaceutical according to any one of claims 1 to 3, wherein the first and / or second antibody is a human antibody, a humanized antibody, or a chimeric antibody.

5. The method of any one of claims 1 to 4, wherein the first and second antigens are not identical.

6. The medicament of any one of claims 1 to 5, wherein the medicament depletes a cell population expressing the first and second antigens.

7. An antibody comprising a human IgG1 Fc region and an antigen-binding region capable of binding to an antigen, The Fc region is (a) E430G, K439E, and G236R, (b) E345K, K439E, and G236R, (c) E345R, K439E, and G236R, or (d) E430G, K439E, and G237A including the substitution of The antibody.

8. The antibody of claim 7, wherein the Fc region comprises an E430G substitution.

9. The antibody of claim 7 or 8, wherein the Fc region comprises a G237A substitution.

10. The antibody of any one of claims 7 to 9, wherein the Fc region comprises a G236R substitution.

11. A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region described in any one of claims 1, 2 and 4 to 6, and the second antibody comprises a second antigen-binding region and a second Fc region described in any one of claims 1 and 3 to 6.

12. A composition comprising a first and a second antibody, wherein the first antibody comprises a first antigen-binding region capable of binding to a first antigen and a first Fc region of human IgG1, and the second antibody comprises a second antigen-binding region capable of binding to a second antigen and a second Fc region of human IgG1; The first Fc region is (a) E430G, K439E, and G236R, (b) E345K, K439E, and G236R, (c) E345R, K439E, and G236R, or (d) E430G, K439E, and G237A including the substitution: The second Fc region containing one substitution selected from the group consisting of E430G, E345K, and E345R; and Contains the S440K substitution, The amino acid positions correspond to those of human IgG1 according to the EU numbering system. The composition.

13. The composition of claim 12, wherein the first and second Fc regions comprise an E430G substitution.

14. The composition of claim 12 or 13, wherein the first Fc region comprises a G236R substitution.

15. The composition of any one of claims 12 to 14, wherein the second Fc region comprises a G237A substitution.

16. The composition of any one of claims 11 to 15, wherein the first and second antigens are not identical.

17. The first antibody and the second antibody are in a molar ratio of about 1:50 to 50:1, e.g., about 1:1 molar ratio, about 1:2 molar ratio, about 1:3 molar ratio, about 1:4 molar ratio, about 1:5 molar ratio, about 1:6 molar ratio, about 1:7 molar ratio, about 1:8 molar ratio, about 1:9 molar ratio, about 1:10 molar ratio, about 1:15 molar ratio, about 1:20 molar ratio, about 1:25 molar ratio, about 1:30 molar ratio, about 1:35 molar ratio, about 1:40 molar ratio, about 1:45 molar ratio, about 1:50 molar ratio, about 1:60 molar ratio, about 1:70 molar ratio, about 1:80 molar ratio, about 1:90 molar ratio, about 1:100 molar ratio, about 1:150 molar ratio, about 1:200 molar ratio, about 1:250 molar ratio, about 1:300 molar ratio, about 1:350 molar ratio, about 1:40 ...

17. The composition of any one of claims 11 to 16, wherein the hydroxyl group is present in the composition in a molar ratio of about 1:50, about 50:1, about 45:1, about 40:1, about 35:1, about 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:

1.

18. The composition of any one of claims 11 to 17, wherein the first antibody and the second antibody are present in the composition in a 1:1 molar ratio.

19. A composition according to any one of claims 11 to 18 for use as a medicament.

20. The pharmaceutical of any one of claims 1 to 6, the antibody of any one of claims 7 to 10, or the composition of any one of claims 11 to 19, wherein the antigen-binding region is capable of binding to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA-DR, CD3, CD5, 4-1BB, and PD1.

21. The antigen-binding region is (a) a VH region comprising the CDR1 sequence shown in SEQ ID NO:196, the CDR2 sequence shown in SEQ ID NO:197, and the CDR3 sequence shown in SEQ ID NO:198, and a VL region [DR4] comprising the CDR1 sequence shown in SEQ ID NO:200, the CDR2 sequence shown in AAT, and the CDR3 sequence shown in SEQ ID NO:201; (b) a VH region comprising the CDR1 sequence shown in SEQ ID NO:50, the CDR2 sequence shown in SEQ ID NO:51, and the CDR3 sequence shown in SEQ ID NO:52, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:54, the CDR2 sequence shown in FAS, and the CDR3 sequence shown in SEQ ID NO:55 [DR5-01-G56T]; (c) a VH region comprising the CDR1 sequence shown in SEQ ID NO:57, the CDR2 sequence shown in SEQ ID NO:58, and the CDR3 sequence shown in SEQ ID NO:59, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:61, the CDR2 sequence shown in RTS, and the CDR3 sequence shown in SEQ ID NO:62 [DR5-05]; (d) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:36, the CDR2 sequence set forth in SEQ ID NO:37, and the CDR3 sequence set forth in SEQ ID NO:38, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:40, the CDR2 sequence set forth in DAS, and the CDR3 sequence set forth in SEQ ID NO:41 [CD20, 7D8]; (e) a VH region comprising 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 a VL region comprising 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 [CD20, 11B8]; (f) a VH region comprising the CDR1 sequence set forth in SEQ ID NO:43, the CDR2 sequence set forth in SEQ ID NO:44, and the CDR3 sequence set forth in SEQ ID NO:45, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO:47, the CDR2 sequence set forth in VAT, and the CDR3 sequence set forth in SEQ ID NO:48 [CD37]; (g) a VH region comprising 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 a VL region comprising 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 [CD52, CAMPATH-1H]; (h) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 161, the CDR2 sequence set forth in SEQ ID NO: 162, and the CDR3 sequence set forth in SEQ ID NO: 163, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 165, the CDR2 sequence set forth in LVS, and the CDR3 sequence set forth in SEQ ID NO: 166 [CD52, h2E8]; (i) a VH region comprising the CDR1 sequence shown in SEQ ID NO: 168, the CDR2 sequence shown in SEQ ID NO: 169, and the CDR3 sequence shown in SEQ ID NO: 170, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 172, the CDR2 sequence shown in AAS, and the CDR3 sequence shown in SEQ ID NO: 173 [HLA-DR, hul243]; (j) a VH region comprising the CDR1 sequence shown in SEQ ID NO: 175, the CDR2 sequence shown in SEQ ID NO: 176, and the CDR3 sequence shown in SEQ ID NO: 177, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 179, the CDR2 sequence shown in DNN, and the CDR3 sequence shown in SEQ ID NO: 180 [HLA-DR, 1D09C3]; (k) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 182, the CDR2 sequence set forth in SEQ ID NO: 183, and the CDR3 sequence set forth in SEQ ID NO: 184, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 186, the CDR2 sequence set forth in DTS, and the CDR3 sequence set forth in SEQ ID NO: 187 [CD3, huCLB T3 / 4]; or (l) a VH region comprising the CDR1 sequence shown in SEQ ID NO: 189, the CDR2 sequence shown in SEQ ID NO: 190, and the CDR3 sequence shown in SEQ ID NO: 191, and a VL region comprising the CDR1 sequence shown in SEQ ID NO: 193, the CDR2 sequence shown in ATS, and the CDR3 sequence shown in SEQ ID NO: 194 [CD5] The medicament according to any one of claims 1 to 6 and 20, the antibody according to any one of claims 7 to 10 and 20, or the composition according to any one of claims 11 to 20.

22. A medicament, antibody or composition according to any one of claims 1 to 21 for depleting a cell population expressing a first antigen and a second antigen.

23. A medicament, antibody or composition according to any one of claims 1 to 21 for inducing proliferation in a cell population expressing the first antigen and the second antigen.

24. A kit comprising a first container comprising a first antibody as defined in any one of claims 1 to 2, 4 to 6 and 20 to 21, and a second container comprising a second antibody as defined in any one of claims 1, 3 to 6 and 20 to 21.

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