Antibody variant combinations and uses thereof

Abstract

To provide antibodies having increased Fc effector functions or decreased Fc effector functions, to provide the use of such antibodies in combination, and to provide compositions comprising such antibodies.SOLUTION: Provided are combinations of first and second antibodies having modified Fc effector functions resulting from amino acid substitutions in an Fc region, the amino acid substitutions allowing for co-dependent activation of effector functions such as CDC or ADCC, or agonistic activity or enhanced agonistic activity.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to antibodies having an altered Fc effector function, i.e., an 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 Art

[0002] Background of the Invention Effector functions mediated by the Fc of monoclonal antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP), contribute to the therapeutic concentration range defined by efficacy and toxicity. CDC is initiated by the binding of C1q to the Fc region of the antibody. C1q is a multimeric protein consisting of six binding globular heads attached to a stalk. Each individual globular binding head has a low affinity for IgG; for the classical complement pathway to be induced, C1q must increase 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 Fc gamma receptors (FcγR) 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 non-covalent 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 an antibody or polypeptide comprising a variant Fc region having one or more amino acid modifications that result in alteration of effector functions such as complement-dependent cytotoxicity (CDC). WO2014 / 108198 (Patent Document 2) discloses a polypeptide such as an antibody comprising a variant Fc region having one or more amino acid modifications that result in an increase in complement-dependent cytotoxicity (CDC).

[0005] WO2012 / 130831 (Patent Document 3) relates to an Fc region-containing polypeptide having an effector function altered 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 as compared to polypeptides comprising a wild-type IgG Fc region, and exhibit antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the polypeptide reduced to at least 20% of that induced by a polypeptide comprising a wild-type human IgG Fc region. WO2012 / 130831 (Patent Document 3) does not disclose an antibody having enhanced Fc-Fc interaction and / or enhanced hexamer formation ability.

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

[0007] However, if the target antigen is widely expressed in vivo in both healthy and pathogenic cells, the antibody can be toxic by killing normal cells. Therefore, it is necessary to make the effector function of an antibody having enhanced effector functions dependent on another antibody in a combination that is selective for pathogenic cells and thus can prevent the death 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 having an enhanced effector function rely only on the antigen-binding region of the antibody to achieve specificity for target cells, which can limit the appropriate target to a target highly selectively expressed in diseased cells. Individually acting antibodies having mutations that reduce the effector function can spare healthy cells expressing the antigen target of those antibodies, but the mutations that reduce the effector function can limit the efficacy. Thus, it is clearly necessary to create combinations of antibodies in which each individual antibody can bind to both pathogenic and normal cells, but the enhanced effector function is activated only when both antibodies bind to the same pathogenic cell or preferentially in that case. This decoupling of effector function activation from individual target binding allows the creation of antibodies that bind to targets that could not previously be optimally used for antibody therapy because of unwanted toxicity to healthy cells or lack of efficacy against diseased cells, since effector function activation becomes selective for cells to which the combination of antibodies binds simultaneously.

[0010] Accordingly, it is an object of the present invention to provide a first antibody and a second antibody modified to provide maximum activity against target cells to which both antibodies bind simultaneously, wherein the first antibody provides no or minimal activity against target cells to which only the first antibody binds, and the second antibody provides minimal or reduced activity against target cells to which only the second antibody binds, as compared to the activity against cells to which both antibodies bind simultaneously.

[0011] Both are a first antibody and a second antibody that include an Fc region and an antigen-binding region of human IgG, both having substitutions that increase Fc-Fc interaction and substitutions that inhibit self-oligomerization, the first antibody further having substitutions for reducing effector functions such as CDC and / or ADCC, the first antibody having reduced effector functions such as CDC and / or ADCC compared to a first parental antibody that does not include substitutions for reducing effector functions, and the combination of the first and second antibodies having enhanced effectors compared to the first and second antibodies individually. It is a further object of the present invention to provide a first antibody and a second antibody.

[0012] A first antibody that includes an Fc region and an antigen-binding region of human IgG and has substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions for reducing effector functions such as CDC and / or ADCC compared to a first parental antibody, and a second antibody that includes an Fc region and an antigen-binding region of human IgG and has substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions for enhancing effector functions such as CDC and / or ADCC compared to a second parental antibody, wherein the activities of the first antibody and the second antibody are co-dependent by complementary substitutions that inhibit self-oligomerization. It is another object of the present invention to provide a first antibody and a second antibody.

[0013] A first antibody comprising an Fc region and an antigen-binding region of human IgG, having substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions for reducing effector functions such as CDC and / or ADCC as compared to a first parental antibody, and a second antibody comprising an Fc region and an antigen-binding region of human IgG, having substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions that induce agonist activity such as an increase in activation of a target receptor upon binding as compared to a second parental antibody, wherein the activities of the first and second antibodies are co-dependent by complementary self-oligomerization-inhibiting substitutions, it is a further object of the present invention to provide the first antibody and the second antibody.

[0014] A first antibody comprising an Fc region and an antigen-binding region of human IgG, having substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions for reducing agonist activity such as a decrease in activation of a target receptor upon binding as compared to a first parental antibody, and a second antibody comprising an Fc region and an antigen-binding region of human IgG, having substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions for reducing agonist activity such as a decrease in activation of a target receptor upon binding as compared to a second parental antibody, wherein the combination of the first and second antibodies has enhanced agonist activity that is co-dependent by complementary self-oligomerization-inhibiting substitutions, it is a further object of the present invention to provide the first antibody and the second antibody.

[0015] A first antibody comprising an Fc region of human IgG and an antigen-binding region, having substitutions that increase Fc-Fc interaction, substitutions that inhibit self-oligomerization, and substitutions that decrease effector functions such as CDC and / or ADCC compared to a first parental antibody, and a second antibody comprising an Fc region of human IgG and an antigen-binding region, having substitutions that inhibit self-oligomerization, increase Fc-Fc interaction, activate signaling when the antigen-binding region of the antibody binds to the corresponding antigen, and optionally induce enhanced signaling compared to a second parental antibody, wherein the activities of the first antibody and the second antibody are co-dependent by complementary self-oligomerization-inhibiting substitutions. It is a further object of the present invention to provide the first antibody and the second antibody.

[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 one Fc-Fc enhancing substitution and one or more substitutions that decrease effector functions such as CDC and / or ADCC, and a second antibody having 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 functions such as CDC and / or ADCC, wherein the first and second antibodies further have complementary self-oligomerization-inhibiting substitutions that co-dependently heterooligomerize the first and second antibodies.

[0017] Although not limited by theory, a combination of a first antibody and a second antibody of the invention having complementary substitutions that co-depend on the effector or signaling function, e.g., agonist activity, of the first and second antibodies is thought to be able to reduce the toxicity of the combination and increase the therapeutic concentration range of the combination. Further, a combination of a first antibody and a second antibody of the invention can be used to specifically deplete a cell population expressing an antigen recognized by the first antibody and the second antibody. Thus, a combination of a first antibody and a second antibody of the invention can specifically deplete a tumor cell population 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 antigen recognized by the first and second antibodies.

[0018] In one aspect, the 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 medicament 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 Fc region comprises (a) 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, and (b) the substitution K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, the substitution is not S440K, and (c) 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, and P329R; the second Fc region comprises (d) 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) When the first Fc region comprises the substitution of K322E or P329R, comprising one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to 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), Provided is a first antibody for use as a medicament in combination with a second antibody.

[0019] In one aspect, the present invention relates to 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 medicament 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 Fc region is (a) Comprising 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, and (b) Comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) Comprising 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 is (d) Comprising 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the Eu numbering system, Provided is a first antibody for use as a medicament in combination with a second antibody.

[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 medicament 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 Fc region is (a) Comprising 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, and (b) Comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) Comprising 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 is (d) includes 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, and (e) includes the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, the first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the Eu numbering system, There is provided a first antibody for use as a medicament in combination with a second antibody.

[0021] In one aspect, the present invention relates to 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 medicament 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 Fc region (a) includes 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, and (b) includes the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) includes one substitution of an amino acid at position P329, or the substitution of K322E; wherein the second Fc region (d) includes 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) Comprising one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the EU numbering system, Provided is a first antibody for use as a medicament in combination with a second antibody.

[0022] In another aspect, the present invention relates to 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 medicament 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 Fc region (a) Comprising the substitutions of K248E and T437R, and (b) Comprising the substitution of K439E or S440K, and (c) Comprising one 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, and L235E; The second Fc region (d) Comprising the substitutions of K248E and T437R, and (e) Comprising the substitution of K439E or S440K, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the Eu numbering system, Provide a first antibody for use as a medicament in combination with a second antibody.

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

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

[0025] Amino acid substitutions 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 regulatory 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 regulatory substitutions according to the present invention and increase effector functions such as CDC and / or ADCC.

[0027] In one aspect, the second Fc region comprises the substitution of G237A.

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

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

[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, wherein the Fc region (a) comprises 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, and (b) comprises the substitution K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) comprises 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, and provides the antibody.

[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, wherein the Fc region (a) Comprising the substitutions K248E and T437R, and (b) Comprising the substitution K439E or S440K, and (c) Comprising one substitution of an amino acid at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A; (d) Comprising K332E, E269K, L234A, L234F, L235A, L235Q, L235E, K326A, K326W, E333A, and E333S, provides an antibody.

[0032] 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 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, wherein the first Fc region (a) Comprises 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, and (b) Comprises the substitution K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) Comprises 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, and P329R; wherein the second Fc region (d) It contains 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, and (e) It contains the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) When the first Fc region contains the substitution of K322E or P329R, it contains one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, and the amino acid positions correspond to human IgG1 according to the EU numbering system, A composition is provided.

[0034] In one aspect, the present invention is 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 is (a) It contains 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, and (b) It contains the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) It contains 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 is (d) comprising 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, and (e) comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, the first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, and the amino acid positions correspond to human IgG1 according to the EU numbering system, A composition is provided.

[0035] In one aspect, the present invention is 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 is (a) comprising 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, and (b) comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) comprising one substitution of an amino acid at position P329, or the substitution of K322E; The second Fc region is (d) comprising 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, and (e) comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) Comprising one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, wherein the first Fc region has a substitution of K439E and the second Fc region has a substitution of S440K, or the first Fc region has a substitution of S440K and the second Fc region has a substitution of K439E, and the amino acid positions correspond to 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 antibody 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) comprises substitutions of K248E and T437R, and (b) comprises a substitution of K439E or S440K, and (c) comprises one 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, and L235E; The second Fc region (d) comprises substitutions of K248E and T437R, and (e) comprises one substitution of K439E or S440K, wherein the first Fc region has a substitution of K439E and the second Fc region has a substitution of S440K, or the first Fc region has a substitution of S440K and the second Fc region has a substitution of K439E; and the amino acid positions correspond to 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 contacting the cell population with a first and a second antibody as defined herein, or a composition of the first and second antibodies, or a composition.

[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 recited in the claims described herein or an effective amount of the 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 present invention relates to the first and second antibodies or compositions described herein for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

[0041] 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 or composition described herein.

[0042] [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 medicament 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 Fc region comprises (a) 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, and (b) includes the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) includes one 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; The second Fc region is (d) includes one substitution of the 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, and (e) includes the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the Eu numbering system, The first antibody. [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 medicament 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 is (a) includes one substitution of the 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, and (b) includes the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) includes one substitution of the amino acid at position P329, or the substitution of K322E; The second Fc region is (d) includes 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, and (e) includes the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) includes one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, the first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the EU numbering system, the first antibody. [Inventive concept 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 medicament 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 Fc region (a) includes the substitutions of K248E and T437R, and (b) includes the substitution of K439E or S440K, and (c) includes one 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, and L235E; wherein the second Fc region (d) includes the substitutions of K248E and T437R, and (e) includes the substitution of K439E or S440K, The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to human IgG1 according to the Eu numbering system. The first antibody. [Inventive concept 1004] A first antibody for use as a medicament in combination with a second antibody of any one of inventive concepts 1001 or 1002, wherein the first and second Fc regions comprise substitutions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [Inventive concept 1005] A first antibody for use as a medicament in combination with a second antibody of any one of inventive concepts 1001 - 1002 and 1004, wherein the first and second Fc regions comprise substitutions selected from the group consisting of E430G, E345K, and E345R. [Inventive concept 1006] A first antibody for use as a medicament in combination with a second antibody of any one of inventive concepts 1001 - 1002 and 1004 - 1005, wherein the first and second Fc regions comprise an E430G substitution. [Inventive concept 1007] A first antibody for use as a medicament in combination with a second antibody of any one of inventive concepts 1001 and 1003 - 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, G237P. [Inventive concept 1008] A first antibody for use as a medicament in combination with a second antibody of any one of inventive concepts 1001 and 1003 - 1007, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R. [Inventive concept 1009] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1001 and 1003 to 1008, wherein the first Fc region comprises the substitution G237Q. [The present invention 1010] A first antibody for use as a medicament in combination with a second antibody, any one 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 medicament in combination with a second antibody, any one of the present inventions 1001, 1003 to 1006, and 1010, wherein the first Fc region comprises the substitution G236R. [The present invention 1012] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1001, 1003 to 1006, and 1010, wherein the first Fc region comprises the substitution E269K. [The present invention 1013] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1001 and 1003 to 1006, wherein the first Fc region comprises the substitution K322A. [The present invention 1014] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1002 to 1006, wherein the first Fc region comprises the substitution P329R. [The present invention 1015] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1002 to 1006, wherein the first Fc region comprises the substitution K322E. [The present invention 1016] A first antibody for use as a medicament in combination with a second antibody, any one of the present inventions 1001 and 1003 to 1013, 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 1017] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 1001, 1003 to 1013, and 1016, wherein the second Fc region contains a G237A substitution. [The present invention 1018] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 1001 to 1015, wherein the second Fc region contains 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 medicament in combination with a second antibody of any one of the present inventions 1001 to 1015 and 1018, wherein the second Fc region contains one substitution selected from the group consisting of K326A, K326W, E333A, and E333S. [The present invention 1020] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 1001, 1003 to 1013, and 1016 to 1017, wherein the second Fc region contains G237A and E333S substitutions. [The present invention 1021] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 1001 to 1019, wherein the second Fc region contains a K326A substitution. [The present invention 1022] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 1001 to 1019, wherein the second Fc region contains an E333S substitution. [The present invention 1023] A first antibody for use as a medicament in combination with a second antibody of any one of the present inventions 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 medicament in combination with a second antibody of any one of the present inventions 1001 to 1016, 1018, and 1023, wherein the second Fc region contains K326W and E333S substitutions. [Invention 1025] A first antibody for use as a medicament in combination with a second antibody of any one of Inventions 1001 to 1016, 1018, and 1023, wherein the second Fc region comprises the substitutions K326A and E333A. [Invention 1026] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and / or second antibody is a human antibody, a humanized antibody, or a chimeric antibody. [Invention 1027] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and / or second antibody is a monoclonal antibody. [Invention 1028] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and / or second antibody is of the human IgG1 isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype. [Invention 1029] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and / or second antibody is of the human IgG1 isotype. [Invention 1030] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein both the first and second antigens are molecules exposed on the cell surface. [Invention 1031] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and second antigens coexist in a cell or tissue that is a target cell or target tissue for the disease or disorder to be treated. [Invention 1032] A first antibody for use as a medicament in combination with a second antibody of any one of the above inventions, wherein the first and second antigens are not identical. [Invention 1033] A first antibody for use as a medicament in combination with a second antibody of any of the present invention, wherein the medicament depletes a cell population expressing a first and a second antigen. [Inventive concept 1034] A first antibody for use as a medicament in combination with a second antibody of inventive concept 1033, wherein the cell population is a tumor cell. [Inventive concept 1035] A first antibody for use as a medicament in combination with a second antibody of any of inventive concepts 1033 - 1034, wherein the cell population is a hematological tumor cell or a solid tumor cell. [Inventive concept 1036] A first antibody for use as a medicament in combination with a second antibody of any of inventive concepts 1033 - 1034, wherein the cell population is a population of white blood cells, lymphocytes, B cells, T cells, regulatory T cells, NK cells, myeloid - derived suppressor cells, tumor - associated macrophage cells. [Inventive concept 1037] An antibody comprising an antigen - binding region capable of binding to an antigen and the Fc region of human IgG, wherein the Fc region (a) comprises 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, and (b) comprises the substitution K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) comprises 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, said antibody. [Inventive concept 1038] An antibody comprising an antigen - binding region capable of binding to an antigen and the Fc region of human IgG, wherein the Fc region (a) comprises the substitutions K248E and T437R, and (b) Comprising the substitution of K439E or S440K, and (c) Comprising one substitution of an amino acid at position G237 or P329, or one or more substitutions selected from the group consisting of G236R, G236K, K322A, (d) Comprising K332E, E269K, L234A, L234F, L235A, L235Q, L235E, K326A, K326W, E333A, and E333S, Said antibody. [Invention 1039] An antibody of Invention 1037, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. [Invention 1040] An antibody of either Invention 1037 or 1039, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, and E345R. [Invention 1041] An antibody of either Invention 1037 or 1039 - 1041, wherein the Fc region comprises the substitution of E430G. [Invention 1042] An antibody of either Invention 1037 - 1041, wherein 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. [Invention 1043] An antibody of either Invention 1037 - 1042, wherein the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R. [Invention 1044] An antibody of either Invention 1037 - 1043, wherein the Fc region comprises the substitution of G237Q or G237A. [Invention 1045] An antibody according to any one of inventions 1037 to 1041, wherein the Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. [Invention 1046] An antibody according to any one of inventions 1037 to 1041 and 1045, wherein the Fc region comprises the substitution G236R. [Invention 1047] An antibody according to any one of inventions 1037 to 1041 and 1045, wherein the Fc region comprises the substitution E269K. [Invention 1048] An antibody according to any one of inventions 1037 to 1041, wherein the Fc region comprises the substitution K322A. [Invention 1049] An antibody according to any one of inventions 1037 to 1048, which is of the human IgG1 isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype. [Invention 1050] An antibody according to any one of inventions 1037 to 1049, which is of the human IgG1 isotype. [Invention 1051] A composition comprising a first antibody and a second antibody, wherein the first antibody comprises a first antigen-binding region and a first Fc region according to any one of inventions 1001 to 1015 and 1026 to 1036, and the second antibody comprises a second antigen-binding region and a second Fc region according to any one of inventions 1001 to 1006 and 1016 to 1036. [Invention 1052] A composition comprising a first antibody 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 is (a) comprises 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, and (b) Comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) 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 is (d) Comprising 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, The amino acid positions correspond to human IgG1 according to the EU numbering system, Said composition. [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 is (a) Comprising 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, and (b) Comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) Comprising one substitution of an amino acid at position P329, or the substitution of K322E; The second Fc region is (d) includes 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, and (e) includes the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) includes one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, and the amino acid positions correspond to human IgG1 according to the EU numbering system, The composition. [Inventive item 1054] A composition comprising a first antibody 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 is (a) includes the substitutions of K248E and T437R, and (b) includes the substitution of K439E or S440K, and (c) includes one 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, and L235E; The second Fc region is (d) includes the substitutions of K248E and T437R, and (e) includes one substitution of K439E or S440K, The first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; the amino acid positions correspond to human IgG1 according to the Eu numbering system. Said composition. [Inventive item 1055] A composition according to any one of Inventive items 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. [Inventive item 1056] A composition according to any one of Inventive items 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. [Inventive item 1057] A composition according to any one of Inventive items 1052 to 1053 and 1055 to 1056, wherein the first and second Fc regions comprise an E430G substitution. [Inventive item 1058] A composition according to any one of Inventive items 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, G237P. [Inventive item 1059] A composition according to any one of Inventive items 1052 to 1058, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237Q, G237R, and G237S. [Inventive item 1060] A composition according to any one of Inventive items 1052 to 1059, wherein the first Fc region comprises a G237Q substitution. [Inventive item 1061] A composition according to any one of Inventive items 1052 to 1057, wherein the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K. [Invention 1062] A composition according to any one of Inventions 1052 to 1057 and 1061, wherein the first Fc region contains the substitution G236R. [Invention 1063] A composition according to any one of Inventions 1052 to 1057 and 1061, wherein the first Fc region contains the substitution E269K. [Invention 1064] A composition according to any one of Inventions 1052, 1054 to 1057, wherein the first Fc region contains the substitution K322A. [Invention 1065] A composition according to any one of Inventions 1053 to 1057, wherein the first Fc region contains the substitution P329R. [Invention 1066] A composition according to any one of Inventions 1053 to 1057, wherein the first Fc region contains the substitution K322E. [Invention 1067] A composition according to any one of Inventions 1052, 1054 to 1064, wherein the second Fc region contains one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S. [Invention 1068] A composition according to any one of Inventions 1052, 1054 to 1064, and 1067, wherein the second Fc region contains the substitution G237A. [Invention 1069] A composition according to any one of Inventions 1052 to 1067, wherein the second Fc region contains one substitution selected from the group consisting of K326A, K326W, E333A, and E333S. [Invention 1070] A composition according to any one of Inventions 1052 to 1067 and 1069, wherein the second Fc region contains two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S. [Invention 1071] A composition according to any one of Inventions 1052 to 1067 and 1069 to 1070, wherein the second Fc region contains the substitutions K326W and E333S. [Invention 1072] A composition according to any one of inventions 1052 - 1067 and 1069 - 1070, wherein the second Fc region comprises the substitutions K326A and E333A. [Invention 1073] A composition according to any one of inventions 1052 - 1067 and 1069, wherein the second Fc region comprises the substitution E333S. [Invention 1074] A composition according to any one of inventions 1052, 1054 - 1067, wherein the second Fc region comprises the substitutions G237A and E333S. [Invention 1075] A composition according to any one of inventions 1051 - 1074, wherein both the first and second antigens are molecules exposed on the cell surface. [Invention 1076] A composition according to any one of inventions 1051 - 1075, wherein the first and second antigens are not identical. [Invention 1077] A composition according to any one of inventions 1051 - 1076, wherein the first antibody and the second antibody are present in the composition at a molar ratio of about 1:50 - 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, 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, 2:1. [Invention 1078] A composition according to any one of aspects 1051 - 1077 of the present invention, wherein the first antibody and the second antibody are present in the composition at a molar ratio of about 1:50 to 50:1, such as a molar ratio of 1:40 to 40:1, such as a molar ratio of about 1:30 to 30:1, such as a molar ratio of about 1:20 to 20:1, such as a molar ratio of about 1:10 to 10:1, such as a molar ratio of about 1:9 to 9:1, such as a molar ratio of about 1:5 to 5:1. [Aspect 1079 of the present invention] A composition according to any one of aspects 1051 - 1078 of the present invention, wherein the first antibody and the second antibody are present in the composition at a molar ratio of 1:1. [Aspect 1080 of the present invention] A composition according to any one of aspects 1051 - 1079 of the present invention, further comprising a pharmaceutical carrier or excipient. [Aspect 1081 of the present invention] A composition according to any one of aspects 1051 - 1080 of the present invention, which is a pharmaceutical composition. [Aspect 1082 of the present invention] Any composition of the present invention for use as a medicament. [Aspect 1083 of the present invention] The first or second antibody according to any one of aspects 1001 - 1036 of the present invention, the antibody according to any one of aspects 1037 - 1050 of the present invention, or the composition according to any one of aspects 1051 - 1082 of the present invention, wherein the antigen - binding region can bind to an antigen selected from the group consisting of DR4, DR5, CD20, CD37, CD52, HLA - DR, CD3, CD5, 4 - 1BB, PD1. [Aspect 1084 of the present invention] 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:196, and the CDR3 sequence shown in SEQ ID NO:198, and a VL region 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 [DR4]; (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 as 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 as RTS, and the CDR3 sequence shown in SEQ ID NO:62 [DR5-05]; (d) 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 as DAS, and the CDR3 sequence shown 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 as DAS, and the CDR3 sequence shown in SEQ ID NO:14 [CD20, 11B8]; (f) 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 as VAT, and the CDR3 sequence shown 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 as NTN, and the CDR3 sequence shown in SEQ ID NO:7 [CD52, CAMPATH-1H]; (h) 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 as LVS, and the CDR3 sequence shown 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 as 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 as DNN, and the CDR3 sequence shown in SEQ ID NO:180 [HLA-DR, 1D09C3]; (k) A VH region comprising the CDR1 sequence shown in SEQ ID NO:182, the CDR2 sequence shown in SEQ ID NO:183, and the CDR3 sequence shown in SEQ ID NO:184, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:186, the CDR2 sequence shown as DTS, and the CDR3 sequence shown 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] A first or second antibody of any of the present inventions 1001 to 1036 and 1083, an antibody of any of the present inventions 1037 to 1050 and 1083, or a composition of any of the present inventions 1051 to 1083, which contains the above. [The present invention 1085] A method for treating an individual having a disease, comprising the step of administering to the individual an effective amount of a first and second antibody of any of the present inventions 1001 to 1037 and 1083 to 1084, an antibody of any of the present inventions 1037 to 1050 and 1083 to 1084, or a composition of any of the present inventions 1051 to 1084. [The present invention 1086] The method of the present invention 1085, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. [The present invention 1087] Any of the methods of the present inventions 1085 to 1086, which further comprises the step of administering an additional therapeutic agent. [The present invention 1088] A method for depleting a cell population expressing a first antigen and a second antigen, comprising the step of contacting the cell population with a first and second antibody, antibody, or composition of any of the present inventions 1001 to 1084. [The present invention 1089] The method of the present invention 1088, wherein the cell population is a tumor cell population such as a hematological 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, comprising the step of contacting the cell population with a first and second antibody, antibody, or composition of any of the present inventions 1001 to 1084. [The present invention 1091] Any of the methods of the present invention 1088 - 1090, wherein the cell population is present in the blood. [The present invention 1092] Any of the methods of the present invention 1088 - 1091, wherein the cell population is a leukocyte. [The present invention 1093] Any of the methods of the present invention 1088 - 1092, wherein the cell population is a subset of the leukocyte cell population. [The present invention 1094] Any of the methods of the present invention 1088 - 1093, wherein the cell population is a lymphocyte cell population. [The present invention 1095] The method of the present invention 1094, wherein the cell population is a B cell population, a T cell population, an NK cell population, a regulatory T cell population, or a myeloid-derived suppressor cell population. [The present invention 1096] Any of the methods of the present invention 1085 - 1095, wherein the first and / or second antigen is a member of TNFR-SF. [The present invention 1097] A kit comprising a first container containing a first antibody as defined in any of the present invention 1001 - 1015, 1026 - 1036, and 1083 - 1084, and a second container containing a second antibody as defined in any of the present invention 1001 - 1006, 1016 - 1036, and 1083 - 1084. These and other aspects of the present invention, specifically, various uses and therapeutic applications of the first and second antibodies, are described in further detail below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0044] Detailed Description of the Invention In the description of the embodiments of the present invention, specific technical terms are used for clarity. However, the present invention is not intended to be limited to the specific terms so selected, and it should be understood that each specific term includes all technical equivalents that function in a similar manner to achieve a similar purpose.

[0045] Definitions The term "parent antibody" is understood to be an antibody that is identical to the antibody according to the present invention, except that it does not have a C1q binding modulating substitution according to the present invention. Thus, the parent antibody may have an Fc-Fc enhancing substitution and a self-oligomerization inhibiting substitution. The term "C1q binding modulating substitution" is understood to be a substitution that can inhibit C1q binding, such as 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, 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 Fc region and a binding region of an immunoglobulin" refers, in the context of the present invention, to a polypeptide comprising an Fc region of an immunoglobulin and a binding region having the ability to bind to any molecule present on, for example, a cell, bacterium, or virion, such as a polypeptide. The Fc region of an immunoglobulin is typically an antibody fragment that can be generated after digestion of an antibody with papain (which is known to those skilled in the art), and is defined as an antibody fragment comprising two CH2-CH3 regions of the immunoglobulin and a linking region, such as a hinge region. The antibody isotype, for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE, is defined by the constant domain of the antibody heavy chain. The Fc region mediates the effector functions of the antibody together with cell surface receptors called Fc receptors and proteins of the complement system. The binding region can be a polypeptide sequence, such as a protein, protein ligand, receptor, antigen-binding region, or ligand-binding region having the ability to bind to a cell, bacterium, or virion. When the binding region is, for example, a receptor, the "polypeptide comprising an Fc region and a binding region of an immunoglobulin" may be prepared as a fusion protein of the Fc region of an immunoglobulin and the binding region. When the binding region is an antigen-binding region, the "polypeptide comprising an Fc region and a binding region of an immunoglobulin" can be an antibody such as a chimeric, humanized, or human antibody or an antibody of only the heavy chain or a ScFv-Fc fusion. The polypeptide comprising an Fc region and a binding region of an immunoglobulin can typically comprise a linking region, such as a hinge region, and two CH2-CH3 regions of the heavy chain of the immunoglobulin, and thus the "polypeptide comprising an Fc region and a binding region of an immunoglobulin" can be a "polypeptide comprising at least an Fc region and a binding region of an immunoglobulin". The term "Fc region of an immunoglobulin" means, in the context of the present invention, that a linking region, such as a hinge and CH2 and CH3 regions, corresponding to the antibody subtype of an immunoglobulin, for example, human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE, is present. The polypeptide is not limited to human origin and can be of any origin, such as mouse or cynomolgus monkey origin.As used herein, the term "wild-type Fc region" means an immunoglobulin Fc region having the amino acid sequence as it occurs in nature.

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

[0048] As used herein, the term "CH2 region" or "CH2 domain" 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 the EU numbering. However, the CH2 region may also be of any other subtype described herein.

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

[0050] The term "immunoglobulin" refers to a structurally related class of glycoproteins consisting of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four of which are strongly interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. Heavy chains are interconnected by disulfide bonds in the so-called "hinge region". Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions are regions of hypervariability (or hypervariable regions, which can be hypervariable in the form of loops defined by sequence and / or structure) and are also referred to as complementarity determining regions (CDRs), and can be further subdivided into intervening, more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise specified or inconsistent with the context, 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 the Internet http address www.imgt.org / ).Unless otherwise specified or inconsistent with the context, references to amino acid positions within the Fc region / Fc domain in this specification 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, having the ability to specifically bind to an antigen. The antibodies of the present invention include the Fc domain of an immunoglobulin and an antigen-binding region. Antibodies generally include two CH2-CH3 regions and a linking region, such as a hinge region, and at least the Fc domain. Thus, the antibodies of the present invention can be those that include an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule include binding domains that interact with an antigen. The constant or "Fc" region of an antibody can mediate the binding of the immunoglobulin to host tissues or elements, such as various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. Also, the antibody can be a multispecific antibody, such as a bispecific antibody or a similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes can be present on the same target or on different targets. When the epitopes are present on different targets, such targets can be present on the same cell or on different cells or cell types. As described above, unless otherwise specified or clearly inconsistent with the context, the term "antibody" in this specification includes antibody fragments that 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 methods. It has been shown that the antigen-binding function of an antibody can also be exerted by fragments of a full-length antibody. Examples of binding fragments included within the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies consisting only of two heavy chains, such as those naturally occurring in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche,WO2011069104), an asymmetric chain exchange modified operation domain (SEED or Seed-body), and a bispecific antibody-like molecule (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), dual variable domain immunoglobulin (Abbott, DVD-Ig, US Patent No. 7,612,181); double domain double head antibody (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone / Eli Lilly), antibody format by Knobs-into-holes (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), dual targeting domain antibody (GSK / Domantis), Two-in-one Antibodies or Dual action Fab that recognize two targets (Genentech,NovImmune, Adimab), bridged Mab (Karmanos Cancer Center), covalently linked fusion mAb (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecificity (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), TIG-body, DIG-body and PIG-body (Pharmabcine), bispecific affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybody (Zyngenia), approach using a common light chain (Crucell / Merus, US7262028) or approach using a common heavy chain (κλBody by NovImmune, WO2012023053), as well as fusion proteins containing polypeptide sequences fused to antibody fragments containing an Fc region, such as scFv fusions, such as BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and ZymoGenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusions by Genetech / Roche, scFv fusions by Novartis, scFv fusions by Immunomedics, scFv fusions by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb by f-Star, 2(WO2008 / 003116), as well as bispecific scFv fusions. Also, the term "antibody" includes, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), for example, antibody mixtures (recombinant polyclonal antibodies) (Oligoclonics) obtained by the techniques utilized by Symphogen and Merus, multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides such as chimeric antibodies and humanized antibodies. The antibodies produced can potentially have any isotype.

[0052] The term "full-length antibody", as used herein, refers to an antibody (e.g., a parental antibody) that includes all of the constant and variable domains corresponding 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 present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-directed mutagenesis in vitro or somatic mutagenesis in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are 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 includes a foreign variable domain (derived from a non-human species or modified from any species including human, synthetic, or otherwise) linked to a constant region of human origin.

[0055] As used herein, the term "humanized antibody" refers to an antibody in which both chain types have been humanized as a result of antibody modification procedures. A humanized chain typically has a complementarity-determining region (CDR) of the variable domain that is foreign (of non-human origin or synthetic), while the remainder of the chain is of human origin. Since the evaluation of humanization is based on the resulting amino acid sequence rather than the methodology itself, it is possible to use protocols other than grafting.

[0056] As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", etc. refer to preparations of Ab molecules of single-molecule composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an Ab having a single binding specificity and having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas, which are transgenic or chromosomally introduced non-human animals, such as transgenic mice, having a genome rearranged to produce functional human antibodies and containing B cells immortalized and fused with cells obtained from a transgenic mouse containing a repertoire of human heavy chain transgenes and a repertoire of light chain transgenes.

[0057] As used herein, the term "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotype thereof, such as IgG1m(za) and IgG1m(f)). Further, each heavy chain isotype can be associated with either a kappa (κ) or lambda (λ) light chain. As used herein, the term "hybrid isotype" refers to the Fc region of an immunoglobulin obtained by combining a structural entity of one isotype with a similar region derived from another isotype, thereby generating a hybrid isotype. The hybrid isotype may include an Fc region having an array composed of two or more isotypes selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, whereby combinations such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA can be generated, for example.

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

[0059] As used herein, the term "target" refers to the molecule to which the antigen-binding region of an antibody binds. Targets include any antigen to which the generated antibody is directed. The terms "antigen" and "target" are used interchangeably in relation to an antibody and can constitute the same meaning and purpose for any aspect or embodiment of the present invention.

[0060] The term "epitope" means a molecular determinant having specific binding ability to an antibody variable domain. An epitope usually consists of a group of surface molecules, such as amino acids, sugar side chains, or combinations thereof, and usually has specific three-dimensional structural features as well as specific charge features. Conformational epitopes and non-conformational epitopes are distinguished in that the binding to the former is lost in the presence of a denaturing solvent while the binding to the latter is not lost. An epitope may include amino acid residues 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] The "antibody" or "antibody variant" or "variant of a parental antibody" of the present invention is an antibody molecule containing one or more mutations as compared to the "parental antibody". These different terms are used interchangeably and may constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary parental 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 constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Amino acid substitutions can be such that a natural amino acid is replaced with another naturally occurring amino acid or an amino acid derivative not naturally occurring. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions can be defined by substitutions within one or more of the three following tables of amino acid classes.

[0062] Classes of Amino Acid Residues for Conservative Substitutions TIFF2025085008000001.tif51132

[0063] Alternative Classes of Conservative Amino Acid Residue Substitutions TIFF2025085008000002.tif38132

[0064] Alternative Physical and Functional Classifications of Amino Acid Residues TIFF2025085008000003.tif68136

[0065] In the context of the present invention, substitutions in variants are indicated as original amino acid - position - substituted amino acid and shown as; amino acid residues are denoted using the three - letter or one - letter code including code Xaa and X. Thus, the notation "E345R" or "Glu345Arg" means that the variant contains a substitution of the amino acid at position 345 of the parental antibody with arginine for glutamic acid at the corresponding amino acid position in the variant.

[0066] The position itself does not exist in the antibody, but when the variant contains an amino acid insertion, for example, the notation "position - substituted amino acid", for example "448E" is used.

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

[0068] Similarly, when the identity of one or more substituted amino acid residues is not important, the original amino acid - position, i.e., denoted as "E345".

[0069] When one or more original amino acids and / or one or more substituted amino acids may contain modifications that include more than one but not all amino acids, and glutamic acid at position 345 becomes arginine, lysine, or tryptophan, "Glu345Arg,Lys,Trp" or "E345R,K,W" or "E345R / K / W" or "E345 to R, K or W" 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 natural amino acids, or with other amino acids such as non-natural amino acids. For example, substitution of the amino acid E at position 345 includes each of 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 denoted as E345A, E345C, etc., or E345A, C, etc., as well as E345A / C / , etc. The same applies 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 immune cells involved in the effector phase of an immune response, as opposed to the recognition or activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, such as cytotoxic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcR) or complement receptors and exhibit specific immune functions. In some embodiments, effector cells such as natural killer cells have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcR 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 antigen. In some embodiments, ADCC is further enhanced by antibody-driven classical complement activation, which can result in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CR), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CR on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. Such C3 cleavage products can directly promote complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcR or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, the expression of FcγRI has been shown to be upregulated by interferon γ (IFN γ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose and lyse target cells. In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. Such C3 cleavage products can directly or indirectly promote phagocytosis by effector cells, either directly or by enhancing antibody-mediated phagocytosis.

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

[0073] As used herein, the term "clustering-dependent function" is intended to refer to a function that results from the formation of an antigen complex after oligomerization of a polypeptide or antibody that optionally binds to an antigen on a cell, on the cell membrane, on a virion, or on another particle. Examples of clustering-dependent effector functions include: (i) antibody oligomer formation, (ii) antibody oligomer stability, (iii) antigen oligomer formation, (iv) antigen oligomer stability, (v) induction of apoptosis, (vi) regulation of proliferation, e.g., reduction, inhibition, or stimulation of proliferation, and (vii) any combination of (i) to (vi).

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

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

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

[0077] As used herein, the term "vector" is intended to refer to a nucleic acid molecule having the ability to induce transcription of a nucleic acid segment ligated therein. An example of a type of vector is a "plasmid", which is in the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which case the nucleic acid segment can be ligated into the viral genome. Some specific vectors have the ability to self-replicate in the host cell into which the vector has been introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) may be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome. Furthermore, some specific vectors have the ability to direct the expression of a gene operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most common form of use of vectors, "plasmid" and "vector" may be used interchangeably herein. However, the present invention is intended to encompass such other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[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 a particular target cell but also to the progeny of such cell. Because such progeny may be modified by either mutation or environmental influences in subsequent generations, they may not in fact be identical to the parental cell, but they are still included within the scope of the term "host cell" as used herein. Examples of recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphoid cells, as well as prokaryotic cells, such as Escherichia coli, and other eukaryotic hosts, such as plant cells and fungi.

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

[0080] The term "preparation", as used herein, refers to a preparation of antibody variants and mixtures of different antibody variants that can have an increased ability to form oligomers when interacting with an antigen associated with a cell, cell membrane, virion, or other structure (e.g., an antigen expressed on the cell surface), and can result in antigen-enhanced signal transduction and / or activation.

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

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

[0083] As used herein, the term "oligomer" refers to a molecule (e.g., an antibody) consisting of more than one but a limited number of monomer units, as opposed to a polymer consisting of, at least in principle, an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. In many cases, Greek prefixes are used to denote the number of monomer units within an oligomer; for example, a tetramer consists of 4 units and a hexamer consists of 6 units.

[0084] The term "oligomerization", as used herein, shall refer to the process of converting monomers to a finite degree of polymerization. In the present specification, it has been observed that an antibody comprising a target-binding region according to the present invention can form an oligomer such as a hexamer, for example, on the cell surface, after target binding, via non-covalent association of the Fc region. For the purposes of the present application, "self-oligomerization" or "homo-oligomerization" shall refer to the process of oligomerization between antibody molecules having the same protein sequence, disregarding post-translational modifications. The term "hetero-oligomerization", as used herein, shall refer to the process of oligomerization between antibody molecules having different protein sequences, disregarding post-translational modifications. The different antibodies involved in hetero-oligomerization can bind to different antigens such as different target proteins, glycoproteins, glycans, or glycolipids.

[0085] The term "self-oligomerization inhibition substitution" refers to a substitution in an antibody comprising an Fc region and an antigen-binding region of an immunoglobulin that inhibits the process of oligomerization between antibody molecules having the same protein sequence, disregarding post-translational modifications. Inhibition of self-oligomerization can result in, for example, an increase in the EC50 of the CDC activity of the polypeptide or a decrease in the maximum CDC lysis activity, as measured by the methods described in Examples 2 and 15. Examples of self-oligomerization inhibition 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 by non-covalent interactions.

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

[0088] Fc-Fc enhancement substitution, as used herein, refers to a substitution at position E430, E345, or S440 corresponding to human IgG1 according to EU numbering, provided that the substitution at position S440 is S440Y or S440W. Thus, Fc-Fc enhancement 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 enhancement substitution is E430G, E345K, or E345R.

[0089] With respect to two antigens, as used herein, the term "co-existing" or grammatical variations thereof shall, on the one hand, refer to a situation where the two antigens are co-expressed in the same cell. The antigens may already be in proximity to each other on the cell, or the antigens may come into proximity via oligomerization of the binding polypeptides of the present invention, such as antibodies. Further, the term "co-existing" shall also refer to a situation where the two antigens are expressed in different cells, but such cells are located extremely close to each other.

[0090] The term "co-dependency", as used herein, shall refer to a functional effect that is dependent on the simultaneous binding of two or more different Fc domain-containing polypeptides, including self-oligomerization inhibitory substitutions, to the same target, cell, or virion. With respect to the present invention, the functional effects that can be co-dependent include functions that are dependent on clustering, effector functions mediated by Fc, and binding of effector molecules such as FcγR or C1, but do not necessarily include the individual binding of the Fc domain-containing polypeptide to the target antigen. As used herein, different Fc domain-containing polypeptides including self-oligomerization inhibitory substitutions can each bind individually to various targets, cells, or virions, but the functional outcome of co-dependency is dependent on the simultaneous binding of two or more different components to the same target, cell, or virion. As used herein, the functional effect of co-dependency is specifically restored by two or more different Fc domain-containing polypeptides including self-oligomerization inhibitory substitutions by the restoration of non-covalent Fc-Fc interactions between different components in a co-dependent Fc-containing polypeptide mixture.

[0091] The term "safety margin", as used herein, shall refer to an index of the efficacy and safety of a drug, and is defined as the range between the minimum therapeutic dose (efficacy against diseased tissue) and the minimum toxic dose (efficacy against healthy tissue) of the drug.

[0092] As used herein, the term "C1q binding" shall refer to the direct interaction between C1q and an antibody. Direct C1q binding can be evaluated, for example, by using an antibody immobilized on an artificial surface. Multivalent interactions that result in high avidity binding of C1q to an antibody oligomer can be evaluated when bound to a given antigen on the cell surface or on the virion surface.

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

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

[0095] As used herein, the term "complement activation" refers to the activation of the classical complement pathway initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on a surface. C1 is a complex consisting of six recognition proteins C1q and a heterotetramer of serine proteases C1r2C1s2. C1 is the first protein complex in the initial events of the classical complement cascade, which involves a series of cleavage reactions starting with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b deposits and together with C2a forms an enzymatic active convertase called C3 convertase, whereby complement component C3 is cleaved into C3b and C3a and a C5 convertase is formed. This C5 convertase splits C5 into C5a and C5b, and the final components deposit on the membrane, thereby inducing later events of complement activation in which the terminal complement components C5b, C6, C7, C8, and C9 are assembled into a membrane attack complex (MAC). The complement cascade results in the generation of pores in the cell membrane that cause cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be evaluated by using C1q efficacy, CDC kinetics CDC assays (such as those described in WO2013 / 004842, WO2014 / 108198), or by the cellular deposition of C3b and C4b, a method 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 the lysis of a cell or virion as a result of pores in the membrane created by the MAC construct when the antibody is bound to a target on the cell or virion.

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

[0098] As used herein, the term "complement-dependent cytotoxicity" ("CDCC") is intended to refer to the mechanism of killing of target cells or virions by cells expressing a complement receptor that recognizes the cleavage products of complement component 3 (C3) covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC can be evaluated in a manner similar to that described for ADCC.

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

[0100] As used herein, the term "plasma clearance rate" is a quantitative measure of the rate at which a polypeptide is removed from the blood when administered to 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] As used herein, the term "antibody-drug conjugate" refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that couples the drug, for example, to the antibody. The linker is cleavable or non-cleavable in the presence of malignant cells; in this case, the antibody-drug conjugate kills the malignant cells.

[0102] As used herein, the term "antibody-drug conjugate uptake" refers to the process by which, after an antibody-drug conjugate binds to a target on a cell, it is taken up / sequestered by the cell membrane and thereby drawn into the cell. Antibody-drug conjugate uptake can be evaluated as described in "antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay" as described in WO 2011 / 157741.

[0103] As used herein, the term "apoptosis" refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events can result in characteristic changes (morphological structures) and death of the cells. Such changes include cytoplasmic blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and fragmentation of chromosomal DNA. Apoptosis can be induced by the binding of an antibody to a specific receptor.

[0104] As used herein, the term "programmed cell death" or "PCD" refers to any form of cell death mediated by an intracellular program. There are different forms of PCD, and it is common to all types of PCD that they are executed by active cellular processes that can be disrupted if intracellular signaling is interfered with. In certain embodiments, the occurrence of any form of PCD in a cell or tissue can be examined by staining the cell or tissue with conjugated annexin V and correlating it with phosphatidylserine exposure.

[0105] As used herein, the term "annexin V" refers to a group of annexin proteins that bind to phosphatidylserine (PS) on the cell surface.

[0106] As used herein, the term "FcRn" is intended to refer to the neonatal Fc receptor, which is an Fc receptor. This was first found in rodents as a unique receptor with the ability to transport IgG from mother's milk through the intestinal epithelium of neonatal rodents into the bloodstream of the neonate. Further studies have revealed a similar receptor in humans. However, in humans, this receptor is found in the placenta, which aids in facilitating the transport of maternal IgG to the growing fetus, and has also been shown to play a role in monitoring IgG catabolism. FcRn binds to IgG at an acidic pH of 6.0 - 6.5 but not at neutral or higher pH. Thus, FcRn can bind to IgG from the slightly acidic intestinal lumen (inside the intestine) and ensure efficient unidirectional transport to the basolateral side (inside the body) where the pH is neutral to basic (pH 7.0 - 7.5). This receptor also plays a role in adult IgG recycling by being present within the endocytosis pathway in endothelial cells. The FcRn receptor within the acidic endosome binds to IgG that has undergone internalization by endocytosis, allows it to be recycled to the cell surface, and releases it into the bloodstream at basic pH, thereby suppressing its degradation by lysosomes. This mechanism may explain why the half-life of IgG in the blood is longer compared to other isotypes.

[0107] The term "Protein A", as used herein, is intended to refer to the 56 kDa MSCRAMM surface protein first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found utility in biochemical research due to its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a 3-helix bundle. Each domain has the ability to bind proteins from many mammalian species, most notably IgG. It binds to the Fc region of the heavy chain of most immunoglobulins (overlapping the conserved binding site for the FcRn receptor), and also interacts with the Fab region of the human VH3 family. Due to such interactions in serum, IgG molecules bind to bacteria by their Fc regions rather than their Fab regions alone, thereby disrupting opsonization, complement activation, and phagocytosis by this bacterium.

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

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

[0110] As shown by the inventors, a first antibody having an Fc region with one Fc - Fc enhancing substitution, one or more substitutions that reduce effector functions such as CDC and / or ADCC, and a complementary self - oligomerization inhibiting substitution, when used as a single antibody, does not show CDC activity or shows only extremely 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 functions such as CDC and / or ADCC, and a complementary self - oligomerization inhibiting substitution, when used as a single antibody, shows only limited CDC activity. A single agent does not show or shows a limited ability to induce CDC. However, 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 medicament 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 Fc region comprises (a) 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, and (b) a substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, the substitution is not S440K, and (c) 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; wherein the second Fc region comprises (d) 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, and (e) a substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, the substitution is not S440K; wherein the first Fc region has a substitution of K439E and the second Fc region has a substitution of S440K, or the first Fc region has a substitution of S440K and the second Fc region has a substitution of K439E; and the amino acid positions correspond to human IgG1 according to the Eu numbering system, and provides a first antibody for use as a medicament in combination with a second antibody.

[0112] In another aspect, the present invention is 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 medicament 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 Fc region comprises (a) 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, and (b) a substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) 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; wherein the second Fc region comprises (d) 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, and (e) a substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) when the first Fc region comprises a substitution of K322E or P329R, one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S; either the first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; the 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), provides a first antibody for use as a medicament in combination with a second antibody.

[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 medicament 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 Fc region (a) comprises 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, and (b) comprises a K439E or S440K substitution, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) comprises 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; wherein the second Fc region (d) comprises 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K; The first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E; the amino acid positions correspond to human IgG1 according to the Eu numbering system, Provided is a first antibody for use as a medicament in combination with a second antibody.

[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 medicament 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 is (a) Comprising 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, and (b) Comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and (c) Comprising one substitution of an amino acid at position P329, or the substitution of K322E; The second Fc region is (d) Comprising 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, and (e) Comprising the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and (f) Comprising one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, either the first Fc region has a K439E substitution and the second Fc region has an S440K substitution, or the first Fc region has an S440K substitution and the second Fc region has a K439E substitution; the amino acid positions corresponding to human IgG1 according to the EU numbering system, Provided is a first antibody for use as a medicament in combination with a second antibody.

[0115] Substitutions at positions corresponding to E430, E345, or substitutions of S440Y or S440W are considered Fc-Fc enhancing substitutions according to the present invention, and such substitutions introduce the effects of enhanced Fc-Fc interaction and oligomerization into the polypeptide or antibody. Enhanced oligomerization occurs when the antigen-binding region of the antibody binds to the corresponding target antigen. Enhanced oligomerization generates an oligomer, such as a hexamer. Generation of an oligomer structure such as a hexamer has the effect of increasing Fc effector functions, such as CDC, by increasing the C1q binding avidity of the polypeptide.

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

[0117] In one aspect of the present invention, the first and second Fc regions comprise substitutions selected from the group consisting of E430G, E345K, and E345R.

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

[0119] The first and second Fc regions further comprise substitutions of K439E or S440K, which are considered complementary oligomerization-inhibiting substitutions according to the present invention. That is, for example, a first antibody having K439E cannot form an oligomer with another antibody having the substitution of K439E, but an antibody having the substitution of K439E can form an oligomer with another antibody having the substitution of S440K. An antibody having the substitution of S440K cannot form an oligomer with another antibody having the substitution of S440K, but can form an oligomer with an antibody having the substitution of K439E. Thus, in one aspect of the present invention, the first Fc region comprises the substitution of K439E and the second Fc region comprises the substitution of S440K. In one aspect of the present invention, the first Fc region comprises the substitution of S440K and the second Fc region comprises the substitution of K439E. When the Fc region comprises S440K which is an oligomerization-inhibiting substitution, the Fc region cannot comprise S440Y or S440W which are Fc-Fc enhancing substitutions. Thus, an Fc region having S440K which is an oligomerization-inhibiting substitution 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 medicament 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 Fc region (a) comprises the substitutions of K248E and T437R, and (b) comprises the substitution of K439E or S440K, and (c) comprises one 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, and L235E; and the second Fc region (d) comprises the substitutions of K248E and T437R, and (e) comprising the substitutions K439E or S440K, wherein the first Fc region has the substitution K439E and the second Fc region has the substitution S440K, or the first Fc region has the substitution S440K and the second Fc region has the substitution K439E; and the amino acid positions correspond to human IgG1 according to the Eu numbering system, There is provided a first antibody for use as a medicament in combination with a second antibody.

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

[0122] In one embodiment of the present invention, the first Fc region comprises substitutions that reduce effector functions such as CDC and / or ADCC. Thus, in one embodiment of the present invention, the first Fc region comprises substitutions that reduce CDC. In one embodiment of the present invention, the first Fc region comprises substitutions that reduce ADCC. In one embodiment of the present invention, the first Fc region comprises substitutions that reduce both CDC and ADCC. In one embodiment of the present invention, the first Fc region comprises one 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. Thereby, in addition to the reduction of CDC activity, Fcγ receptor binding is also reduced, and thus, there is provided an embodiment in which the first Fc region comprises a substitution that can also reduce effector functions mediated by Fcγ receptors.

[0123] In one embodiment of the present 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 inventors have found that by 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, the ability of the antibody to induce CDC is decreased. Therefore, substitution of G237 with another natural amino acid is thought to decrease the ability of the antibody to induce CDC. Substitutions at amino acid position G237 can decrease the ability of antibodies with Fc-Fc enhancing substitutions and self-oligomerization inhibiting substitutions to induce CDC alone.

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

[0126] In one aspect of the invention, the first Fc region comprises one or more substitutions selected from the group consisting of G236R, G236K, E269K, and P329R. In one aspect of the invention, the first Fc region comprises the substitutions L234F and L235E. Thus, there is provided an aspect in which the first Fc region comprises substitutions that, in addition to decreasing CDC activity, also decrease Fcγ receptor binding and thereby decrease effector functions mediated by the Fcγ receptor.

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

[0128] In one aspect of the present invention, the first Fc region comprises one substitution selected from the group consisting of K322A and K322E. Thus, an aspect is provided 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 the Fcγ receptor.

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

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

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

[0132] In one aspect 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 aspect of the present invention, the first Fc region comprises the substitution of P329R. In one aspect of the present invention, the first Fc region comprises the substitution of P329R. In one aspect of the present invention, the first Fc region comprises the substitution of P329K. In one aspect of the present invention, the first Fc region comprises the substitution of P329E. In one aspect of the present invention, the first Fc region comprises the substitution of P329D. In one aspect of the present invention, the first Fc region comprises the substitution of P329A.

[0133] There is provided an embodiment in which a first antibody comprises a first Fc region having an Fc-Fc enhancing substitution that introduces the effect of enhanced Fc-Fc interaction and oligomerization of the antibody. Enhanced oligomerization occurs when the antigen-binding region of the antibody binds to the corresponding target antigen. Enhanced oligomerization produces an oligomer such as a hexamer, for example. The production of an oligomer structure such as a hexamer has the effect of increasing Fc effector functions, such as CDC, by increasing the C1q-binding avidity of the polypeptide. However, by introducing a self-oligomerization inhibiting substitution and a substitution that reduces effector functions such as CDC and / or ADCC, an antibody having reduced effector functions such as CDC and / or ADCC is produced, which may enable improved modulation of the toxicity profile of the antibody in combination with a second antibody. That the first antibody has reduced effector functions such as CDC and / or ADCC is understood as when the first antibody is compared with a parental antibody having the same Fc-Fc enhancing substitution and self-oligomerization inhibiting substitution but no substitution that reduces effector functions.

[0134] In one embodiment of the invention, the second Fc region comprises a substitution that increases effector functions such as CDC and / or ADCC. Thus, in one embodiment, the second Fc region comprises a substitution that increases CDC. In one embodiment, the second Fc region comprises a substitution that increases ADCC. In one embodiment, the second Fc region comprises a substitution that increases both CDC and ADCC. That the second antibody has increased effector functions such as CDC and / or ADCC is understood as when the second antibody is compared with a parental antibody having the same Fc-Fc enhancing substitution and self-oligomerization inhibiting substitution but no substitution that enhances effector functions.

[0135] In one aspect of the present 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 aspect of the present invention, the second Fc region comprises the substitution of G237A. In one aspect of the present invention, the second Fc region comprises a substitution that reduces Fcγ receptor binding, such as G237A.

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

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

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

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

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

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

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

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

[0145] Thus, there is provided an aspect in which the second antibody comprises a second Fc region having an Fc-Fc enhancing substitution that introduces an enhanced Fc-Fc interaction and oligomerization effect of the antibody. The enhanced oligomerization occurs when the antigen-binding region of the antibody binds to the corresponding target antigen. The enhanced oligomerization produces an oligomer, such as a hexamer. The production of an oligomer structure such as a hexamer has the effect of increasing the Fc effector function, such as CDC, by increasing the C1q binding avidity of the polypeptide. However, by introducing substitutions that increase effector functions such as CDC and / or ADCC, antibodies are produced that have increased oligomerization and increased effector functions such as CDC and / or ADCC, which may enable improved efficacy of the antibody in combination with the first antibody. That the second antibody has reduced effector functions such as CDC and / or ADCC is understood as being compared to a parental antibody that has the same Fc-Fc enhancing substitution and self-oligomerization inhibiting substitution but does not have a substitution that increases the effector function.

[0146] The following table provides a non-limiting list of aspects that describe combinations of a first polypeptide and a second polypeptide that include specific substitutions. Thus, for example, aspect 1 of the following table is a combination of a first antibody that includes substitutions at positions corresponding to E430G, K439E, and G236R of human IgG, respectively, with a second antibody that includes the substitutions E430G and S440K of human IgG, respectively. As described herein, all of the first and second antibodies of aspects 1-177 may optionally include additional substitutions.

[0147] TIFF2025085008000004.tif115129TIFF2025085008000005.tif214129TIFF2025085008000006.tif214129TIFF2025085008000007.tif214129TIFF2025085008000008.tif214129TIFF2025085008000009.tif214129TIFF2025085008000010.tif214129TIFF2025085008000011.tif66129

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

[0149] TIFF2025085008000012.tif87128TIFF2025085008000013.tif206120TIFF2025085008000014.tif42128

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

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

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

[0153] Aspects described herein with respect to antibodies refer to antibodies that comprise an Fc region of an immunoglobulin and an antigen-binding region, and the antibody may be a multispecific antibody such as a bispecific antibody having a second polypeptide or antibody having a first Fc region and a first antigen-binding region of an immunoglobulin, and a second Fc region and a second antigen-binding region of an immunoglobulin.

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

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

[0156] In one aspect of the present 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 a preferred aspect, 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 aspect of the present invention, the antibody, or the first and / or the second antibody, comprises a first and / or a second constant region comprising a sequence selected from Table 1.

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

[0160] In one aspect of the present invention, the first and / or the second antibody comprises a first and / or a 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 the second heavy chain sequences are independently selected from the group.

[0161] In one aspect 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 up to 5 additional substitutions, such as up to 4 additional substitutions, such as up to 3 additional substitutions, such as up to 2 additional substitutions, such as up to 1 additional substitution, have been introduced.

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

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

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

[0165] In one aspect of the present invention, the first and second antibodies deplete a cell population expressing the first and second antigens.

[0166] In one aspect of the present invention, the cell population is a tumor cell.

[0167] In one aspect of the present invention, the cell population is a hematological tumor cell or a solid tumor cell.

[0168] In one aspect of the present invention, the cell population is a leukocyte such as a leukocyte cell population.

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

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

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

[0172] In one aspect of the present invention, the cell population is an NK cell. In one aspect of the present invention, the cell population is a myeloid-derived suppressor cell. In one aspect of the present invention, the cell population is a tumor-associated macrophage.

[0173] As a medicament for depleting a specific cell population expressing a first and a second antigen recognized by a first and a second antibody, an embodiment in which the first antibody and the second antibody according to the present invention are used is described herein. 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 may be particularly useful in the depletion of specific subsets of lymphocytes, such as specific cell populations of the immune system such as B cells or T cells or even subsets of B cells or subsets of 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] Antibodies having a natural, e.g., human Fc domain may be antibodies having mutations other than the mutations of the present invention, such as mutations that affect glycosylation or that allow the antibody to be a bispecific antibody. The term "natural antibody" means an antibody that does not contain genetically introduced mutations. Thus, naturally occurring modifications, e.g., antibodies containing different allotypes, should be understood as "natural antibodies" in the sense of the present invention and may thus be understood as parental antibodies. Natural antibodies can serve as templates for at least 3 substitutions in the first antibody according to the present invention or at least 2 substitutions in the second antibody to provide the first and second antibodies of the present invention. Examples of parental antibodies containing substitutions other than the mutations of the present invention are bispecific antibodies such as those described in WO2011 / 131746 (Genmab) that utilize reducing conditions to facilitate the exchange of halves of two antibody molecules containing an IgG4-like CH3 region, thus forming bispecific antibodies without aggregates being formed simultaneously. Other examples of parental antibodies include 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), but are not limited thereto. Other exemplary parental antibody formats include, without limitation, 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 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 is comprising 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, and comprising the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, 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.

[0177] In another aspect, the antibody comprises an antigen-binding region capable of binding to an antigen and the Fc region of human IgG, wherein the Fc region comprises the substitutions of K248E and T437R, and comprises the substitution of K439E or S440K, and comprises one 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 present 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 present invention, the Fc region comprises a substitution selected from the group consisting of E430G, E345K, and E345R.

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

[0181] In one aspect of the present 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 aspect of the present 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 aspect of the present invention, the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R. In one aspect of the present invention, the Fc region comprises the substitution G237Q.

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

[0185] In one aspect of the present invention, the Fc region comprises the substitution G236R.

[0186] In one aspect of the present invention, the Fc region comprises the substitution E269K.

[0187] In one aspect of the present invention, the Fc region comprises the substitution K322A.

[0188] Targets and methods of use The first and / or second antibodies according to the present invention can bind to a target expressed in the same cell. In one aspect, the target is a target that activates, inhibits, modulates, and / or controls a signaling 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 according to the present invention: tumor necrosis factor receptor superfamily, GPI-anchored proteins, lipid-added proteins, hydrolases (EC 3.), and regulatory factor superfamily, B7 family-related proteins, immunoglobulin superfamily, interleukin receptor family, integrins, Ig-like cell adhesion molecule family, receptor-type protein tyrosine phosphatases, C-type lectins, tetraspanins, transmembrane 4 domain, activated leukocyte immunoglobulin-like receptor, C-C motif chemokine receptor, G protein-coupled receptor, toll-like receptor, receptor-type tyrosine kinase. In one aspect of the present invention, the first and second antigen-binding regions can bind to target antigens from the same protein class. In one aspect of the present invention, the first and second antigen-binding regions can bind to target antigens from different protein classes. In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of GPI-anchored proteins, and the second antigen-binding region can bind to a target antigen from the protein class of tetraspanins. In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of tetraspanins, and the second antigen-binding region can bind to a target antigen from the protein class of GPI-anchored proteins. In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of GPI-anchored proteins, and the second antigen-binding region can bind to a target antigen from the protein class of transmembrane 4 domain. In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of transmembrane 4 domain, and the second antigen-binding region can bind to a target antigen from the protein class of GPI-anchored proteins.

[0191] In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of the tumor necrosis factor receptor superfamily, and the second antigen-binding region can bind to a target antigen from the protein class of the tumor necrosis factor receptor superfamily.

[0192] In one aspect of the present invention, the first antigen-binding region can bind to a target antigen from the protein class of the tumor necrosis factor receptor superfamily, and the second antigen-binding region can bind to a target antigen from the protein class of the immunoglobulin superfamily.

[0193] Cell surface receptors include receptors belonging to receptor families such as, for example, the hematopoietic factor receptor family, the cytokine receptor family, the tyrosine kinase receptor family, the serine / threonine kinase receptor family, the TNF receptor family, the G protein-coupled receptor family, the GPI anchor receptor family, the tyrosine phosphatase receptor family, the adhesion factor family, and the hormone receptor family. Various references regarding the receptors belonging to these receptor families and their characteristics are available, for example, Cooke B A., King R J B., van der Molen H J.ed.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 aspect 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 aspect of the invention, the first antibody and / or the second antibody binds to a cell surface receptor, which includes, for example, hormone receptors and cytokine receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation regulatory factor receptors, and the like. Examples of cytokine receptors are 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, 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, stem cell factor (SCF) receptor, vascular endothelial 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, leukemia 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 the ability to bind ligands of the tumor necrosis factor superfamily (TNFSF) via an extracellular cysteine-rich domain. TNF receptors form trimeric complexes in the cell membrane. The TNFRSF includes the following list of 29 proteins; TNFR1 (Uniprot P19438), FAS (Uniprot P25445), DR3 (Uniprot Q93038), DR4 (Uniprot O00220), DR5 (Uniprot O14763), DR6 (Uniprot O75509), NGFR (Uniprot P08138), EDAR (Uniprot Q9UNE0), DcR1 (Uniprot O14798), DcR2 (Uniprot Q9UBN6), DcR3 (Uniprot O95407), OPG (Uniprot O00300), TROY (Uniprot Q9NS68), XEDAR (Uniprot Q9HAV5), LTbR (Uniprot P36941), HVEM (Uniprot Q92956), TWEAKR (Uniprot Q9NP84), CD120b (Uniprot P20333), OX40 (Uniprot P43489), CD40 (Uniprot P25942), CD27 (Uniprot P26842), CD30 (Uniprot P28908), 4-1BB (Uniprot Q07011), RANK (Uniprot Q9Y6Q6), TACI (Uniprot O14836), BLySR (Uniprot Q96RJ3), BCMA (Uniprot Q02223), GITR (Uniprot Q9Y5U5), RELT (Uniprot Q969Z4).

[0197] In one aspect 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 aspect, the antigen-binding region can bind to DR4. In one aspect, the antigen-binding region can bind to DR5. In one aspect, the antigen-binding region can bind to CD20. In one aspect, the antigen-binding region can bind to CD37. In one aspect, the antigen-binding region can bind to CD52. In one aspect, the antigen-binding region can bind to HLA-DR. In one aspect, the antigen-binding region can bind to CD3. In one aspect, the antigen-binding region can bind to CD5.

[0198] In one aspect of the present invention, the antibody, or the first and / or second antibody, a VH region comprising the CDR1 sequence shown in SEQ ID NO:196, the CDR2 sequence shown in SEQ ID NO:196, and the CDR3 sequence shown in SEQ ID NO:198, and a VL region 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 [DR4]; 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 as 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 as 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 as 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 as 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 as 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 shown in SEQ ID NO:182, the CDR2 sequence shown in SEQ ID NO:183, and the CDR3 sequence shown in SEQ ID NO:184, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:186, the CDR2 sequence shown in DTS, and the CDR3 sequence shown 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] Comprising an antigen-binding region containing the same.

[0199] In one aspect, the present 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 is 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, wherein the first Fc region comprises 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, and comprises the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and 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; wherein the second Fc region comprises 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, and comprises the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and when the first Fc region comprises the substitution of K322E or P329R, comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, wherein the first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, corresponding to human IgG1 according to the EU numbering system, 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 comprises 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 comprises the substitution K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and 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 comprises 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 comprises the substitution K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K; either the first Fc region has the substitution K439E and the second Fc region has the substitution S440K, or the first Fc region has the substitution S440K and the second Fc region has the substitution K439E, corresponding to human IgG1 according to the EU numbering system for amino acid positions, and provides a composition.

[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 comprises 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, and comprises the substitution of K439E or S440K, provided that when the substitution in (a) is S440Y or S440W, it is not S440K, and comprises one substitution of an amino acid at position P329, or the substitution of K322E; The second Fc region comprises 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, and comprises the substitution of K439E or S440K, provided that when the substitution in (d) is S440Y or S440W, it is not S440K, and comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S, either the first Fc region has the substitution of K439E and the second Fc region has the substitution of S440K, or the first Fc region has the substitution of S440K and the second Fc region has the substitution of K439E, where the amino acid positions correspond to human IgG1 according to the EU numbering system, A composition is provided.

[0203] In one aspect 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 aspect 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 aspect of the present invention, the composition comprising the first and second Fc regions comprises the substitution E430G.

[0206] In one aspect of the present invention, the composition comprising 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.

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

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

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

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

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

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

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

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

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

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

[0217] In one aspect of the present invention, the composition comprising the 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 aspect of the present invention, the composition comprising the first Fc region comprises one substitution selected from the group consisting of P329R, P329K, P329E, P329D, and P329A.

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

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

[0221] In one aspect of the present invention, the composition comprising the first Fc region comprises the substitution E269K.

[0222] In one embodiment of the present invention, the composition comprising the 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 present invention, the composition comprising the 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 present invention, the composition comprising the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S.

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

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

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

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

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

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

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

[0232] In one aspect of the present invention, a composition comprising a first antibody and a second antibody is present in the composition at a molar ratio of 1:50 to 50:1, such as 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, a molar ratio of 1:40, a molar ratio of 1:45, a molar ratio of 1:50, a molar ratio of 50:1, a molar ratio of 45:1, a molar ratio of 40:1, a molar ratio of 35:1, a molar ratio of 30:1, a molar ratio of 25:1, a molar ratio of 20:1, a molar ratio of 15:1, a molar ratio of 10:1, a molar ratio of 9:1, a molar ratio of 8:1, a molar ratio of 7:1, a molar ratio of 6:1, a molar ratio of 5:1, a molar ratio of 4:1, a molar ratio of 3:1, a molar ratio of 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, such as, for example, a molar ratio of about 1:40 to 40:1, such as, for example, a molar ratio of about 1:30 to 30:1, such as, for example, a molar ratio of about 1:20 to 20:1, such as, for example, a molar ratio of about 1:10 to 10:1, such as, for example, a molar ratio of about 1:9 to 9:1, such as, for example, a molar ratio of about 1:5 to 5:1.

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

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

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

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

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

[0239] In one aspect of the present invention, the cell population is a leukocyte such as a leukocyte cell population.

[0240] In one aspect of the present invention, the cell population is a subset of the leukocyte cell population.

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

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

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

[0244] In one aspect of the present invention, the cell population is an NK cell population.

[0245] In one aspect of the present invention, the cell population is a myeloid-derived suppressor cell.

[0246] Therapeutic uses The first and second antibodies, bispecific antibodies, or compositions according to any aspect or embodiment of the present invention can be used as a medicament, i.e., for therapeutic use.

[0247] In one aspect, the present 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 present invention provides an antibody or composition according to any aspect or embodiment disclosed herein for use in the treatment of cancer, autoimmune disease, inflammatory disease, or infectious disease.

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

[0250] In an aspect, the present invention is 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 embodiment of the present invention, the disease is selected from the group consisting of cancer, autoimmune disease, inflammatory disease, and infectious disease.

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

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

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

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

[0256] The present invention also relates to a parts kit for simultaneous, separate, or sequential use in therapy, comprising the first and second antibodies described herein. Furthermore, such first and second antibodies can be obtained according to any method described herein.

[0257] In one aspect, the present invention is a parts kit comprising an antibody or composition according to any aspect or embodiment described herein, wherein the first and second antibodies or compositions are present in one or more containers, for example vials.

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

[0259] In another aspect, the invention relates to the use of the first and second antibodies, compositions, or part kits according to any of the embodiments described herein for use in a diagnostic method.

[0260] In another aspect, the invention relates to a diagnostic method comprising administering to at least a part of the body of a human or other mammal the first and second antibodies, compositions, or part kits according to any of the embodiments described herein.

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

[0262] In another aspect, the invention relates to a method for imaging at least a part of the body of a human or other mammal, comprising administering the first and second antibodies, compositions, or part kits according to any of the embodiments described herein.

[0263] Further uses It is understood that the embodiments described below with respect to the first and second antibodies or antibody refer to the first and second antibodies or antibody each comprising an Fc region and an antigen-binding region of an immunoglobulin.

[0264] In a further aspect, the invention relates to the first and second antibodies of the invention as described above for use as a medicament, in particular for use as a medicament for the treatment of a disease or disorder. Examples of such diseases and disorders include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, infections by bacteria, viruses, or fungi.

[0265] In another aspect, the invention relates to the first and second antibodies, bispecific antibodies, compositions, and part kits described herein for the treatment of a disease, such as cancer.

[0266] In another aspect, the present invention relates to a method for treating human diseases, including administration of the first and second antibodies, compositions, or part kits described herein.

[0267] In another aspect, the present invention relates to a method for treating human cancer, including administration of the first and second antibodies, compositions, or part kits.

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

[0269] "Effective amount" or "therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result at the required dosage and period. The 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 induce a desired response in the individual. Also, the therapeutically effective amount is an amount in which the therapeutic beneficial effect outweighs any toxic or adverse effects of the antibody or a portion of the antibody.

[0270] Dosage The embodiments described below with respect to the antibody are understood to refer to an antibody comprising an Fc region and an antigen-binding region of an immunoglobulin, and 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 depend on the disease or condition to be treated and can be determined by those skilled in the art. Exemplary and non-limiting ranges of the therapeutically effective amount of the antibody of the present invention are from about 0.1 to 100 mg / kg, such as from about 0.1 to 50 mg / kg, such as from about 0.1 to 20 mg / kg, such as from about 0.1 to 10 mg / kg, such as about 0.5, such as about 0.3, about 1, about 3, about 5 or about 8 mg / kg.

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

[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 radiotherapy and / or surgery.

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

[0275] The present invention also provides an isolated nucleic acid and vector encoding an antibody according to any one of the above aspects, as well as a vector and expression system encoding the antibody. Nucleic acid constructs, vectors, and expression systems suitable for antibodies and their variants are known in the art and are described in this example. In aspects where the variant antibody includes not only the heavy chain (or its Fc-containing fragment) but also the light chain, the nucleotide sequences encoding the heavy chain portion and the light chain portion may be present in the same nucleic acid or vector, or in different nucleic acids or vectors.

[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 the Fc region of the heavy chain, and the following steps: a) preparing a nucleotide construct encoding the Fc region of the variant; b) expressing the nucleotide construct in a host cell, and c) recovering the antibody variant from the cell culture of the host cell also provides a method comprising.

[0277] In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody also includes a light chain, and thus the host cell further expresses a light chain coding construct on a vector that is either the same or different.

[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 occur, such as a eukaryotic cell, such as a mammalian cell, such as a human cell. In a further embodiment, the host cell is a non-human cell genetically modified to produce a glycoprotein having human-like glycosylation or human-type glycosylation. Examples of such cells are genetically modified methylotrophic yeast (Pichia pastoris) (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325) and genetically modified duckweed (Lemna minor) (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

[0279] In one aspect, the host cell is a host cell that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists several such antibody production systems, such as Sp2 / 0, NS / 0, or the mammary gland of transgenic animals (goats), in which only partial C-terminal lysine removal is obtained. In one aspect, the host cell is a host cell having a modified glycosylation mechanism. Such cells have been reported in the art and can be used as host cells for expressing the variants of the present invention and thereby producing antibodies having modified glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03 / 035835; and WO99 / 54342. Further methods for generating modified glycoforms are known in the art and include, without limitation, those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation modification technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

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

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

[0282] The present invention is further illustrated by the following examples which should not be construed as further limitations.

[0283] (Table 1) Sequence Listing TIFF2025085008000015.tif207149TIFF2025085008000016.tif216149TIFF2025085008000017.tif214149TIFF2025085008000018.tif212149TIFF2025085008000019.tif216149TIFF2025085008000020.tif217149TIFF2025085008000021.tif213149TIFF2025085008000022.tif211149TIFF2025085008000023.tif211149TIFF2025085008000024.tif207149TIFF2025085008000025.tif220149TIFF2025085008000026.tif217149TIFF2025085008000027.tif216149TIFF2025085008000028.tif216149TIFF2025085008000029.tif216149TIFF2025085008000030.tif205149TIFF2025085008000031.tif220149TIFF2025085008000032.tif222149TIFF2025085008000033.tif216149TIFF2025085008000034.tif216149TIFF2025085008000035.tif216149TIFF2025085008000036.tif210149TIFF2025085008000037.tif222149TIFF2025085008000038.tif216149TIFF2025085008000039.tif216149TIFF2025085008000040.tif216149TIFF2025085008000041.tif210149TIFF2025085008000042.tif217149TIFF2025085008000043.tif216149TIFF2025085008000044.tif216149TIFF2025085008000045.tif210149TIFF2025085008000046.tif215149TIFF2025085008000047.tif217149TIFF2025085008000048.tif216149TIFF2025085008000049.tif212149TIFF2025085008000050.tif213149TIFF2025085008000051.tif212149TIFF2025085008000052.tif216149TIFF2025085008000053.tif216149TIFF2025085008000054.tif171149.

[0284] Table defining the substitutions tested in Examples 5 - 23 TIFF2025085008000055.tif198163

[0285] Table defining self - oligomerization inhibitory substitutions TIFF2025085008000056.tif17128

[0286] Table defining FcγR binding inhibitory and C1q binding inhibitory substitutions TIFF2025085008000057.tif77163

Example

[0287] Example 1 Antibody expression construct For the expression of human antibodies and humanized antibodies used in this specification, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific) and cloned into the pcDNA3.3 expression vector (ThermoFisher Scientific) containing the constant region of the human IgG heavy chain (constant region human IgG1m(f)HC: SEQ ID NO 22; constant region human IgG2 HC: SEQ ID NO 31; constant region human IgG3 HC: SEQ ID NO 32; or constant region human IgG4 HC: SEQ ID NO 33) and / or the constant region 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 (WO2004 / 035607; VH: SEQ ID NO 8; VL: SEQ ID NO 12) derived from the previously described CD20 antibody IgG1-CD20-11B8. The CD52 antibody variants in this application have VH and VL sequences (Crowe et al., 1992 Clin Exp Immunol. 87(1):105-110; VH: SEQ ID NO 1; VL: SEQ ID NO 5) derived from the previously described CD52 antibody CAMPATH-1H and VH and VL sequences (US2014 / 0127236; VH: SEQ ID NO 160; VL: SEQ ID NO 164) derived from the previously described CD52 antibody h2E8. The CD37 antibody variants in this application have VH and VL sequences (WO2011 / 112978; VH: SEQ ID NO 42; VL: SEQ ID NO 46) derived from the previously described CD37 antibody IgG1-CD37-37.3. The DR4 antibody variants in this application have VH and VL sequences (US2009 / 0136503A1; VH: SEQ ID NO 200; VL: SEQ ID NO 204) derived from the previously described DR4 antibody DR4-chCTB007.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 (WO2014 / 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 VH and VL sequences derived from 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, which is 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] Transient expression antibody construct A plasmid DNA mixture encoding both the heavy and light chains of the antibody was 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). The antibody concentration in the supernatant was measured by absorbance at 280 nm. The supernatant containing the antibody was used directly in the in vitro assay or the antibody was purified as described below.

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

[0290] Example 2: Selectivity of CDC activity by a mixed antibody variant by introduction of the P329R mutation into anti-CD52 IgG1-CAMPATH-1H-K439E + anti-CD20 IgG1-11B8-S440K with 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. Purified antibodies at different concentrations (final concentration range 0.001 - 60.0 μg / mL) were tested in an in vitro CDC assay against Wien 133 cells using 20% NHS. Different mutations were introduced into antibodies IgG1-11B8 and IgG1-CAMPATH-1H: E430G (which induces enhanced Fc-Fc interaction); P329R (which inhibits direct C1q binding to the antibody); and either mutation K439E or S440K (which inhibits the formation of homohexameric antibody complexes through inhibition of intermolecular Fc-Fc interaction and promotes the formation of heterohexameric antibody complexes through cross-complementary Fc-Fc interaction). As a control, single antibodies were also mixed 1:1 with the non-binding isotype control antibody IgG1-b12 or IgG1-b12-E430G so that a direct comparison of the concentrations of the individual components and the mixtures composed of them was possible. For the CDC assay, 0.1×10 in RPMI (Lonza, Cat No. BE12-115F) containing 0.2% bovine serum albumin (BSA; Roche, Cat No. 10735086001) 6Individual Wien 133 cells (kindly provided by Dr. Geoff Hale (BioAnaLab Limited, Oxford, UK)) were pre-incubated for 15 minutes at RT on a shaker in a polystyrene round-bottom 96-well plate (Greiner bio-one Cat # 650101) with a concentration series of purified antibodies and a total volume of 80 μL. Next, 20 μL of normal human serum (NHS; Sanquin, Reference No. M0008) was added as a complement source and incubated for 45 minutes in an incubator at 37 °C (final NHS concentration 20%; antibody final concentration 0.001 - 10.0 μg / mL at 3-fold dilution). After stopping the reaction by placing the plate on ice, the cells were pelleted by centrifugation and the supernatant was replaced by exchanging it with 20 μL of a 2 μg / mL propidium iodide solution (PI; Sigma Aldrich, Cat No. P4170). The number of PI-positive cells was determined by flow cytometry using an Intellicyt iQue screener (Westburg), and the lysis rate was calculated as (number of PI-positive cells / total number of cells) × 100%. The data were analyzed using the best-fit values of non-linear dose-response fitting using log-transformed concentrations in GraphPad PRISM, and the area under the dose-response curve for three experimental replicates was calculated. The relative area under the curve (AUC) values represent normalization against minimal lysis (0% with IgG1-b12) and maximal lysis (100% with a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G).

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

[0292] Figure 2 shows the selectivity of CDC activity by the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K upon introduction of the K322E mutation. Wiens 133 cells were incubated with an antibody concentration series in the presence of 20% NHS. CDC efficacy was presented by normalizing the lysis rate determined by the percentage of PI-positive cells and (B) the AUC to the unbound 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. The in vitro CDC assay was performed using 30 μg / mL of the antibody in the presence of 20% NHS with Burkitt lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C) and Ramos (D), acute lymphoblastic leukemia (ALL) cell line REH (E), multiple myeloma cell line U266B1 (F), and B cell lymphoma cell line U-698-M (G). The CDC activity is presented as the lysis rate determined by the percentage of PI-positive cells, normalized for each cell line against the unbound 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 variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K having C1q binding inhibitory mutations (G236R, K322A, E269K, K322E or P329R). Wien 133 cells were incubated with an antibody concentration series in the presence of 20% NHS. The CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. The normalization was performed against the unbound 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 a mixed antibody variant of anti-CD20 IgG1-11B8-E430G-S440K with anti-CD52 IgG1-CAMPATH-1H-E430G-K439E+ a C1q-binding enhancing mutation (E333S, K326W or K326A / E333A). Wien 133 cells were incubated with a series of antibody concentrations 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 non-binding control antibody mix IgG1-b12-K439E+IgG1-b12-S440K (0%) and a mixture of IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0296] Figure 6 shows the selectivity of CDC activity by a mixed antibody variant of anti-CD20 IgG1-11B8-E430G-S440K with anti-CD52 IgG1-CAMPATH-1H-E430G-K439E+ a C1q-binding enhancing mutation (E333S, K326W or K326A / E333A) and a C1q-binding inhibitory mutation G236R (A), K322A (B), E269K (C), K322E (D) or P329R (E). Wien 133 cells were incubated with a series of antibody concentrations 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 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 antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q-binding inhibitory mutations G236R or K322A (A, B) and anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding enhancing mutation E333S (C, D) to human lymphoma cell lines Wien 133 (A, C) and Raji (B, D). Antibody binding was tested by flow cytometry. Binding is represented as the geometric mean of fluorescence intensity (MFI). As a negative control for binding, samples without primary antibody or non-binding anti-gp120 antibody IgG1-b12 were used.

[0298] Figure 8 shows the ADCC ability of antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q-binding inhibitory mutations G236R or K322A (A, C) and anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding enhancing mutation E333S (B, D). (A, B) An ADCC reporter bioassay was performed in which Raji target cells were co-incubated with an 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) An in vitro europium TDA (EuTDA) ADCC assay was performed in which Wien 133 target cells were co-incubated with an antibody dilution series and human PBMC (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 a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-K439E with enhanced Fc-Fc interaction having a C1q binding inhibitory mutation G236R or K322A and anti-CD20 IgG1-11B8-S440K with enhanced Fc-Fc interaction with or without the C1q binding enhancing mutation E333S. The Fc-Fc interaction enhancing mutations tested were E430G, E345K, E345R, and E345R-E430G. Wien 133 cells were incubated with a series of antibody concentrations in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC and maximum lysis of the percentage of PI-positive cells. 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 a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with a C1q binding regulatory mutation or double mutation G237A-K322A at position G236 (G236R or G236K) or G237 (G237A, G237T, G237Q, or G237R) and anti-CD20 IgG1-11B8-E430G-S440K with or without the C1q binding regulatory mutations E333S, E333A, K326A, K326W-E333S, G237A, or G237A-E333S. Wien 133 cells were incubated with a series of antibody concentrations in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC and maximum lysis of the percentage of PI-positive cells. 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-CD20 11B8-E430G-S440K with or without the C1q-binding inhibitory mutation G236R and with or without the C1q-binding enhancing mutation E333S of anti-CD52 CAMPATH-1H-E430G-K439E. Wien 133 cells were incubated with a series of antibody concentrations 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 + IgG1-11B8-E430G (100%).

[0302] Figure 12 shows the selectivity of CDC activity against (A) Daudi cells and (B) Wien 133 cells by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without the C1q-binding inhibitory 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 series of antibody concentrations 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 + 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 inhibitory mutation G236R and IgG1-DR5-05-E430G-S440K with or without the C1q-binding enhancing mutation E333S against BxPC-3 human pancreatic cancer cells. A 3-day survival assay was performed, and cell viability was determined using the CellTiter-Glo kit.

[0304] Figure 14 shows the CDC activity by antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G having C1q-binding regulatory mutations G237A, G236R, A327K, K322E or P329R. Wien 133 cells were incubated with antibody concentration series 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 non-binding control antibodies IgG1-b12-S440K (0%; not shown) and IgG1-CAMPATH-1H-E430G (100%).

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

[0306] Figure 16 shows the clearance rate of 500 μg of antibody administered intravenously in SCID mice. (A - C) Total human IgG in plasma samples was determined by ELISA and plotted against concentration-time curves for (A) IgG1-CAMPATH-1H variants, (B) IgG1-11B8 variants, and (C) a combination of IgG1-CAMPATH-1H variants + IgG1-11B8 variants. Each data point represents the mean + / - standard deviation of triplicate samples. (D) Clearance up to day 21 after antibody administration was determined according to the formula D*1,000 / AUC (where D is the injection 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 inhibitory mutations G236R or G237T and IgG1-11B8-E430G-S440K variants with C1q-binding enhancing mutations K326A or E333S to the 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) as tested by ELISA assay. Binding is presented relative to an antibody-free control (background) and binding to IgG1-11B8-E430G-S440K (100%) for a 20 μg / mL antibody sample. Detection was performed using streptavidin-poly HRP and ABTS.

[0308] Figure 18 shows the selectivity of CDC activity by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without C1q-binding inhibitory mutations (G236R or G237T) and anti-CD20 IgG1-11B8-E430G-S440K with or without C1q-binding enhancing mutation E333S and with or without FcγR-binding inhibitory mutation G237A. Wien 133 cells were incubated with antibody concentration series 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 non-binding control antibody IgG1-b12 (0%) and a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0309] Figure 19 shows the selective activity of combinations of variants of IgG1-CAMPATH-1H and IgG1-11B8 in whole blood determined by flow cytometric 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 an IgG1-CAMPATH-1H-E430G-K439E variant containing an additional G237 mutation, mixed with IgG1-11B8-G237A-E430G-S440K. (C) Selective activity of an IgG1-CAMPATH-1H-E430G-K439E variant 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 a co-dependent antibody combination of IgG1-CAMPATH-1H-E430G-K439E with additional mutations G236R, G237Q or G237R mixed with IgG1-11B8-G237A-E430G-S440K. Y-axis: Log scale representation of the B cell fraction determined as described 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 having a C1q binding inhibitory mutation in the anti-CD52 component and a C1q binding enhancing mutation in the anti-CD20 component. The in vitro CDC assay was performed using antibodies at 0.01 - 40 μg / mL in the presence of 20% NHS, using Burkitt lymphoma cell lines Daudi (A), Raji (B) and Ramos (C), ALL cell line REH (D), and B cell lymphoma cell line U-698-M (E). CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells and as maximum lysis. Normalization was performed against unbound 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 mixed antibody variants of anti-CD37 IgG1-CD37-37.3-E430G-K439E with or without the C1q binding inhibitory 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 antibody concentration series 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 unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37.3-E430G + IgG1-11B8-E430G (100%).

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

[0313] Figure 23 shows the binding of antibody variants of anti-CD20 IgG1-11B8 having the Fc-Fc interaction enhancing mutation E430G and the self-oligomerization inhibiting mutation S440K, in combination with either the C1q binding regulatory mutation 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 presented normalized to the Bmax value (100%) of wild-type IgG1-11B8. As a negative control for binding, the non-binding anti-gp120 antibody IgG1-b12 was used. (C, D) The maximum binding (Bmax) to Raji cells by IgG1-11B8 antibody variants having the mutations E430G and S440K, in combination with either the C1q binding regulatory mutation K326A or E333A (C) or E333S, G237A or G237A-E333S (D), is shown normalized to the binding of wild-type IgG1-11B8. (E, F) The apparent Kd values of IgG1-11B8 antibody variants having the mutations E430G and S440K, in combination with either the C1q binding regulatory mutation K326A or E333A (E) or E333S, G237A or G237A-E333S (F), that bind to Raji cells.

[0314] Figure 24 shows the FcRn binding of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibody variants. (A, C) Binding to human FcRn of variants of 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 anti-CD20 antibody IgG1-11B8 with 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 a coating of 2 μg / mL of the recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and a series of antibody dilutions. 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 the total human IgG (hIgG) concentration as measured in blood samples collected from mice injected with anti-CD52 IgG1-CAMPATH-1H or anti-IgG1-11B8 antibody variants or mixtures thereof. (A) Total hIgG concentration 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 concentration 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 concentration 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 bearing the mutations as in (A) and (B). In all panels, the dotted line represents the predicted IgG1 concentration over time for wild-type IgG1 antibody in SCID mice. (D) Clearance up to day 21 after antibody administration was determined according to the formula D*1000 / AUC, where D is the injection dose and AUC is the area under the concentration-time curve.

[0316] Figure 26 shows the concentration (in μg / ml) of C4d detected in samples incubated with antibody variants of IgG1-CAMPATH-1H, IgG1-11B8 and IgG1-b12 bearing the mutations E430G, K439E or S440K and G236R, G237A, G237Q or G237R, after subtraction of the mean C4d concentration detected in antibody-free negative control samples. Positive control samples include antibody variants bearing the Fc-Fc interaction enhancing mutations E345R, E430G and S440Y (RGY).

[0317] Figure 27 shows the C1q binding to Wien 133 cells incubated on ice with normal human serum as a complement source, 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, detected by flow cytometry. Mean fluorescence intensity values were normalized to the antibody-free control reaction (0%) and the highest level (100%) of the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G, as estimated by fitting a logarithmic agonist response model. (A) C1q binding of the control reaction. (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 unbound control IgG1-b12 or different IgG1-11B8 variants.

[0318] Figure 28 shows the 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 of (A) high-affinity allotype FcγRIIA 131H, (B) low-affinity allotype FcγRIIA 131R, (C) FcγRIIB, (D) high-affinity allotype FcγRIIIA 158V, or (E) low-affinity allotype FcγRIIIA 158F, as tested in an ELISA assay. (F) Binding of immobilized FcγRIa to the antibody variants tested in ELISA. Binding is presented for 20 μg / mL antibody samples and was normalized for each experiment to the mean signal (100%) observed for wild-type IgG1-CAMPATH-1H, after subtraction of the signal in wells incubated without primary antibody. Detection was performed using streptavidin-poly HRP 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 by introduction of mutations that enhance Fc-Fc interaction, inhibit self-oligomerization, and regulate C1q binding. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy was measured in three independent experiments and presented as (A) the mean AUC normalized to non-binding control antibodies IgG1-b12 (0%) and a 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] Figures 30 (A, B) show 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 self-oligomerization-inhibiting mutations and C1q-binding-regulating mutations as indicated. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibodies IgG1-b12 (0%) and a 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 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 for each experiment to the luminescence values observed for IgG1-b12 (0%) and the luminescence values observed for wild-type IgG1-Campath-1H + wild-type IgG1-11B8 (100%) and then averaged over three (FcγRIIa) or two (FcγRIIIa) experimental replicates.

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

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

[0324] Figure 34 shows the selective co-dependent CDC activity of a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 by introduction of mutations that enhance Fc-Fc interaction, inhibit self-oligomerization, and inhibit C1q binding. CDC efficacy is shown for variants of IgG1-CAMPATH-1H-E430G-K439E having 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 the AUC normalized against the non-binding control antibody IgG1-b12 (0%) and 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 introduction of mutations that enhance Fc - Fc interaction, inhibit self - oligomerization, and inhibit C1q binding. (A) CDC efficacy as a single agent or as a mixture of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345K, E345Q, E345R or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (B) CDC efficacy as a single agent or as a mixture of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (C) CDC efficacy as a single agent or as a mixture of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the matching Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (D) CDC efficacy as a single agent or as a mixture of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G or K248E - T437R, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - modulating mutations G236R, G237A or E333S. CDC efficacy is presented as the AUC normalized to the non - binding control antibodies IgG1 - b12 (0%) and the mixture of IgG1 - CAMPATH - 1H - E430G+IgG1 - 11B8 - E430G (100%).

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

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

[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 antibody variants of IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-CD5-INSERM that carry mutations that enhance Fc-Fc interaction, 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. The fraction was calculated as [100%×(number of cells in sample / number of cells in "sample without antibody")×(number of granulocytes in "sample without antibody" / 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 that carry mutations that enhance Fc-Fc interaction, inhibit self-oligomerization, and inhibit (G237T) or enhance (K326W-E333S) C1q binding. (A) Survival rate of BxPC-3 human pancreatic cancer cells after 72-hour incubation with the indicated antibody variants. (B) Survival rate of COLO 205 human colon cancer cells after 72-hour 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] A, which was set to 100% in FIG. 1, shows the selectivity of CDC activity by a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K by introduction of the P329R mutation. Wien 133 cells were incubated with a concentration antibody concentration series in the presence of 20% pooled normal human serum (NHS). The CDC efficacy was presented by normalizing the lysis rate determined by the percentage of propidium iodide (PI)-positive cells (A) and the area under the dose-response curve (AUC) (B) to the unbound control antibody IgG1-b12 (0%) and the mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0331] FIG. 2 shows the selectivity of CDC activity by a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K by introduction of the K322E mutation. Wien 133 cells were incubated with an antibody concentration series in the presence of 20% NHS. The CDC efficacy was presented by normalizing the lysis rate determined by the percentage of PI-positive cells (A) and the AUC (B) to the unbound 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 introduction of the K322E mutation into the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20-11B8-E430G-S440K. The in vitro CDC assay was performed using 30 μg / mL of the antibody in the presence of 20% NHS with Burkitt lymphoma cell lines Wien 133 (A), Daudi (B), Raji (C) and Ramos (D), acute lymphoblastic leukemia (ALL) cell line REH (E), multiple myeloma cell line U266B1 (F), and B cell lymphoma cell line U-698-M (G). The CDC activity is presented as the lysis rate determined by the percentage of PI-positive cells, normalized for each cell line against the non-binding control antibody IgG1-b12 (0%) and for REH, U266B1 and Wien 133 IgG1-CAMPATH-1H-E430G (100%) or for Daudi, Raji, Ramos and U-698-M IgG1-11B8-E430G (100%).

[0333] Figure 4 shows the selectivity of CDC activity by the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K having a C1q binding inhibitory mutation (G236R, K322A, E269K, K322E or P329R). Wien 133 cells were incubated with a series of antibody concentrations in the presence of 20% NHS. The CDC efficacy is presented as the normalized AUC of the percentage of PI-positive cells. The 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 a mixed antibody variant of anti-CD20 IgG1-11B8-E430G-S440K having anti-CD52 IgG1-CAMPATH-1H-E430G-K439E+C1q binding enhancing mutations (E333S, K326W or K326A / E333A). Wien 133 cells were incubated with a series of antibody concentrations 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 non-binding control antibody mix IgG1-b12-K439E+IgG1-b12-S440K (0%) and a mixture of IgG1-CAMPATH-1H-E430G+IgG1-11B8-E430G (100%).

[0335] Figure 6 shows the selectivity of CDC activity by a mixed antibody variant of anti-CD20 IgG1-11B8-E430G-S440K having anti-CD52 IgG1-CAMPATH-1H-E430G-K439E+C1q binding enhancing mutations (E333S, K326W or K326A / E333A) and C1q binding inhibitory mutations G236R (A), K322A (B), E269K (C), K322E (D) or P329R (E). Wien 133 cells were incubated with a series of antibody concentrations 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 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 antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q-binding inhibitory mutations G236R or K322A (A, B) and anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding enhancing mutation E333S (C, D) to human lymphoma cell lines Wien 133 (A, C) and Raji (B, D). Antibody binding was tested by flow cytometry. Binding is represented as the geometric mean of fluorescence intensity (MFI). As a negative control for binding, samples without primary antibody or unbound anti-gp120 antibody IgG1-b12 were used.

[0337] Figure 8 shows the ADCC activity of antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with C1q-binding inhibitory mutations G236R or K322A (A, C) and anti-CD20 IgG1-11B8-E430G-S440K with C1q-binding enhancing mutation E333S (B, D). (A, B) An ADCC reporter bioassay was performed in which Raji target cells were incubated with an 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) An in vitro europium TDA (EuTDA) ADCC assay was performed in which Wien 133 target cells were incubated with an antibody dilution series and human PBMC (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 a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-K439E with enhanced Fc-Fc interaction having a C1q-binding inhibitory mutation G236R or K322A and anti-CD20 IgG1-11B8-S440K with enhanced Fc-Fc interaction with or without a C1q-binding enhancing mutation E333S. The Fc-Fc interaction enhancing mutations tested were E430G, E345K, E345R, and E345R-E430G. Wien 133 cells were incubated with an antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC and maximum lysis of the percentage of PI-positive cells. 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 a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with a C1q-binding modulating mutation or double mutation G237A-K322A at position G236 (G236R or G236K) or position G237 (G237A, G237T, G237Q, or G237R) and anti-CD20 IgG1-11B8-E430G-S440K with or without a C1q-binding modulating mutation E333S, E333A, K326A, K326W-E333S, G237A, or G237A-E333S. Wien 133 cells were incubated with an antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC and maximum lysis of the percentage of PI-positive cells. 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 anti-CD52 CAMPATH-1H-E430G-K439E with or without the C1q-binding inhibitory mutation G236R and anti-CD20 11B8-E430G-S440K with or without the C1q-binding enhancing mutation E333S, in the form of mixed antibody isotype variants (IgG1, IgG2, IgG3, and hinge-stabilized IgG4). Wien 133 cells were incubated with a series of antibody concentrations 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 + IgG1-11B8-E430G (100%).

[0341] Figure 12 shows the selectivity of CDC activity against (A) Daudi cells and (B) Wien 133 cells by a mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without the C1q-binding inhibitory 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 series of antibody concentrations 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 + IgG1-CD37-37.3-E430G (100%).

[0342] Figure 13 shows DR5-mediated cytotoxicity against BxPC-3 human pancreatic cancer cells by (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 inhibitory mutation G236R and IgG1-DR5-05-E430G-S440K with or without the C1q-binding enhancing mutation E333S. A 3-day survival assay was performed and cell viability was determined using the CellTiter-Glo kit.

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

[0344] Figure 15 shows the binding of antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E having C1q-binding inhibitory mutations G236R or K322A (A, B, C) and antibody variants of anti-CD20 IgG1-11B8-E430G-S440K having C1q-binding enhancing mutation E333S (D, E, F) to human FcRn. FcRn ELISA was performed using 5 μg / mL of the coated recombinant extracellular domain of human FcRn (FcRnhsECDHis-B2M-BIO) and a series of antibody dilutions. The amount of bound antibody was determined using an HRP-conjugated goat anti-human IgG1 antibody and the chemiluminescent substrate ABTS. The 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 against the concentration-time curve for (A) IgG1-CAMPATH-1H variants, (B) IgG1-11B8 variants, and (C) a combination of IgG1-CAMPATH-1H variants + IgG1-11B8 variants. Each data point represents the mean + / - standard deviation of triplicate samples. (D) The clearance up to day 21 after antibody administration was determined according to the formula D*1,000 / AUC, where D is the injection 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 inhibitory mutations G236R or G237T and IgG1-11B8-E430G-S440K variants with C1q-binding enhancing mutations K326A or E333S to the 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) as tested by ELISA assay. Binding is presented relative to the antibody-free control (background) and binding to IgG1-11B8-E430G-S440K (100%) for a 20 μg / mL antibody sample. Detection was performed using streptavidin-poly HRP and ABTS.

[0347] Figure 18 shows the selectivity of CDC activity by mixed antibody variants of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E with or without C1q-binding inhibitory mutations (G236R or G237T) and anti-CD20 IgG1-11B8-E430G-S440K with or without C1q-binding enhancing mutation E333S and with or without FcγR-binding inhibitory mutation G237A. Wien 133 cells were incubated with an antibody concentration series 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 a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G (100%).

[0348] Figure 19 shows the selective activity of combinations of variants of IgG1-CAMPATH-1H and IgG1-11B8 in whole blood as determined by flow cytometric 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 variant containing an additional G237 mutation, mixed with IgG1-11B8-G237A-E430G-S440K. (C) Selective activity of IgG1-CAMPATH-1H-E430G-K439E variant 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 co-dependent antibody combinations of IgG1-CAMPATH-1H-E430G-K439E with additional mutations G236R, G237Q or G237R mixed with IgG1-11B8-G237A-E430G-S440K. Y-axis: Log scale representation of the B cell fraction determined as described above.

[0349] Figure 20 shows the selectivity of CDC activity against different cell lines with different expression levels of CD20 and CD52 by a combination of IgG1-CAMPATH-1H-E430G-K439E and IgG1-11B8-E430G-S440K antibody variants having a C1q binding inhibitory mutation in the anti-CD52 component and a C1q binding enhancing mutation in the anti-CD20 component. An in vitro CDC assay was performed using antibodies at 0.01 - 40 μg / mL in the presence of 20% NHS with Burkitt lymphoma cell lines Daudi (A), Raji (B) and Ramos (C), ALL cell line REH (D), and B cell lymphoma cell line U-698-M (E). CDC efficacy is presented as the normalized AUC of the percentage of PI positive cells and as the maximum lysis. Normalization was performed against unbound 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 mixed antibody variants of anti-CD37 IgG1-CD37-37.3-E430G-K439E with or without the C1q binding inhibitory 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 an antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the normalized AUC of the percentage of PI positive cells and as the lysis at 40 μg / mL of IgG. Normalization was performed against unbound control antibody IgG1-b12 (0%) and a mixture of IgG1-CD37-37.3-E430G + IgG1-11B8-E430G (100%).

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

[0352] Figure 23 shows the binding of antibody variants of anti-CD20 IgG1-11B8 having the Fc-Fc interaction enhancing mutation E430G and the self-oligomerization inhibiting mutation S440K, in combination with either the C1q binding regulatory mutation 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 presented as normalized to the Bmax value (100%) of wild-type IgG1-11B8. As a negative control for binding, the non-binding anti-gp120 antibody IgG1-b12 was used. (C, D) The maximum binding (Bmax) of IgG1-11B8 antibody variants having the mutations E430G and S440K, in combination with either the C1q binding regulatory mutation K326A or E333A (C) or E333S, G237A or G237A-E333S (D), to Raji cells is shown normalized to the binding of wild-type IgG1-11B8. (E, F) The apparent Kd values of IgG1-11B8 antibody variants having the mutations E430G and S440K, in combination with either the C1q binding regulatory mutation K326A or E333A (E) or E333S, G237A or G237A-E333S (F), that bind to Raji cells.

[0353] Figure 24 shows the FcRn binding of anti-CD52 IgG1-CAMPATH-1H and anti-CD20 IgG1-11B8 antibody variants. (A, C) Binding to human FcRn of variants of 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 anti-CD20 antibody IgG1-11B8 with 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 a coating of 2 μg / mL of the recombinant extracellular domain of human FcRn (FcRnECDHis-B2M-BIO) and a series of antibody dilutions. 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 the total human IgG (hIgG) concentration as measured in blood samples collected from mice injected with anti-CD52 IgG1-CAMPATH-1H or anti-IgG1-11B8 antibody variants or mixtures thereof. (A) Total hIgG concentration 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 concentration 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 concentration 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 the mutations as in (A) and (B). In all panels, the dotted line represents the predicted IgG1 concentration over time for wild-type IgG1 antibody in SCID mice. (D) Clearance up to day 21 after antibody administration was determined according to the formula D*1000 / AUC, where D is the injection dose and AUC is the area under the concentration-time curve.

[0355] Figure 26 shows the concentration (in μg / ml) of C4d detected in samples incubated with antibody variants of IgG1-CAMPATH-1H, IgG1-11B8 and IgG1-b12 carrying the mutations E430G, K439E or S440K and G236R, G237A, G237Q or G237R, after subtraction of the mean C4d concentration detected in antibody-free negative control samples. Positive control samples included 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, 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, detected by flow cytometry. Mean fluorescence intensity values were normalized relative to the antibody-free control reaction (0%) and the highest level (100%) of a mixture of IgG1-CAMPATH-1H-E430G + IgG1-11B8-E430G, as estimated by fitting a logarithmic agonist response model. (A) C1q binding in the control reaction. (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 unbound 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 ECDs of (A) high-affinity allotype FcγRIIA 131H, (B) low-affinity allotype FcγRIIA 131R, (C) FcγRIIB, (D) high-affinity allotype FcγRIIIA 158V, or (E) low-affinity allotype FcγRIIIA 158F, as tested in an ELISA assay. (F) Binding of immobilized FcγRIa to the antibody variants tested in ELISA. Binding is presented for 20 μg / mL antibody samples and was normalized for each experiment relative to the mean signal (100%) observed for wild-type IgG1-CAMPATH-1H, after subtraction of the signal in wells incubated without primary antibody. Detection was performed using streptavidin-poly HRP 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 by introduction of mutations that enhance Fc-Fc interaction, inhibit self-oligomerization, and regulate C1q binding. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy was measured in three independent experiments and presented as the mean AUC normalized to non-binding control antibodies IgG1-b12 (0%) and a 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] Figures 30 (A, B) show 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 self-oligomerization-inhibiting and C1q-binding-regulating mutations as indicated. Wien 133 cells were incubated with antibody concentration series in the presence of 20% NHS. CDC efficacy is presented as the AUC normalized to non-binding control antibodies IgG1-b12 (0%) and a 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 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 for each experiment to the luminescence values observed for IgG1-b12 (0%) and the luminescence values observed for wild-type IgG1-Campath-1H + wild-type IgG1-11B8 (100%) and then averaged over three (FcγRIIa) or two (FcγRIIIa) experimental replicates.

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

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

[0363] Figure 34 shows the selective co - dependent CDC activity of a mixed antibody variant of anti - CD52 IgG1 - CAMPATH - 1H and anti - CD20 IgG1 - 11B8 by introduction of mutations that enhance Fc - Fc interaction, inhibit self - oligomerization and inhibit C1q binding. CDC efficacy is shown for variants of IgG1 - CAMPATH - 1H - E430G - K439E having 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 the AUC normalized to the non - binding control antibodies IgG1 - b12 (0%) and 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 introduction of mutations that enhance Fc - Fc interaction, inhibit self - oligomerization, and inhibit C1q binding. (A) CDC efficacy as a single agent or a mixture thereof of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345K, E345Q, E345R or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (B) CDC efficacy as a single agent or a mixture thereof of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (C) CDC efficacy as a single agent or a mixture thereof of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the matching Fc - Fc interaction - enhancing mutations E430G, E430N, E430T, E430V, E430Y, E345A, E345Q, E345V or E345Y, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - inhibiting mutations G236R or G237A. (D) CDC efficacy as a single agent or a mixture thereof of antibody variants of IgG1 - CAMPATH - 1H and IgG1 - 11B8 carrying the Fc - Fc interaction - enhancing mutations E430G or K248E - T437R, the self - oligomerization - inhibiting mutations K439E or S440K, and the C1q - binding - modulating mutations G236R, G237A or E333S. CDC efficacy is presented as the AUC normalized to the non - binding control antibodies IgG1 - b12 (0%) and the mixture of IgG1 - CAMPATH - 1H - E430G+IgG1 - 11B8 - E430G (100%).

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

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

[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 antibody variants of IgG1-CAMPATH-1H, IgG1-huCLB-T3 / 4, and IgG1-CD5-INSERM that carry mutations that enhance Fc-Fc interaction, 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. The fraction was calculated as [100% × (number of cells in sample / number of cells in "sample without antibody") × (number of granulocytes in "sample without antibody" / 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 that carry mutations that enhance Fc-Fc interaction, inhibit self-oligomerization, and inhibit (G237T) or enhance (K326W-E333S) C1q binding. (A) Survival rate of BxPC-3 human pancreatic cancer cells after incubation with the indicated antibody variants for 72 hours. (B) Survival rate of COLO 205 human colon cancer cells after incubation with the indicated antibody variants for 72 hours. 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 separate single antibodies (Figure 1 shows the selectivity of CDC activity by the mixed antibody variant of anti-CD52 IgG1-CAMPATH-1H-E430G-K439E + anti-CD20 IgG1-11B8-E430G-S440K by introduction of the P329R mutation. Wien 133 cells were incubated with a concentration antibody concentration series in the presence of 20% pooled normal human serum (NHS). CDC efficacy was presented by normalizing the lysis rate determined by the percentage of (A) propidium iodide (PI)-positive cells and (B) the area under the dose-response curve (AUC) to the unbo...

Claims

1. 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 medicament 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 is (a) comprising 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; 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 an 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) 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; (e) a substitution of K439E or S440K, except that when the substitution in (d) is S440Y or S440W, the substitution is not 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 first antibody.

2. 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 medicament 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 is (a) comprising 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; 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 P329, or a substitution of K322E; The second Fc region (d) 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; (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) 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.

3. 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 medicament 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 is (a) contains the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing an 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.

4. 3. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 or 2, 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.

5. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 2 and 4, wherein the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, and E345R.

6. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 2 and 4 to 5, wherein the first and second Fc regions comprise an E430G substitution.

7. 7. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 6, 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, G237P.

8. 8. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 7, wherein the first Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R.

9. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 8, wherein the first Fc region comprises a G237Q substitution.

10. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 6, wherein the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K.

11. 11. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1, 3-6 and 10, wherein the first Fc region comprises a G236R substitution.

12. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1, 3-6 and 10, wherein the first Fc region comprises an E269K substitution.

13. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 6, wherein the first Fc region comprises a K322A substitution.

14. 7. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 2 to 6, wherein the first Fc region comprises a P329R substitution.

15. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 2 to 6, wherein the first Fc region comprises a K322E substitution.

16. 14. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 and 3 to 13, wherein the second Fc region comprises one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S.

17. 20. The first antibody of any one of claims 1, 3-13 and 16, for use as a medicament in combination with a second antibody, wherein the second Fc region comprises a G237A substitution.

18. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 15, wherein the second Fc region comprises one or more substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

19. 20. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 15 and 18, wherein the second Fc region comprises one substitution selected from the group consisting of K326A, K326W, E333A, and E333S.

20. 18. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1, 3-13 and 16-17, wherein the second Fc region comprises the substitutions G237A and E333S.

21. 20. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 19, wherein the second Fc region comprises a K326A substitution.

22. 20. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 19, wherein the second Fc region comprises an E333S substitution.

23. 20. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 16 and 18, wherein the second Fc region comprises two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

24. 24. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 16, 18 and 23, wherein the second Fc region comprises the substitutions K326W and E333S.

25. 24. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 1 to 16, 18 and 23, wherein the second Fc region comprises the substitutions K326A and E333A.

26. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and / or second antibody is a human antibody, a humanized antibody or a chimeric antibody.

27. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and / or second antibody is a monoclonal antibody.

28. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and / or second antibody is of human IgG1, IgG2, IgG3 or IgG4 isotype.

29. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and / or second antibody is of human IgG1 isotype.

30. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and second antigens are both cell surface exposed molecules.

31. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and second antigens co-exist in a cell or tissue which is a target cell or target tissue for the disease or disorder to be treated.

32. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the first and second antigens are not identical.

33. A first antibody for use as a medicament in combination with a second antibody according to any one of the preceding claims, wherein the medicament depletes a cell population expressing the first and second antigens.

34. 34. The first antibody for use as a medicament in combination with a second antibody according to claim 33, wherein the cell population is tumor cells.

35. A first antibody for use as a medicament in combination with a second antibody according to any one of claims 33 to 34, wherein the cell population is blood tumor cells or solid tumor cells.

36. 35. The first antibody for use as a medicament in combination with a second antibody according to any one of claims 33 to 34, 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.

37. 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 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; 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 an 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.

38. 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) contains the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) comprising 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.

39. 38. The antibody of claim 37, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

40. The antibody of any one of claims 37 and 39, wherein the Fc region comprises a substitution selected from the group consisting of E430G, E345K, and E345R.

41. The antibody of any one of claims 37 and 39 to 41, wherein the Fc region comprises an E430G substitution.

42. 42. The antibody of any one of claims 37 to 41, wherein 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.

43. 43. The antibody of any one of claims 37 to 42, wherein the Fc region comprises one substitution selected from the group consisting of G237A, G237T, G237S, G237Q, G237R.

44. The antibody of any one of claims 37 to 43, wherein the Fc region comprises a G237Q or G237A substitution.

45. The antibody of any one of claims 37 to 41, wherein the Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K.

46. The antibody of any one of claims 37-41 and 45, wherein the Fc region comprises a G236R substitution.

47. The antibody of any one of claims 37 to 41 and 45, wherein the Fc region comprises an E269K substitution.

48. The antibody of any one of claims 37 to 41, wherein the Fc region comprises a K322A substitution.

49. The antibody of any one of claims 37 to 48, which is of a human IgG1, IgG2, IgG3, or IgG4 isotype.

50. 50. The antibody of any one of claims 37 to 49, which is of human IgG1 isotype.

51. 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 claims 1 to 15 and 26 to 36, and the second antibody comprises a second antigen-binding region and a second Fc region of any one of claims 1 to 6 and 16 to 36.

52. 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 is (a) comprising 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; 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 an 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) 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; (e) a substitution of K439E or S440K, except that when the substitution in (d) is S440Y or S440W, the substitution is not 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.

53. 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 is (a) comprising 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; 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 P329, or a substitution of K322E; The second Fc region (d) 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; (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) 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.

54. 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 is (a) contains the substitutions K248E and T437R; and (b) contains a substitution of K439E or S440K; and (c) containing an 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.

55. 54. The composition of any one of claims 52-53, 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.

56. 56. The composition of any one of claims 52-53 and 55, wherein the first and second Fc regions comprise a substitution selected from the group consisting of E430G, E345K, and E345R.

57. The composition of any one of claims 52-53 and 55-56, wherein the first and second Fc regions comprise an E430G substitution.

58. 58. The composition of any one of claims 52-57, 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, G237P.

59. The composition of any one of claims 52-58, wherein the first Fc region comprises a substitution selected from the group consisting of G237A, G237T, G237Q, G237R, and G237S.

60. The composition of any one of claims 52 to 59, wherein the first Fc region comprises a G237Q substitution.

61. The composition of any one of claims 52-57, wherein the first Fc region comprises one or more substitutions selected from the group consisting of G236R and E269K.

62. The composition of any one of claims 52-57 and 61, wherein the first Fc region comprises a G236R substitution.

63. The composition of any one of claims 52-57 and 61, wherein the first Fc region comprises an E269K substitution.

64. The composition of any one of claims 52, 54 to 57, wherein the first Fc region comprises a K322A substitution.

65. The composition of any one of claims 53 to 57, wherein the first Fc region comprises a P329R substitution.

66. The composition of any one of claims 53 to 57, wherein the first Fc region comprises a K322E substitution.

67. The composition of any one of claims 52, 54-64, wherein the second Fc region comprises one or more substitutions selected from the group consisting of G237A, K326A, K326W, E333A, and E333S.

68. The composition of any one of claims 52, 54-64, and 67, wherein the second Fc region comprises a G237A substitution.

69. The composition of any one of claims 52-67, wherein the second Fc region comprises a substitution selected from the group consisting of K326A, K326W, E333A, and E333S.

70. The composition of any one of claims 52-67 and 69, wherein the second Fc region comprises two substitutions selected from the group consisting of K326A, K326W, E333A, and E333S.

71. The composition of any one of claims 52-67 and 69-70, wherein the second Fc region comprises the substitutions K326W and E333S.

72. The composition of any one of claims 52-67 and 69-70, wherein the second Fc region comprises the following substitutions: K326A and E333A.

73. The composition of any one of claims 52-67 and 69, wherein the second Fc region comprises an E333S substitution.

74. The composition of any one of claims 52, 54 to 67, wherein the second Fc region comprises the substitutions G237A and E333S.

75. The composition of any one of claims 51-74, wherein the first and second antigens are both cell surface exposed molecules.

76. The composition of any one of claims 51 to 75, wherein the first and second antigens are not identical.

77. 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 ...

77. The composition of any one of claims 51 to 76, 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.

78. 78. The composition of any one of claims 51 to 77, wherein the first antibody and the second antibody are present in the composition in a molar ratio of about 1:50 to 50:1, such as a molar ratio of about 1:40 to 40:1, such as a molar ratio of about 1:30 to 30:1, such as a molar ratio of about 1:20 to 20:1, such as a molar ratio of about 1:10 to 10:1, such as a molar ratio of about 1:9 to 9:1, such as a molar ratio of about 1:5 to 5:

1.

79. The composition of any one of claims 51 to 78, wherein the first antibody and the second antibody are present in the composition in a 1:1 molar ratio.

80. The composition of any one of claims 51-79, further comprising a pharmaceutical carrier or excipient.

81. The composition of any one of claims 51 to 80, which is a pharmaceutical composition.

82. A composition according to any one of the preceding claims for use as a medicament.

83. The first or second antibody of any one of claims 1 to 36, the antibody of any one of claims 37 to 50, or the composition of any one of claims 51 to 82, 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, PD1.

84. 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 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 first or second antibody of any one of claims 1 to 36 and 83, the antibody of any one of claims 37 to 50 and 83, or the composition of any one of claims 51 to 83, comprising:

85. A method for treating an individual having a disease, comprising administering to the individual an effective amount of a first and a second antibody of any one of claims 1 to 37 and 83 to 84, an antibody of any one of claims 37 to 50 and 83 to 84, or a composition of any one of claims 51 to 84.

86. 86. The method of claim 85, wherein the disease is selected from the group of cancer, an autoimmune disease, an inflammatory disease, and an infectious disease.

87. The method of any one of claims 85-86, comprising administering a further therapeutic agent.

88. 85. A method for depleting a cell population expressing a first antigen and a second antigen, comprising contacting said cell population with a first and a second antibody, antibody or composition of any one of claims 1 to 84.

89. 90. The method of claim 88, wherein the cell population is a tumor cell population, such as a blood tumor cell population or a solid tumor cell population.

90. 85. A method of inducing proliferation in a cell population expressing a first antigen and a second antigen, comprising contacting the cell population with a first and a second antibody, antibody or composition of any one of claims 1 to 84.

91. The method of any one of claims 88-90, wherein the cell population is present in blood.

92. The method of any one of claims 88-91, wherein the cell population is white blood cells.

93. The method of any one of claims 88-92, wherein the cell population is a subset of a white blood cell population.

94. The method of any one of claims 88 to 93, wherein the cell population is a lymphocyte cell population.

95. 95. The method of claim 94, 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.

96. The method of any one of claims 85 to 95, wherein the first and / or second antigen is a member of the TNFR-SF.

97. A kit comprising a first container comprising a first antibody as defined in any one of claims 1 to 15, 26 to 36 and 83 to 84, and a second container comprising a second antibody as defined in any one of claims 1 to 6, 16 to 36 and 83 to 84.

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