Polypeptide variants and their uses

Abstract

Described herein are polypeptides and antibodies having an Fc region and an antigen-binding region, wherein the Fc region has an Fc-Fc enhancing mutation and a C1q binding enhancing mutation, wherein the C1q binding enhancing mutation provides a polypeptide or antibody with increased CDC activity and / or agonist activity.

Description

Field of the Invention

[0001] The present invention relates to polypeptides (eg, antibodies) comprising a binding region containing an Fc region, which have at least two amino acid substitutions in the Fc region compared to a parent polypeptide or antibody. Background of the Invention

[0003] The Fc-mediated effector functions of monoclonal antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP), contribute to a therapeutic window defined by efficacy and toxicity. CDC is initiated by the binding of C1q to the Fc region of an antibody. C1q is a multimeric protein composed of six globular binding heads attached to a stem.

[0004] It has been shown that IgG hexamerization after target binding on the cell surface is enhanced by point mutations in the Fc region. Hexamerization is mediated by intermolecular non-covalent Fc-Fc interactions, and Fc-Fc interactions can be enhanced by point mutations in the CH3 domain, including E345R and E430G.

[0005] WO 2013 / 004842 discloses antibodies or polypeptides comprising variant Fc regions with one or more amino acid modifications resulting in modified effector functions, such as complement dependent cytotoxicity (CDC).

[0006] WO 2014 / 108198 discloses polypeptides such as antibodies comprising a variant Fc region having one or more amino acid modifications that result in increased complement dependent cytotoxicity (CDC).

[0007] WO2016 / 164480 discloses antigen-binding complexes with agonistic activity.

[0008] Enhanced Fc-Fc interactions between antibodies can be used to amplify the effect of antibody binding to its target on the cell surface. However, simply enhancing Fc-Fc interactions between Fc regions is not always sufficient to create a strong enough signal to activate a signaling pathway resulting from, for example, binding to a receptor.

[0009] Therefore, one object of the present invention is to provide a polypeptide or antibody comprising an Fc region and an antigen binding region of human IgG, which polypeptide has increased Fc-Fc interaction and agonistic activity, such as increased target receptor activation upon binding, when compared to a parent polypeptide, wherein the parent polypeptide is a human IgG of the same isotype and has the same antigen binding region, but without any mutations in the Fc region, i.e., the parent polypeptide or parent antibody.

[0010] Another object of the present invention is to provide a polypeptide that activates signaling, optionally induces enhanced signaling, when the antigen binding region of the polypeptide (e.g., polypeptide) binds to the corresponding antigen compared to the parent polypeptide, wherein the parent polypeptide does not have any mutations in the Fc region.

[0011] Another object of the present invention is to provide polypeptides with enhanced Fc-Fc interaction properties and enhanced effector functions such as CDC.Another object of the present invention is to provide polypeptides with enhanced Fc-Fc interaction and enhanced C1q binding properties when compared to a parent polypeptide without any mutations in the Fc region. SUMMARY OF THE INVENTION

[0013] As described herein, the present invention relates to polypeptides or antibodies having an Fc region and an antigen binding region, wherein the Fc region has an Fc-Fc enhancing mutation and a C1q binding mutation, wherein the C1q binding mutation provides a polypeptide or antibody with increased CDC activity and / or agonist activity.

[0014] Without being limited by theory, it is believed that when the polypeptide or antibody of the present invention binds to a target on the cell surface, a stable binding interaction occurs between the Fc regions of the two polypeptide or antibody molecules, which leads to enhanced oligomerization, such as hexamer formation, thereby providing an affinity surface. The polypeptide or antibody of the present invention further has an increased Fc effector response compared to a parent polypeptide or parent antibody without any mutations in the Fc region, i.e., a parent polypeptide or antibody of the same isotype.

[0015] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one Fc-Fc enhancing substitution at a position selected from E430, E345 or an S440Y or S440W substitution, and b) at least one C1q binding substitution, wherein the position corresponds to human IgG1 according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition 1991 NIH Publication No. 91-3242).

[0016] In one aspect of the invention, a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen binding region is provided, wherein the Fc region comprises a) a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0017] Substitutions at positions corresponding to E430, E345, or S440Y or S440W substitutions are considered Fc-Fc enhancing substitutions according to the present invention.

[0018] Substitutions at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, 1332, E333, and P396 are considered to be CIq binding substitutions according to the present invention.

[0019] That is, in the first aspect of the present invention, the inventors discovered that introducing a first mutation that enhances Fc-Fc interaction and a second mutation that enhances C1q binding provides a polypeptide or antibody with agonistic activity and / or enhanced CDC.

[0020] In one aspect, the invention provides a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0021] That is, the inventors discovered that Fc-Fc enhancing mutations and one or more C1q binding substitutions at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333, and P396 can provide agonistic activity.

[0022] The inventors further discovered that Fc-Fc enhancing mutations and one or more C1q binding substitutions at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333, and P396 can provide enhanced Fc-mediated effector function, such as enhanced CDC.

[0023] The combination of Fc-Fc enhancing mutations and C1q binding substitutions in a polypeptide or antibody further has the surprising effect of generating a polypeptide or antibody with agonistic properties when compared to the parent polypeptide or parent antibody.

[0024] In one embodiment of the invention, the polypeptide or antibody comprises at least one substituent selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y.

[0025] In one embodiment of the invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T.

[0026] In one embodiment of the invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of E345K, E345Q, E345R and E345Y.

[0027] In one embodiment of the invention, the polypeptide or antibody comprises at least the substitution E430G. In one embodiment of the invention, the polypeptide or antibody comprises at least the substitution E345K. In one embodiment of the invention, the polypeptide or antibody comprises at least the substitution S440Y.

[0028] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at one or more positions selected from the group consisting of K326, E333 and P396.

[0029] In one embodiment of the invention, the polypeptide or antibody comprises substitutions at one or more positions selected from the group consisting of K326A, K326W, E333S, E333A and P396L, such as substitutions at two or three positions.

[0030] In one embodiment of the invention, the polypeptide or antibody comprises substitutions at one or more positions selected from the group consisting of K326A, K326W, E333S, E333A, E333T and P396L, for example substitutions at two or three positions.

[0031] In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and E333S.

[0032] In another aspect, the invention relates to a method for increasing the agonist activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of human IgG, the method comprising a) introducing a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) introducing a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0033] In another aspect, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region and an antigen-binding region of human IgG, the method comprising a) introducing a substitution at a position selected from E430, E345 or an S440Y or S440W substitution, and b) introducing a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0034] In another aspect, the invention relates to a composition comprising at least one polypeptide or antibody as described herein.

[0035] In another aspect, the invention relates to a polypeptide, an antibody or a composition as described herein for use as a medicament.

[0036] In another aspect, the invention relates to a polypeptide, antibody or composition as described herein for use in treating cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

[0037] In another aspect, the present invention relates to a method of treating an individual suffering from a disease, comprising administering to the individual an effective amount of a polypeptide, antibody or composition as described herein.

[0038] These and other aspects of the invention, in particular various uses and therapeutic applications of the polypeptides or antibodies, are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown are the effects of E430G, K326A / E333A / P396L, and K326A / E333A / P396L / E430G on the efficacy of anti-DR5 antibodies IgG1-hDR5-01-G56T (A), IgG1-hDR-05 (B), and antibody combinations (C) on adhesion to human BxPC-3 pancreatic cancer cells, as determined in a 3-day viability assay (CellTiter-Glo). Representative examples of two experiments are shown.

[0041] Figure 2Shown is the efficacy of a monovalent anti-DR5 antibody with K326A / E333A / P396L / E430G against adherent human BxPC-3 pancreatic cancer (A) and COLO 205 colon cancer (B) cells, as determined in a 3-day viability assay (CellTiter-Glo). As a monovalent anti-DR5 antibody, a bispecific antibody with one DR5-specific arm derived from IgG1-hDR5-01-G56T and one nonspecific arm against the HIV protein gp120 derived from IgG1-b12 was generated by controlled Fab arm exchange. Representative examples of three experiments are shown.

[0042] Figure 3 Shown are the effects of E430G in combination with K326A / E333A / P396L or two of these substitutions, E333A / P396L, K326A / E333A, or K326A / P396L, on the efficacy of the anti-DR5 antibody IgG1-hDR5-01-G56T on adhesion to human BxPC-3 pancreatic cancer (A) and COLO 205 colon cancer (B) cancer cells, as determined in a 3-day viability assay (CellTiter-Glo), and on C1q binding, as determined in an ELISA assay (C). Representative examples of three experiments are shown.

[0043] Figure 4 Shown are the effects of E430G in combination with K326A / E333A or K326W / E333S on the binding of C1q to the anti-DR5 antibody IgG1-CONA-C49W, as determined in an ELISA assay (A), and on the efficacy of the anti-DR5 antibody IgG1-hDR5-01-G56T on adhesion to human BxPC-3 pancreatic cancer (B) and COLO 205 colon cancer (C) cancer cells, as determined in a 3-day viability assay (CellTiter-Glo). Representative examples of three experiments are shown.

[0044] Figure 5 Shown are the effects of E430G in combination with the C1q binding substitutions S267E / H268F / S324T or the IgG1 / IgG3 chimeric isotype IgG1 variant 113F on C1q binding to the anti-DR5 antibody IgG1-hDR5-01-G56T, as determined in an ELISA assay (A), and on the potency of the anti-DR5 antibody IgG1-hDR5-01-G56T on adhesion to human BxPC-3 pancreatic cancer (B) and COLO 205 colon cancer (C) cells, as determined in a 3-day viability assay (CellTiter-Glo). Representative examples of three experiments are shown.

[0045] Figure 6Shown is a summary of a 3-day viability assay (CellTiter-Glo) of adherent human BxPC-3 pancreatic cancer cells using 10 μg / mL IgG1-hDR5-01-G56T variants containing the indicated mutations. Effects are expressed and ranked by the percentage of viable cells relative to WT IgG1-hDR5-01-G56T (which was set to 100%). Significant effects on cell viability compared to WT are indicated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA with Dunnett's multiple comparison test).

[0046] Figure 7 Shown are the agonistic effects of anti-DR5 antibodies IgG1-CONA-C49W-K326A / E333A / P396L / E430G (A) and IgG1-hDR5-01-G56T-K326W / E333S / E430G (B) on WIL2-S SF suspension cells in serum-free medium in the presence or absence of 2.5 μg / mL purified human C1q, as determined in a 24-hour viability assay. The percentage of viable cells is represented by the percentage of TO-PRO-3 negative cells.

[0047] Figure 8 Shown are the agonistic effects of 2.5 μg / mL of the anti-DR5 antibody variant IgG1-hDR5-01-G56T with a combination of E430G (Fc-Fc enhancing substitution) and C1q binding substitutions (A) and the antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G (B) on WIL2-S SF suspension cells in serum-free medium in the presence or absence of a concentration series of purified human C1q, as determined in a 24-hour viability assay. The percentage of viable cells is represented by the percentage of TO-PRO-3 negative cells. Data from four different experiments are presented with error bars indicating standard deviation.

[0048] Figure 9 The efficacy of 2.5 μg / mL agonist anti-DR5 IgG1-hDR5-01-G56T-K326W / E333S / E430G (A) and the antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G (B) on WIL2-S SF suspension cells in serum-free medium with or without purified human C1q and anti-C1q neutralizing antibodies, as determined in a 24-hour viability assay. The percentage of viable cells is represented by the percentage of TO-PRO-3 negative cells.

[0049] Figure 10Shown are solution-phase complement activation as measured by quantification of C4d deposition for IgG1-hDR5-01-G56T antibody variants containing the 430q Fc-Fc enhancing substitution in combination with the C1q binding substitutions K326W / E333S, K326A / E333A, or K326A / E333A / P396L when antibody samples were incubated in NHS. HAGG (heat-aggregating gamma globulin) and IgG1-CONA-RGY were tested as positive controls for solution-phase complement activation.

[0050] Figure 11 Shown are the effects of introducing K326W, E333S, or K326W / E333S substitutions into IgG-CONA-C49W and IgG1-CONA-C49W-E430G on C1q binding, as determined in ELISA assays (A, B), on C1q binding to antibodies bound to DR5-positive WIL2-S SF cells, as determined by flow cytometry (C, D), and on the reduction of cell viability of WIL2-S SF suspension cells, as determined in a 3-day viability assay (CellTiter-Glo) (E, F, G). Standard deviations were calculated from two independent experiments.

[0051] Figure 12 Shown is the effect of introducing K326W, E333S or K326W / E333S substitutions into IgG-CONA-C49W and IgG1-CONA-C49W-E430G (2.5 μg / mL) on the viability of WIL2-S SF suspension cells in serum-free medium in the presence of a concentration series of purified human C1q, as determined in a 24-hour viability assay. The percentage of viable cells was determined in the CellTiter-Glo assay.

[0052] Figure 13 Shown is the effect of adding anti-C1q antibody on the potency of anti-DR5 IgG1-CONA-C49W antibody variants with C1q binding enhancing mutations and / or Fc-Fc interaction enhancing mutations in a 24-hour viability assay on WIL2-S SF suspension cells in serum-free medium supplemented with purified human C1q. The percentage of viable cells was determined in a CellTiter-Glo assay.

[0053] Figure 14The effect of adding peptides that inhibit Fc-Fc interactions between anti-DR5 IgG1-CONA-C49W antibody variants opsonized on WIL2-S SF suspension cells in serum-free medium supplemented with purified human C1q is shown in a 24-hour viability assay. The percentage of viable cells was determined in the CellTiter-Glo assay. The scrambled peptide WCDLEGVTWHACL was used as a nonspecific control peptide.

[0054] Figure 15 Shown are the effects of combining the CIq binding substitutions K326W / E333S with the Fc-Fc enhancing mutations E345K, E345R or S440Y on the agonistic activity of the anti-DR5 antibody IgG1-CONA-C49W towards adherent human BxPC-3 pancreatic cancer cells as determined in a 3-day viability assay (CellTiter-Glo).

[0055] Figure 16 Shown is the effect of mutation E430G introduced into anti-DR5 antibody IgG1-CONA-C49W in combination with the C1q binding enhancing mutations S267E / H268F / S324T or the IgG1 / IgG3 chimeric isotype IgG1 variant 113F on the viability of adherent human BxPC-3 pancreatic cancer cells as determined in a 3-day viability assay (CellTiter-Glo).

[0056] Figure 17 Shown is the effect of a functional monovalent anti-DR5 antibody with K326W / E333S / E430G mutations on the viability of WIL2-S SF suspension cells, as measured in a 1-day viability assay (CellTiter-Glo). A functional monovalent anti-DR5 antibody was generated as a bispecific antibody by controlled Fab arm exchange of IgG1-CONA-C49W-F405L-K326W / E333S / E430G (DR5-specific arm) and IgG1-b12-K409R-K326W / E333S / E430G (nonspecific arm, directed against HIV protein gp120). RLU: relative luminescence unit.

[0057] Figure 18 Shown is the effect of introducing K326W / E333S / E430G substitutions on the agonist activity of IgG1 and IgG3 isotype variants of anti-DR5 antibodies (IgG1-CONA-C49W and IgG3-CONA-C49W-R345H) as measured in a 1-day viability assay on WIL2-S SF cells (A) and a 3-day viability assay on BxPC-3 (B), HPAF-II (C), and HT-29 cells (D). Viability was determined using the CellTiter-Glo kit.

[0058] Figure 19 Shown are the effects of introducing both the E430G hexamerization-enhancing mutation and the C1q binding-enhancing mutations K326W / E333T or K326W / E333S into the anti-DR5 antibody IgG1-CONA-C49W on WIL2-S suspension cells as determined in a 24-hour viability assay (CellTiterGlo).

[0059] Figure 20 Figure 2 shows the clearance of 450 μg of antibody administered iv in SCID mice. (A) Total human IgG in serum samples was determined by ELISA and plotted in a concentration versus time curve. Each data point represents the mean + / - standard deviation of triplicate samples. (B) Clearance was determined to day 21 after antibody administration using the formula D*1.000 / AUC, where D is the injected dose and AUC is the area under the curve of the concentration-time curve. Representative examples of two independent ELISA experiments are shown.

[0060] Figure 21 Shown are the effects of the E430G Fc-Fc enhancing mutation in combination with C1q binding enhancing mutations (K326A / E333A or K326W / E333S) on human FcRn binding of IgG1-7D8 antibody variants, as measured by ELISA at pH 6.0 and 7.4 using coated FcRnECDHis-B2M-BIO. IgG1-7D8-I235A / H310A / H435A was used as a negative control for FcRn binding (FcRn knockout) at pH 6.0; IgG1-7D8-M252Y / S254T / T256E was used as a control for enhanced FcRn binding at pH 7.4.

[0061] Figure 22 Chromium release ADCC assay using WIL2-S SF as target cells and human PBMC (3 donors) as effector cells (E:T ratio 100:1) in serum-free medium with and without the addition of purified human C1q. Chromium-labeled WIL2-S SF cells were incubated with a concentration series of antibodies in the absence and presence of C1q to compare the ADCC activity of IgG1-7D8-F405L-K326W / E333S / E430G with that of IgG1-7D8-E430G and WT IgG1-7D8. Nonspecific antibody IgG1-b12 was used as a negative control.

[0062] Figure 23Shown are the effects of K326W / E333S / E430G on the agonistic activity of anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 on WIL2-S SF suspension cells, as determined in a 24-hour viability assay (CellTiterGlo).

[0063] Figure 24 Shown are the effects of complementary Fc mutation pairs K439E; S440K on the agonistic activity of the anti-DR5 dual-epitope targeting antibody combination IgG1-hDR5-01-G56T-K326W / E333S / E430G + IgG1-hDR5-05-K326W / E333S / E430G on WIL2-S SF suspension cells, as determined in a 24-hour viability assay (CellTiterGlo).

[0064] Figure 25 Shown are the results of a CDC assay on Wien 133 cells testing IgG1-Campath antibody variants with the hexamerization-enhancing mutations E430G or K248E / T437R, as well as the hexamerization-enhancing mutation K248E / T437R in combination with the C1q binding-enhancing mutations K326W / E333S. Wien 133 cells were incubated with a concentration series of the antibody variants in the presence of 20% pooled normal human serum (NHS).

[0065] Figure 26 Shown is the effect of combining the hexamerization-enhancing mutation E430G and the C1q-binding-enhancing mutations K326W / E333S on the efficacy of the anti-DR4 antibody IgG1-DR4-chCTB007 on adhesion to human BxPC-3 pancreatic cancer cells, as determined in a 3-day viability assay (CellTiterGlo).

[0066] Figure 27 CDC assay using WIL2S SF human B lymphocytes with anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B), and IgG1-CD95-HFE7A (C) in the presence of 20% normal human serum is shown. WIL2-S SF cells were incubated with a concentration series of antibody variants for 45 minutes in the presence of 20% pooled normal human serum (NHS). IgG1-b12 was used as a non-binding control antibody.

[0067] Figure 28Shown is a 45-minute viability assay (CellTiter-Glo) using WIL2-S SF cells incubated with anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B), and IgG1-CD95-HFE7A (C) in serum-free medium without C1q.

[0068] Figure 29 Shown is a 24-hour viability assay (CellTiter-Glo) using WIL2-S SF cells in serum-free medium with C1q as cross-linker using the anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B) and IgG1-CD95-HFE7A (C).

[0069] Figure 30 Shown is a 24-hour viability assay (CellTiter-Glo) using WIL2-S SF cells incubated with anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B), and IgG1-CD95-HFE7A (C) in serum-free medium without C1q.

[0070] Figure 31 Shown are the activities of IgG1-CD134-SF2 (WT), IgG1-CD134-SF2-E345R, IgG1-CD134-SF2-E430G, and IgG1-CD134-SF2-K326W / E333S / E430G antibodies in the OX40 Jurkat reporter assay. Thaw-and-Use GloResponse NFκB-luc2 / OX40 Jurkat was incubated with a range of antibody concentrations for 5 hours in the presence of 8% fetal bovine serum. OX40 assay responses were recorded as luminescence detected after stimulation of OX40 with an anti-OX40 antibody that induces expression of a luciferase reporter gene. RLU: relative luminescence unit.

[0071] Figure 32Shown are the effects of the Fc-Fc enhancing mutation E430G, combined with the C1q binding enhancing mutations K326W / E333S, on the CD40 responses of IgG1-CD40-SGN40 and IgG1-CD40-CP870893. Thaw-and-Use GloResponse NFκB-luc2 / CD40 Jurkat cells were incubated with a range of antibody concentrations for 5 hours in the presence of 8% fetal bovine serum. CD40 assay responses were recorded by luminescence detected after stimulation of CD40 with either an anti-CD40 antibody or a CD40 ligand that induces expression of a luciferase reporter gene. RLU: relative luminescence unit.

[0072] Figure 33 Shown are the effects of the Fc-Fc enhancing mutation E430G, combined with the C1q binding enhancing mutations K326W / E333S, on the 4-1BB response of IgG1-CD137-MOR7480 and IgG1-BMS-663513. Thaw-and-Use GloResponse™ NFκB-luc2 / 4-1BB Jurkat cells were incubated with a range of antibody concentrations for 5 hours in the presence of 1% fetal bovine serum. 4-1BB assay responses were recorded by luminescence detected after stimulation of 4-1BB with either an anti-4-1BB antibody or a 4-1BB ligand with an anti-His antibody, which induces expression of a luciferase reporter gene. IgG-b12-K326W / E333S / E430G was used as a negative control. RLU: relative luminescence unit.

[0073] Figure 34 Shown are the effects of the Fc-Fc enhancer mutation E430G, combined with the C1q binding enhancer mutations K326W / E333S, on the GITR response of IgG1-GITR-INCAGN01876. Thaw-and-UseGloResponse NFκB-luc2 / GITR Jurkat cells were incubated with a range of antibody concentrations for 6 hours in the presence of 1% fetal bovine serum. The GITR assay response was recorded by luminescence detected after GITR stimulation with an anti-GITR antibody that induced expression of a luciferase reporter gene. RLU: relative luminescence unit.

[0074] Figure 35Shown are the effects of the Fc-Fc enhancing mutation E430G, combined with the C1q binding enhancing mutations K326W / E333S, on the GITR response of the antibody GITR-36E5 in the IgG1, IgG2, IgG3, and IgG4 subclasses. Thaw-and-Use GloResponse NFκB-luc2 / GITR Jurkat cells were incubated with a final concentration of 111 ng / mL of antibody for 6 hours in the presence of 1% fetal bovine serum. GITR assay responses were recorded by luminescence detected after stimulation of GITR with an anti-GITR antibody that induces expression of a luciferase reporter gene. Antibody IgG1-b12 was used as a non-binding control. RLU: relative luminescence unit. Detailed Description of the Invention

[0076] In describing the embodiments of the present invention, specific terms will be employed for the sake of 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 operate in a similar manner to achieve a similar purpose.

[0077] definition

[0078] The term "parent polypeptide" or "parent antibody" is to be understood as a polypeptide or antibody which is identical to a polypeptide or antibody according to the invention, but wherein the parent polypeptide or parent antibody does not have the Fc-Fc enhancing mutation and the C1q binding mutation according to the invention.

[0079] The term "polypeptide comprising an Fc region of an immunoglobulin and a binding region" in the context of the present invention refers to a polypeptide comprising an Fc region of an immunoglobulin and a binding region capable of binding to any molecule, such as a polypeptide, present on a cell, a bacterium, or a virion. The Fc region of an immunoglobulin is defined as an antibody fragment typically produced after digestion of an antibody with papain (which is known to those skilled in the art), which includes the two CH2-CH3 regions of the immunoglobulin and a connecting region, such as a hinge region. The constant domains of the antibody heavy chains define the antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates the effector functions of the antibody via cell surface receptors called Fc receptors and proteins of the complement system. The binding region can be a polypeptide sequence capable of binding to a cell, a bacterium, or a virion, such as a protein, a protein ligand, a receptor, an antigen binding region, or a ligand binding region. If the binding region is, for example, a receptor, then the "polypeptide comprising an Fc region of an immunoglobulin and a binding region" can be prepared as a fusion protein of the Fc region of an immunoglobulin and the binding region. If the binding region is an antigen binding region, the "polypeptide comprising the Fc region of an immunoglobulin and the binding region may be an antibody, such as a chimeric antibody, a humanized antibody or a human antibody, or a heavy chain-only antibody or a ScFv-Fc fusion. A polypeptide comprising the Fc region of an immunoglobulin and the binding region may generally comprise a connecting region, such as a hinge region, and two CH2-CH3 regions of an immunoglobulin heavy chain, and thus a "polypeptide comprising the Fc region of an immunoglobulin and the binding region" may be a "polypeptide comprising at least the Fc region of an immunoglobulin and the binding region". The term "Fc region of an immunoglobulin" in the context of the present invention means the connecting region, such as the hinge region, and the presence of the CH2 and CH3 regions of an immunoglobulin according to the antibody subclass, for example, human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM or IgE. The polypeptide is not limited to human origin, but may be of any origin, for example mouse or macaque origin. The term "wild-type Fc region" in the context of the present invention refers to an immunoglobulin Fc region having a naturally occurring amino acid sequence.

[0080] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

[0081] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, according to EU numbering, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340. However, the CH2 region may also be of any other subtype as described herein.

[0082] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, according to EU numbering, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447. However, the CH3 region may also be of any other subtype as described herein.

[0083] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, i.e., a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, with all four chains potentially interconnected by disulfide bonds. The structure of immunoglobulins is well established. See, for example, Fundamental Immunology, Chapter 7 (Paul, W. ed., Chapter 2, Raven Press, NY (1989)). In short, each heavy chain typically comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically comprises three domains: CH1, CH2, and CH3. The heavy chains are interconnected in the so-called "hinge region" via disulfide bonds. Each light chain typically comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which are highly variable in the form of sequence- and / or structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or conflicting with context, the CDR sequences herein were identified 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); also available on the Internet at http address www.imgt.org / ) according to IMGT rules. Unless otherwise stated or conflicting with context, amino acid positions in the Fc region / Fc domain of the present invention are referred to according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1): 78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition-1991 NIH Publication No. 91-3242).

[0084] 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 any of them that has the ability to specifically bind to an antigen. The antibodies of the present invention comprise the Fc domain of an immunoglobulin and an antigen-binding region. Antibodies typically contain two CH2-CH3 regions and a connecting region, such as a hinge region, for example, at least an Fc domain. Thus, the antibodies of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. The constant region or "Fc" region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Antibodies can also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term "bispecific antibody" refers to an antibody that is specific for at least two different, usually non-overlapping, epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be in the same cell or different cells or cell types. As indicated above, unless otherwise indicated or clearly conflicting with context, the term antibody herein includes fragments of antibodies that comprise at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, which consist of only two heavy chains and occur naturally in, for example, camels (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), chain exchange engineered domains (SEED or Seed-body), which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No.7,612,181); dual-domain bimanual antibodies (Unilever; Sanofi Aventis, WO20100226923), di-diabody (ImClone / Eli Lilly), knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), dual-targeting domain antibodies (GSK / Domantis), two-in-one antibodies or dual-action Fabs that recognize two targets (Genentech, NovImmune, Adimab), cross-linked Mabs (Karmanos Cancer Center), covalently fused monoclonal antibodies (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecific (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p.65-74), TIG-body, DIG-body and PIG-body (Pharmabcine), dual affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybodies (Zyngenia), approaches with a common light chain (Crucell / Merus, US7262028) or with a common heavy chain (kλ Bodies, NovImmune, WO2012023053), and fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc region, such as scFv fusions, such as BsAb from ZymoGenetics / BMS, Biogen HERCULES (US007951918) of Idec, SCORPIONS of Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p.672-92), scFv fusions of Genetech / Roche, scFv fusions of Novartis, scFv fusions of Immunomedics, scFv fusions of Changzhou Adam Biotech Inc (CN102250246), TvAb of Roche (WO 2012025525, WO 2012025530), mAb of f-Star. 2 (WO2008 / 003116) and double scFv fusions. It should also be understood that, unless otherwise specified, the term antibody also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal) such as those produced by the technology developed by Symphogen and Merus (Oligoclonics), such as the multimeric Fc proteins described in WO2015 / 158867, fusion proteins and antibody-like polypeptides such as those described in WO2014 / 031646, such as chimeric antibodies and humanized antibodies. The antibodies generated can potentially have any isotype.

[0085] The term "full length antibody" as used herein refers to an antibody (e.g., a parent antibody) that contains all heavy and light chain constant and variable domains corresponding to those normally found in a wild-type antibody of that isotype.

[0086] As used herein, the term "human antibody" is intended to include antibodies with 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 by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human framework sequences.

[0087] As used herein, the term "chimeric antibody" refers to an antibody in which the two chain types (i.e., heavy and light chains) are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (derived from a non-human species, or synthesized or engineered from any species including humans) linked to a constant region of human origin.

[0088] As used herein, the term "humanized antibody" refers to an antibody in which two chain types are humanized due to antibody engineering. Humanized chains are typically those in which the complementary determining regions (CDRs) of the variable domains are foreign (derived from species other than humans or synthesized), while the remainder of the chain is a chain of human origin. Humanization assessment is based on the resulting amino acid sequence, rather than on the method itself, which allows the use of schemes other than implantation.

[0089] As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "monoclonal antibody" and the like refer to antibody molecule preparations having a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice) having a genome comprising a repertoire of human heavy chain transgenes and a repertoire of light chain transgenes (rearranged to produce functional human antibodies) and fused to immortalized cells.

[0090] As used herein, "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)). In addition, each heavy chain isotype can be combined with a kappa (k) or lambda (λ) light chain. The term "mixed isotype" as used herein refers to the Fc region of an immunoglobulin produced by combining the structural features of one isotype with similar regions from another isotype, thereby producing a hybrid isotype. The mixed isotype may comprise an Fc region having a sequence comprising two or more isotypes selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, thereby creating combinations such as, for example, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0091] As used herein, the terms "antigen binding region," "antigen binding region," "binding region," or antigen binding domain refer to the region of an antibody that is capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in the form of sequence and / or structurally defined loops), also known as complementarity determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs). An antigen can be any molecule, such as a polypeptide, present, for example, on a cell, bacterium, or virion.

[0092] As used herein, the term "target" refers to a molecule that binds to the antigen-binding region of an antibody. Targets include any antigen against which an antibody is prepared. With respect to antibodies, the terms "antigen" and "target" are used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0093] The term "epitope" refers to a protein determinant that is capable of specific binding to an antibody variable domain. Epitopes are typically composed of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that, in the presence of denaturing solvents, binding to the former, but not the latter, is lost. An epitope can include amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding.

[0094] An “antibody” or “antibody variant” or “variant of a parent antibody” of the present invention is an antibody molecule comprising one or more mutations compared to the “parent antibody”. The different terms are used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary parent antibody forms include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof. Similarly, a “polypeptide” or “variant of a polypeptide comprising an Fc region and a binding region of an immunoglobulin” or “variant of a parent polypeptide comprising an Fc region and a binding region of an immunoglobulin” of the present invention is a “polypeptide comprising an Fc region and a binding region of an immunoglobulin” that comprises one or more mutations compared to the “parent polypeptide comprising an Fc region and a binding region of an immunoglobulin”. The different terms are used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Amino acid substitutions can exchange a natural amino acid for another naturally occurring amino acid, or a non-naturally occurring amino acid derivative. Amino acid substitutions can be conservative or non-conservative. In the context of the present invention, conservative substitutions can be defined by substitutions within the amino acid classes reflected in one or more of the following three tables:

[0095] Categories of amino acid residues for conservative substitutions

[0096]

[0097] Alternative conservative amino acid residue substitution categories

[0098] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0099] Alternative physical and functional classifications of amino acid residues

[0100]

[0101] In the context of the present invention, substitutions in variants are represented by:

[0102] Original amino acid-position-substituted amino acid

[0103] Amino acid residues are indicated using either three-letter or one-letter codes, including the codes Xaa and X. Thus, the designation "E345R" or "Glu345Arg" means a variant comprising a substitution of arginine for glutamic acid in the variant amino acid position corresponding to amino acid 345 in the parent antibody.

[0104] When the position itself does not exist in the antibody, but the variant contains an amino acid insertion, e.g.

[0105] Position - substituted amino acid; use a label, e.g. "448E".

[0106] Such notations are particularly relevant in the context of modifications in a series of homologous polypeptides or antibodies.

[0107] Similarly, when the identity of the substituted amino acid residue is not important:

[0108] Initial amino acid-position; or "E345".

[0109] The original amino acid and / or the substituted amino acid may contain modifications of more than one but not all amino acids, such as substitution of glutamic acid at position 345 with arginine, lysine or tryptophan:

[0110] "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.

[0111] In addition, the term "substitution" encompasses substitution with any of the other 19 naturally occurring amino acids, or with other amino acids, such as non-natural amino acids. For example, substitution of amino acid E at position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345P, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is equivalent to the designation 345X, where X refers to any amino acid. These substitutions may also be referred to as E345A, E345C, etc., or E345A, C, etc., or E345A / C / , etc. This applies similarly to each position mentioned herein, so that any such substitution is expressly included herein.

[0112] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response in contrast to the recognition and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic 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 play a specific immune function. In some embodiments, effector cells such as natural killer cells can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells expressing FcR participate in the specific killing of target cells and present antigens to other components of the immune system, or are combined with cells presenting antigens. In some embodiments, ADCC can be further enhanced by the classical complement activation driven by antibodies, causing activated C3 fragments to be deposited on target cells. C3 cleavage products are ligands for complement receptors (CR) (e.g., 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 leads to C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of cells carrying FcγRI against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct phagocytosis by effector cells or indirectly promote phagocytosis by enhancing antibody-mediated phagocytosis.

[0113] As used herein, the term "Fc effector function" refers to a function that is a result of binding of a polypeptide or antibody to its target (such as an antigen) on a cell membrane, wherein the Fc effector function is attributable 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-γ receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated binding of opsonized antibodies to complement receptors, (x) opsonization, and (xi) a combination of any one of (i) to (x).

[0114] As used herein, the term "clustering-dependent function" refers to a function that results from the formation of an antigen complex after binding of a polypeptide or antibody to its antigen, optionally after oligomerization on a cell, on a 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) proliferation regulation, for example, as a decrease, inhibition or stimulation of proliferation, and (vii) a combination of any one of (i) to (vi).

[0115] As used herein, the term "clustering-dependent function" means a function that is a result of the formation of an antigen complex after oligomerization of a polypeptide or antibody that binds to its antigen (optionally on a cell, on a 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) apoptosis induction, (vi) proliferation regulation, such as proliferation reduction, inhibition or stimulation, and (vii) a combination of any one of (i) to (vi).

[0116] As used herein, the term "agonism" is understood to mean stimulation or activation of receptors on the cell membrane, resulting in a biological response, such as intracellular signaling. Such agonistic effects can lead to the induction of apoptosis (programmed cell death) or activation of immune cells, or activation of intracellular pathways.

[0117] As described in Example 2, agonistic activity or increased agonistic activity can be determined in a viability assay of antibodies against targets expressing an intracellular death domain using the following steps:

[0118] i) Inoculate the cell line expressing the target of the antibody (e.g. DR5) in a polystyrene 96-well flat-bottom plate at 37°C overnight,

[0119] ii) adding serial dilutions of an antibody (e.g., anti-DR5 antibody) within a certain range (0.0003 to 20,000 ng / mL) and incubating at 37°C for 3 days,

[0120] iii) determining cell viability by quantifying the presence of ATP, for example by using the CellTiler-Glo luminescent cell viability assay,

[0121] iv) Calculate viable cells using the following formula: % viable cells = [(luminescence of the antibody sample - luminescence of the staurosporine sample) / (luminescence of the no-antibody sample - luminescence of the staurosporine sample)] * 100.

[0122] As described in Examples 29, 30, 31 and 32, agonistic activity or increased agonistic activity can be determined in a reporter assay for antibodies against targets that activate an intracellular signaling pathway using the following steps:

[0123] i) inoculating Jurkat cells stably transfected with a target (e.g., OX40, 4-1BB, CD40, or GITR) and a luciferase reporter gene downstream of an NFAT response element, and incubating the cells in a 96-well flat-bottom plate at 37° C.,

[0124] ii) adding serial dilutions of an antibody (e.g., anti-OX4, anti-CD1 antibody, anti-CD40 antibody, or anti-GITR antibody) ranging from, for example, 19.5 to 5,000 ng / mL and incubating for 5 hours,

[0125] iii) adding firefly luciferase substrate (5'-fluoroluciferin) to the cells and incubating for 5-10 minutes,

[0126] iv) Luminescence was measured using an Envision MultiLable plate reader.

[0127] As used herein, the term "vector" means a nucleic acid molecule that can induce transcription of a nucleic acid segment connected to a vector. One type of vector is a "plasmid," which is a form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment can be connected to a viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thereby replicate together with the host genome. In addition, certain vectors can direct the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to encompass such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

[0128] As used herein, the term "recombinant cell" (or simply "host cell") means a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell, but also to the progeny of such cells. Because certain modifications may occur during serial passage due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli (E. coli) and other eukaryotic hosts such as plant cells and fungi.

[0129] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi, including yeast cells, that express the antibody or target antigen.

[0130] The term "preparation" refers to preparations of antibody variants and mixtures of different antibody variants that have an increased ability to form oligomers upon interaction with an antigen associated with a cell (e.g., an antigen expressed on the surface of a cell), a cell membrane, a virion, or other structure, which can result in enhanced signaling and / or activation by the antigen.

[0131] As used herein, the term "affinity" refers to the strength of binding of one molecule (eg, an antibody) to another (eg, a target or antigen) at a single site, such as the monovalent binding of an antibody to an individual antigen-binding site of an antigen.

[0132] As used herein, the term "avidity" refers to the combined strength of binding between two structures, such as multiple antigen binding sites of an antibody that interact simultaneously with a target, or multiple binding sites, such as between an antibody and C1q. When there is more than one binding interaction, the two structures will dissociate only when all binding sites dissociate, and therefore, the off-rate will be slower than the off-rate of an individual binding site, thereby providing a greater effective total binding strength (avidity) compared to the strength of binding (affinity) of the individual binding sites.

[0133] As used herein, the term "oligomer" refers to a molecule consisting of more than one, but finite number of monomeric units (e.g., antibodies), as opposed to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are often used to indicate the number of monomeric units in an oligomer, e.g., a tetramer consists of four units and a hexamer consists of six units.

[0134] As used herein, the term "oligomerization" refers to the process of converting monomers into a limited degree of polymerization. In this context, it is observed that polypeptides, antibodies and / or other dimeric proteins comprising a target binding region according to the present invention can form oligomers, such as hexamers, via non-covalent binding of the Fc region upon target binding (e.g., at the cell surface).

[0135] As used herein, the term "clustering" means the oligomerization of antibodies, polypeptides, antigens or other proteins through non-covalent interactions.

[0136] As used herein, the term "Fc-Fc enhancement" means increasing the binding strength between the Fc regions of two Fc region-containing antibodies or polypeptides or stabilizing the interaction between the Fc regions, so that the polypeptides form oligomers after target binding.

[0137] As used herein, Fc-Fc enhancing substitutions refer to substitutions in the following positions corresponding to human IgG1 according to EU numbering: E430, E345, or S440, with the proviso that the substitution at position S440 is S440Y or S440W. Thus, as used herein, Fc-Fc enhancing substitutions refer to the following amino acid substitutions: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y. In a preferred embodiment, the Fc-Fc enhancing substitution is E430G or E345K.

[0138] As used herein, the term "CIq binding" refers to a direct interaction between CIq and a polypeptide or antibody. Direct CIq binding can be assessed, for example, using immobilized antibodies on artificial surfaces (as described in Examples 4, 5, and 6). When bound to a predetermined antigen on the surface of a cell or virion, the multivalent interaction resulting in high-affinity binding of CIq to the antibody oligomer can be assessed.

[0139] Binding of C1q to a polypeptide or antibody can be measured in an ELISA assay using the following steps: i) coat a 96-well Microlon ELISA plate with 1 μg / mL of polypeptide or antibody in 100 μl PBS overnight at 4°C, ii) incubate the plate at 37°C for 1 hour with 100 μL / well of a serial dilution series of C1q, with a final C1q concentration range of 30-0.01 μg / mL, diluted 3-fold, iii) incubate the plate with 100 μL / well of rabbit anti-human C1q for 1 hour at room temperature, iv) incubate the plate with 100 μL / well of pig anti-rabbit IgG-HRP for 1 hour at room temperature, v) incubate the plate with 100 μL / well of substrate with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for 15 minutes at room temperature, vi) stop the reaction by adding 100 μL / well of 2% oxalic acid. Absorbance was measured at 405 nm in a BioTek EL808 microplate reader.

[0140] As used herein, the term C1q binding substitution refers to a substitution in a polypeptide comprising an immunoglobulin Fc region and an antigen binding region that enhances direct interaction with C1q. Enhanced C1q binding can, for example, result in a decrease in the EC50 of the interaction between C1q and a polypeptide comprising an immunoglobulin Fc region and an antigen binding region, as measured according to the method for determining C1q binding described above.

[0141] As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on the surface. C1 is a complex composed of six recognition proteins, C1q, and a heterotetramer, C1r2C1s2, of a serine protease. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions that begin with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves the complement component C3 into C3b and C3a, forming the C5 convertase. This C5 convertase splits C5 into C5a and C5b, and the last component is deposited on the membrane and, in turn, triggers the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be assessed by using C1q efficacy, CDC kinetics CDC assays (as described in WO2013 / 004842, WO2014 / 108198) or by the cellular deposition of C3b and C4b as described in Beurskens et al., April 1, 2012, vol. 188 no. 73532-3541.

[0142] As used herein, the term "complement dependent cytotoxicity" ("CDC") refers to the process of antibody-mediated complement activation that results in lysis of cells or virions when antibodies bind to their targets on cells or virions due to pores in the membrane created by MAC assemblies.

[0143] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to a mechanism by which antibody-coated target cells or virions are killed by cells expressing Fc receptors that recognize the constant region of a bound antibody. As used herein, the term "antibody-dependent cellular phagocytosis" ("ADCP") refers to a mechanism by which antibody-coated target cells or virions are eliminated by internalization by phagocytes. The internalized antibody-coated target cells or virions are contained in a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP can be assessed by using an in vitro cytotoxicity assay using macrophages as effector cells and video microscopy, as described by van Bij et al. in Journal of Hepatology, Vol. 53, No. 4, October 2010, pp. 677-685.

[0144] As used herein, the term "complement-dependent cellular cytotoxicity" ("CDCC") refers to a mechanism by which target cells or virions are killed by cells expressing complement receptors that recognize complement 3 (C3) cleavage products covalently bound to the target cells or virions due to antibody-mediated complement activation. CDCC can be assessed in a manner similar to that described for ADCC.

[0145] As used herein, the term "plasma half-life" refers to the time it takes for the concentration of a polypeptide in plasma to decrease to half of its initial concentration during the elimination period (after the distribution phase). For antibodies, the distribution phase is typically 1-3 days, during which the plasma concentration decreases by about 50% due to redistribution between plasma and tissues. The plasma half-life can be measured by methods well known in the art.

[0146] As used herein, the term "plasma clearance" is a quantitative measure of the rate at which a polypeptide is removed from the blood when administered to a living organism. Plasma clearance can be calculated as dose / AUC (mL / day / kg), where the AUC value (area under the curve) is determined from the concentration-time curve.

[0147] As used herein, the term "antibody-drug conjugate" refers to an antibody or Fc-containing polypeptide that is specific for at least one type of malignant cell, a drug, and a linker that couples the drug to, for example, the antibody. The linker may be cleavable or non-cleavable in the presence of malignant cells; wherein the antibody-drug conjugate kills the malignant cells.

[0148] As used herein, the term "antibody-drug conjugate uptake" refers to the process by which an antibody-drug conjugate binds to its target on a cell and is subsequently taken up / phagocytosed by the cell membrane, thereby being absorbed into the cell. As described in WO 2011 / 157741, antibody-drug conjugate uptake can be assessed as "antibody-mediated internalization and cell killing by an anti-TF ADC in an in vitro killing assay."

[0149] As used herein, the term "apoptosis" refers to the process of programmed cell death (PCD) that can occur in cells. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. The binding of antibodies to certain receptors can induce apoptosis.

[0150] 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 what the various types of PCD have in common is that they are executed by active cellular processes that can be intercepted by interfering with intracellular signaling. In a specific embodiment, the occurrence of any form of PCD in cells or tissues can be determined by staining the cells or tissues with conjugated Annexin V, which is associated with phosphatidylserine exposure.

[0151] As used herein, the term "Annexin V" refers to a protein in the annexin group that binds phosphatidylserine (PS) on the cell surface.

[0152] As used herein, the term "FcRn" refers to the neonatal Fc receptor, which is an Fc receptor. It was first discovered in rodents as a unique receptor that can transport IgG from breast milk to the neonatal bloodstream through newborn rodent intestinal epithelial cells. Further studies have revealed similar receptors in humans. However, in humans, it is found in the placenta to help promote the transport of maternal IgG to the growing fetus, and it has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at an acidic pH of 6.0-6.5, but not at a neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal lumen (inside the intestine) at a slightly acidic pH and ensure effective unidirectional transport to the basal side from a neutral to alkaline pH (pH 7.0-7.5). This receptor also plays a role in the rescue of IgG in adults through its occurrence in the endothelial cell endocytosis pathway. FcRn receptors in acidic endosomes bind IgG internalized by pinocytosis, recycling it to the cell surface and releasing it at the alkaline pH of the blood, thereby preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the greater half-life of IgG in the blood compared to other isotypes.

[0153] As used herein, the term "protein A" means a 56kDa MSCRAMM surface protein initially found in the cell wall of bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cell osmotic pressure, and a two-component system called ArlS-ArlR. Due to its ability to bind immunoglobulins, it is also used in biochemical research. It consists of 5 homologous Ig binding domains folded into a three-helical bundle. Each domain can bind to proteins from many mammalian species, most notably IgG. It binds to the heavy chain Fc region (overlapping with the conserved binding site of the FcRn receptor) of most immunoglobulins and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, the IgG molecule binds to bacteria by its Fc region rather than simply by its Fab region, thus destroying bacteria conditioning, complement activation, and phagocytosis.

[0154] As used herein, the term "Protein G" refers to an immunoglobulin binding protein expressed in group C and G Streptococcus bacteria that is very similar to Protein A but has a different specificity. It is a 65 kDa (G148 Protein G) and 58 kDa (C40 Protein G) cell surface protein that has been used to purify antibodies through its binding to the Fc region.

[0155] Specific embodiments of the present invention

[0156] As described herein, surprisingly, amino acid substitutions in the Fc region of a polypeptide or antibody provide the polypeptide or antibody with enhanced effector function, such as CDC and / or agonist activity. The present inventors have discovered that by introducing substitutions that enhance Fc-Fc interactions, such as substitutions at positions selected from the group consisting of E430, E345, and S440, and C1q binding substitutions, the Fc effector function of the polypeptide or antibody can be enhanced. Furthermore, the present inventors have discovered that the combination of Fc-Fc enhancing mutations and C1q binding substitutions can generate polypeptides, such as antibodies, having agonistic properties or enhanced agonistic properties.

[0157] In one aspect, the invention provides a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one Fc-Fc enhancing substitution at a position selected from the group consisting of E430, E345 and S440, with the proviso that the mutation in S440 is S440Y or S440W, and b) at least one C1q binding substitution, wherein the position corresponds to human IgG1 according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 1991 NIH Publication No. 91-3242).

[0158] In one aspect of the invention, a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen binding region is provided, wherein the Fc region comprises a) a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0159] Substitutions at positions corresponding to E430, E345 or S440Y or S440W substitutions are considered Fc-Fc enhancing substitutions according to the present invention.

[0160] Substitutions at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396 are considered to be CIq binding substitutions according to the present invention.

[0161] Mutations in one of the following positions, E430, E345, or S440, introduce enhanced Fc-Fc interactions and oligomerization into a polypeptide or antibody, provided that the mutation in S440 is S440Y or S440W. When the antigen-binding region of the polypeptide or antibody binds to the corresponding target antigen, enhanced oligomerization occurs. Enhanced oligomerization produces oligomers, such as hexamers. The generation of oligomeric structures (such as hexamers) has the effect of increasing Fc effector functions, such as CDC, by increasing the C1q binding affinity of the polypeptide. The combination of Fc-Fc enhancing mutations and C1q binding substitutions (such as one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396) produces polypeptides or antibodies with enhanced effector functions. The combination of Fc-Fc enhancing substitutions and C1q binding substitutions further has the effect of producing polypeptides or antibodies with agonistic activity. In one embodiment, the polypeptide or antibody may have increased agonist activity when compared to a parent polypeptide or parent antibody.

[0162] The polypeptides or antibodies according to the invention are of particular interest when activating intracellular signaling pathways by binding to cell surface receptors.

[0163] In one embodiment according to the invention, increased or enhanced Fc effector function or activity of a polypeptide or antibody having an Fc-Fc enhancing substitution and a C1q binding substitution is understood when the polypeptide or antibody is compared to a parent polypeptide or parent antibody, that is, the parent polypeptide or parent antibody does not have the substitution according to the invention but is otherwise identical.

[0164] The present invention allows for novel polypeptide- or antibody-based therapeutic agents to have increased properties such as CDC, agonist activity. That is, the polypeptides or antibodies according to the present invention have increased properties based on the Fc region (such as CDC), and they also have increased properties based on the antigen-binding region (such as agonist activity).

[0165] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, or b) a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333, and P396, wherein the positions correspond to human IgG1 according to EU numbering. In one embodiment of the invention, the polypeptide or antibody comprises at least one substitution at a position selected from E430, E345 or a S440Y or S440W substitution. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333, and P396.

[0166] In one embodiment of the invention, the polypeptide or antibody comprises substitutions at two or three positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0167] In one embodiment of the invention, the polypeptide or antibody comprises a C1q binding substitution selected from the group consisting of:

[0168] i) Two C1q binding substitutions at positions K326 and E333

[0169] ii) three C1q binding substitutions at positions K326, E333, and P396, and

[0170] iii) Three C1q binding substitutions at positions S267, H268 and S324.

[0171] In one embodiment of the invention, one or more C1q binding substitutions are at a position selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396, provided that the substitution at position G236 is not G236F, G236R, or G236Y.

[0172] In one embodiment of the invention, one or more C1q binding substitutions are at a position selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396, provided that the substitution at position S267 is not S267H, S267I, S267K, S267G.

[0173] In one embodiment of the invention, one or more C1q binding substitutions are at a position selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396, provided that the substitution at position H268 is not H268K, H268D, H268E.

[0174] In one embodiment of the invention, at least one Fc-Fc enhancing substitution is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, and S440Y.

[0175] In one embodiment of the invention, the at least one Fc-Fc enhancing substitution is selected from the group consisting of E430G, E430S, E430F, and E430T.

[0176] In one embodiment of the invention, the at least one Fc-Fc enhancing substitution is selected from the group consisting of E345K, E345Q, E345R, and E345Y.

[0177] In one embodiment of the invention, the polypeptide or antibody has at least an E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345R substitution. In one embodiment of the invention, the polypeptide or antibody has at least an S440Y substitution.

[0178] In one embodiment of the invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0179] In one embodiment of the invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T and substitutions at one or more positions selected from the group consisting of K326, E333 and P396.

[0180] In one embodiment of the invention, the Fc region comprises an E430G substitution and substitutions at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333, and P396.

[0181] In one embodiment of the invention, the polypeptide or antibody comprises at least a substitution selected from the group consisting of E345K, E345Q, E345R and E345Y, and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0182] In one embodiment of the invention, the polypeptide or antibody comprises at least a substitution selected from the group consisting of E345K, E345Q, E345R and E345Y and a substitution at one or more positions selected from the group consisting of K326, E333 and P396.

[0183] In one embodiment of the invention, the Fc region comprises an E345K substitution and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, 1332, E333, and P396.

[0184] In one embodiment of the invention, the Fc region comprises an E345R substitution and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, 1332, E333, and P396.

[0185] In one embodiment of the invention, the Fc region comprises at least a substitution selected from the group consisting of S440Y or S440W and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[0186] In one embodiment of the present invention, the Fc region comprises at least a substitution selected from the group consisting of S440Y or S440W and a substitution at one or more positions selected from the group consisting of K326, E333 and P396.

[0187] In one embodiment of the invention, the Fc region comprises an S440Y substitution and a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396.

[0188] Provided herein are embodiments that allow for enhanced C1q binding and / or agonistic properties of polypeptides or antibodies following cell surface antigen binding. In one embodiment, the polypeptide or antibody comprises enhanced agonistic properties. In one embodiment, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the above substitutions may be present in the first and / or second heavy chain.

[0189] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at one or more positions selected from the group consisting of K326, E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at one or more positions selected from the group consisting of K326, E333 and P396, such as two or three substitutions: K326, E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions P396 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions K326, E333 and P396.

[0190] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at one or more positions selected from the group consisting of K326, E333, and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at one or more positions selected from the group consisting of K326, E333, and P396, such as substitutions at two or three positions. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions K326, E333, and P396.

[0191] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at one or more positions selected from the group consisting of K326, E333, and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at one or more positions selected from the group consisting of K326, E333, and P396, such as substitutions at two or three positions. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions K326, E333, and P396.

[0192] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W and a substitution at one or more positions selected from the group consisting of K326, E333, and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W and a substitution at one or more positions selected from the group consisting of K326, E333, and P396, such as a substitution at two or three positions. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W and a substitution at K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W and a substitution at K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W, a substitution at E333, and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W, substitutions at positions K326, E333 and P396.

[0193] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position K326 selected from the group consisting of K326W, K326A, K326D, K326N, K326G, K326F, K326E, K326F, K326Y, K326H and K326M.

[0194] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E333 selected from the group consisting of E333S, E333A, E333T and E333G.

[0195] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E396 selected from the group consisting of E396L, E396I, E396V, E396Q, E396N and E396A.

[0196] In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three, substitutions selected from the group consisting of K326W, E333S, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three substitutions selected from the group consisting of K326W, E333A, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three substitutions selected from the group consisting of K326W, E333T, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three substitutions selected from the group consisting of K326A, E333S, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three substitutions selected from the group consisting of K326A, E333A, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises a K326A substitution. In one embodiment of the invention, the polypeptide or antibody comprises a K326W substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333T substitution. In one embodiment of the invention, the polypeptide or antibody comprises a P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326W and E333S. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326W and E333T. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326W and E333A. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326W and P396L. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326A and E333A. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326A and E333S. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326A and E333T. In one embodiment of the invention, the polypeptide or antibody comprises substitutions K326A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises substitutions E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions E333S and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326S, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333S and P396L.In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333T and P396L.

[0197] In one embodiment of the invention, the polypeptide or antibody comprises one or more C1q binding substitutions at positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises one or more substitutions, such as two or three substitutions, at positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267, H268, and S324.

[0198] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at one or more positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at one or more positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and a substitution at positions S267, H268, and S324.

[0199] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at one or more positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at one or more positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and a substitution at positions S267, H268, and S324.

[0200] In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at one or more positions selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at one or more positions selected from the group consisting of S267, H268, and S324, such as a substitution at two or three positions. In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises the substitution S440Y or S440W and a substitution at positions S267, H268, and S324.

[0201] In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises one or more substitutions, such as two or three substitutions, selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises an S267E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an H268F substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S324T substitution. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and H268F. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions H268F and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E, H268F, and S324T.

[0202] In one embodiment of the invention, the polypeptide or antibody comprises a) at least one substitution at a position selected from the group consisting of: E430, E345, or S440Y or S440W substitutions and b) at least one substitution selected from the group consisting of:

[0203] i.K326A,

[0204] ii.E333A,

[0205] iii.E333T,

[0206] iv.P396L,

[0207] v.E333S,

[0208] vi.K326W、E333S,

[0209] vii.K326W, E333T

[0210] viii.K326A, E333A,

[0211] ix.K326A, K333A, P396L,

[0212] x.S267E, H268F,

[0213] xi.S267E、S324T,

[0214] xii.H268F、S324T,

[0215] xiii.S267E, H268F, S324T, and

[0216] xiv.S324, I332.

[0217] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and at least one substitution selected from the group consisting of:

[0218] i.K326A,

[0219] ii.E333A,

[0220] iii.E333T,

[0221] iv.P396L,

[0222] v.E333S,

[0223] vi.K326W、E333S,

[0224] vii.K326W, E333T,

[0225] viii.K326A, E333A,

[0226] ix.K326A, K333A, P396L,

[0227] x.S267E, H268F,

[0228] xi.S267E、S324T,

[0229] xii.H268F、S324T,

[0230] xiii.S267E, H268F, S324T, and

[0231] xiv.S324, I332.

[0232] Provided herein are embodiments wherein one substitution is at position E430. In one embodiment, the substitution at position E430 is selected from the group consisting of E430G, E430S, E430F, E430T.

[0233] In one embodiment of the invention, the polypeptide or antibody comprises an E430G substitution and a substitution selected from one of the following groups:

[0234] i.K326A,

[0235] ii.E333A,

[0236] iii.E333T,

[0237] iv.P396L,

[0238] v.E333S,

[0239] vi.K326W、E333S,

[0240] vii.K326W, E333T,

[0241] viii.K326A, E333A,

[0242] ix.K326A, K333A, P396L,

[0243] x.S267E, H268F,

[0244] xi.S267E、S324T,

[0245] xii.H268F、S324T,

[0246] xiii.S267E, H268F, S324T, and

[0247] xiv.S324, I332.

[0248] In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, E333A, and P396L substitutions.

[0249] In one embodiment of the invention, the polypeptide or antibody comprises E430S, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430S, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430S, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430S, K326A, E333A, and P396L substitutions.

[0250] In one embodiment of the invention, the polypeptide or antibody comprises E430F, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430F, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises an E430F, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises E430F, K326A, E333A, and P396L substitutions.

[0251] In one embodiment of the invention, the polypeptide or antibody comprises E430T, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430T, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430T, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430T, K326A, E333A, and P396L substitutions.

[0252] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and at least one substitution selected from the group consisting of:

[0253] i.K326A,

[0254] ii.E333A,

[0255] iii.E333T,

[0256] iv.P396L,

[0257] v.E333S,

[0258] vi.K326W、E333S,

[0259] vii.K326W, E333T,

[0260] viii.K326A, E333A,

[0261] ix.K326A, K333A, P396L,

[0262] x.S267E, H268F,

[0263] xi.S267E、S324T,

[0264] xii.H268F、S324T,

[0265] xiii.S267E, H268F, S324T, and

[0266] xiv.S324, I332.

[0267] Provided herein are embodiments wherein one substitution is at position E345. In one embodiment, the substitution at position E345 is selected from the group consisting of E345K, E345Q, E345R, E345Y.

[0268] In one embodiment of the invention, the polypeptide or antibody comprises an E345K substitution and at least one substitution selected from the group consisting of:

[0269] i.K326A,

[0270] ii.E333A,

[0271] iii.E333T,

[0272] iv.P396L,

[0273] v.E333S,

[0274] vi.K326W、E333S,

[0275] vii.K326W, E333T,

[0276] viii.K326A, E333A,

[0277] ix.K326A, K333A, P396L,

[0278] x.S267E, H268F,

[0279] xi.S267E、S324T,

[0280] xii.H268F、S324T,

[0281] xiii.S267E, H268F, S324T, and

[0282] xiv.S324, I332.

[0283] In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, E333A, and P396L substitutions.

[0284] In one embodiment of the invention, the polypeptide or antibody comprises an E345R substitution and at least one substitution selected from the group consisting of:

[0285] i.K326A,

[0286] ii.E333A,

[0287] iii.E333T,

[0288] iv.P396L,

[0289] v.E333S,

[0290] vi.K326W、E333S,

[0291] vii.K326W, E333T,

[0292] viii.K326A, E333A,

[0293] ix.K326A, K333A, P396L,

[0294] x.S267E, H268F,

[0295] xi.S267E、S324T,

[0296] xii.H268F、S324T,

[0297] xiii.S267E, H268F, S324T, and

[0298] xiv.S324, I332.

[0299] In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, E333A, and P396L substitutions.

[0300] In one embodiment of the invention, the polypeptide or antibody comprises E345Q, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Q, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Q, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Q, K326A, E333A, and P396L substitutions.

[0301] In one embodiment of the invention, the polypeptide or antibody comprises E345Y, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Y, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Y, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345Y, K326A, E333A, and P396L substitutions.

[0302] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and at least one substitution selected from the group consisting of:

[0303] i.K326A,

[0304] ii.E333A,

[0305] iii.E333T,

[0306] iv.P396L,

[0307] v.E333S,

[0308] vi.K326W、E333S,

[0309] vii.K326W, E333T,

[0310] viii.K326A, E333A,

[0311] ix.K326A, K333A, P396L,

[0312] x.S267E, H268F,

[0313] xi.S267E、S324T,

[0314] xii.H268F、S324T,

[0315] xiii.S267E, H268F, S324T, and

[0316] xiv.S324, I332.

[0317] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y substitution and at least one substitution selected from the group consisting of:

[0318] i.K326A,

[0319] ii.E333A,

[0320] iii.E333T,

[0321] iv.P396L,

[0322] v.E333S,

[0323] vi.K326W、E333S,

[0324] vii.K326W, E333T,

[0325] viii.K326A, E333A,

[0326] ix.K326A, K333A, P396L,

[0327] x.S267E, H268F,

[0328] xi.S267E、S324T,

[0329] xii.H268F、S324T,

[0330] xiii.S267E, H268F, S324T, and

[0331] xiv.S324, I332.

[0332] In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326W, and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326A, and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326A, E333A, and P396L substitutions.

[0333] In one embodiment of the invention, the polypeptide or antibody comprises an S440W, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440W, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440W, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440W, K326A, E333A, and P396L substitution.

[0334] In one embodiment of the invention, the polypeptide or antibody further comprises one or more substitutions selected from the group consisting of G236A, I332E, S239D and I332E.

[0335] In one embodiment of the invention, the polypeptide or antibody further comprises at least two substitutions selected from the group consisting of:

[0336] i.G236A, I332E, and

[0337] ii.S239D, I332E.

[0338] In one embodiment of the invention, the polypeptide or antibody comprises a) at least one substitution at a position selected from the group consisting of: E430, E345, or S440Y or S440W substitutions and b) a substitution selected from one of the groups consisting of:

[0339] i.H268F, S324T, G236A, I332E,

[0340] ii.H268F, S324T, S239D, I332E,

[0341] iii. S267E, H268F, S324T, G236A, I332E, and

[0342] iv.S267E, H268F, S324T, S239D, I332E.

[0343] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and at least one substitution selected from the group consisting of:

[0344] i.H268F, S324T, G236A, I332E,

[0345] ii.H268F, S324T, S239D, I332E,

[0346] iii. S267E, H268F, S324T, G236A, I332E, and

[0347] iv.S267E, H268F, S324T, S239D, I332E.

[0348] In one embodiment of the invention, the polypeptide or antibody comprises an E430G substitution and at least one substitution selected from the group consisting of:

[0349] i.H268F, S324T, G236A, I332E,

[0350] ii.H268F, S324T, S239D, I332E,

[0351] iii. S267E, H268F, S324T, G236A, I332E, and

[0352] iv.S267E, H268F, S324T, S239D, I332E.

[0353] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and at least one substitution selected from the group consisting of:

[0354] i.H268F, S324T, G236A, I332E,

[0355] ii.H268F, S324T, S239D, I332E,

[0356] iii. S267E, H268F, S324T, G236A, I332E, and

[0357] iv.S267E, H268F, S324T, S239D, I332E.

[0358] In one embodiment of the invention, the polypeptide or antibody comprises an E345K substitution and at least one substitution selected from the group consisting of:

[0359] i.H268F, S324T, G236A, I332E,

[0360] ii.H268F, S324T, S239D, I332E,

[0361] iii. S267E, H268F, S324T, G236A, I332E, and

[0362] iv.S267E, H268F, S324T, S239D, I332E.

[0363] In one embodiment of the invention, the polypeptide or antibody comprises an E345R substitution and at least one substitution selected from the group consisting of:

[0364] i.H268F, S324T, G236A, I332E,

[0365] ii.H268F, S324T, S239D, I332E,

[0366] iii. S267E, H268F, S324T, G236A, I332E, and

[0367] iv.S267E, H268F, S324T, S239D, I332E.

[0368] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and at least one substitution selected from the group consisting of:

[0369] i.H268F, S324T, G236A, I332E,

[0370] ii.H268F, S324T, S239D, I332E,

[0371] iii. S267E, H268F, S324T, G236A, I332E, and

[0372] iv.S267E, H268F, S324T, S239D, I332E.

[0373] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y substitution and at least one substitution selected from the group consisting of:

[0374] i.H268F, S324T, G236A, I332E,

[0375] ii.H268F, S324T, S239D, I332E,

[0376] iii. S267E, H268F, S324T, G236A, I332E, and

[0377] iv.S267E, H268F, S324T, S239D, I332E.

[0378] In one embodiment of the invention, the polypeptide or antibody comprises one or more further substitutions. That is, in one embodiment of the invention, the polypeptide or antibody according to any aspect or embodiment described herein comprises one or more further substitutions in the Fc region.

[0379] In one embodiment of the invention, the polypeptide or antibody comprises a further substitution corresponding to position K439 or, where the Fc region does not comprise a substitution at position S440, the further substitution may be at position S440.

[0380] In one embodiment of the invention, the polypeptide or antibody comprises a further substitution in the polypeptide or antibody corresponding to position K439 or S440, with the proviso that the substitution at S440 is not S440Y or S440W.

[0381] A polypeptide or antibody comprising an Fc-Fc enhancing substitution according to the invention and a C1q binding substitution and a further substitution at position S440, such as S440K, does not form oligomers with a polypeptide or antibody comprising a substitution at S440, such as S440K. A polypeptide or antibody comprising an Fc-Fc enhancing substitution according to the invention and a C1q binding substitution and a further substitution at position K439, such as K439E, does not form oligomers with a polypeptide or antibody comprising a mutation at K439, such as K439E. In one embodiment of the invention, the further substitution is selected from S440K or K439E.

[0382] In one embodiment of the invention, the Fc region comprises a further substitution that is a hexamerization-inhibiting substitution corresponding to K439E or S440K in human IgG1 EU numbering. That is, in one embodiment of the invention, the Fc region comprises an Fc-Fc enhancing substitution such as E430G and a hexamerization-inhibiting substitution K439E. In one embodiment of the invention, the Fc region comprises an Fc-Fc enhancing substitution such as E345K and a hexamerization-inhibiting substitution K439E. In another embodiment of the invention, the Fc region comprises an Fc-Fc enhancing substitution such as E430G and a hexamerization-inhibiting substitution S440K. In one embodiment of the invention, the Fc region comprises an Fc-Fc enhancing substitution such as E345K and a hexamerization-inhibiting substitution S440K. Provided herein are embodiments that allow exclusive hexamerization between a combination of an antibody comprising a K439E substitution and an antibody comprising an S440K substitution. That is, the inhibitory substitutions K439E and S440K can be considered as complementary substitutions. The combination of antibodies with two different complementary hexamerization-inhibiting substitutions may be of particular interest in compositions with two different antibodies with different specificities.

[0383] In one embodiment of the invention, the polypeptide or antibody comprises a) a substitution at a position selected from the group consisting of E430, E345 and S440, with the proviso that the substitution of S440 is not S440Y or S440W and b) a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396 and c) a K439E substitution.

[0384] In one embodiment of the invention, the polypeptide or antibody comprises a) a substitution at a position selected from the group consisting of E430 and E345 and b) a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396 and c) an S440K substitution.

[0385] In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326W, E333S and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, E333A and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K333A, P396L and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, E333A, P396L and K439E substitutions.

[0386] In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326W, E333S, and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, E333A, and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K333A, P396L, and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, E333A, P396L, and K439E substitutions.

[0387] In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326W, E333S and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, E333A and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K333A, P396L and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, E333A, P396L and K439E substitutions.

[0388] In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326W, E333S and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326A, E333A and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K333A, P396L and K439E substitutions. In one embodiment of the invention, the polypeptide or antibody comprises S440Y, K326A, E333A, P396L and K439E substitutions.

[0389] In one embodiment of the invention, the polypeptide or antibody comprises a) a substitution at a position selected from the group consisting of: E430 and E345, and b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, and c) an S440K substitution.

[0390] In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326W, E333S, and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, E333A, and S440S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K333A, P396L, and S440S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E430G, K326A, E333A, P396L, and S440K substitutions.

[0391] In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326W, E333S, and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, E333A, and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K333A, P396L, and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345K, K326A, E333A, P396L, and S440K substitutions.

[0392] In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326W, E333S and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, E333A and S440S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K333A, P396L and S440K substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E345R, K326A, E333A, P396L and S440K substitutions.

[0393] The polypeptides or antibodies according to the present invention have at least an Fc-Fc enhancing substitution and one or more C1q binding substitutions, but may also have additional substitutions as described above to introduce additional functionality into the polypeptide or antibody. In one embodiment, the polypeptide or antibody comprises up to 10 substitutions, such as 9 substitutions, such as 8 substitutions, such as 7 substitutions, such as 6 substitutions, such as 5 substitutions, such as 4 substitutions, such as 3 substitutions, or such as 2 substitutions.

[0394] Thus, embodiments are provided that allow the polypeptides or antibodies of the invention to have additional substitutions that introduce additional features into the polypeptide or antibody. In addition, additional substitutions also allow for changes in the Fc region at positions that are not involved in Fc-Fc interactions and at positions that are not involved in Fc effector functions. In addition, additional substitutions can also be due to allelic variation.

[0395] In one embodiment of the invention, the polypeptide or antibody has at least 20% increased Fc effector function compared to a parent polypeptide or parent antibody that is identical to the antibody, except that it does not comprise the Fc-Fc enhancing substitutions and the C1q binding substitutions in the Fc region.

[0396] In one embodiment of the invention, the polypeptide or antibody has increased Fc effector function by at least 40%, at least 50%, or at least 60% compared to a parent polypeptide or parent antibody that is identical to the antibody, except that it does not comprise the Fc-Fc enhancing substitutions and the C1q binding substitutions in the Fc region.

[0397] In one embodiment of the invention, the polypeptide or antibody comprises increased Fc effector function.

[0398] In one embodiment of the present invention, the Fc effector function is selected from the group consisting of complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis, C1q binding, and FcγR binding. In one embodiment, the Fc effector function is FcγRIIIa signaling. That is, the second mutation according to the present invention is capable of reducing at least one Fc effector function.

[0399] In one embodiment of the invention, the polypeptide or antibody comprises agonist activity. That is, the polypeptide or antibody comprises agonist activity when compared to a parent polypeptide or parent antibody.

[0400] In one embodiment of the invention, the polypeptide or antibody comprises enhanced agonist activity. That is, the polypeptide or antibody comprises enhanced agonist activity compared to a parent polypeptide or parent antibody. In one embodiment of the invention, the polypeptide or antibody comprises enhanced agonist activity when compared to a polypeptide or antibody comprising the same Fc-Fc enhancing mutation but without the C1q binding mutation.

[0401] The agonist activity of TNFR-SF receptors requires exogenous cross-linking to achieve agonist activity. For example, this can be measured in an alternative assay using HEK293 cells containing a secretory reporter gene driven by NF-kB (e.g., pMetLuc-Reporter gene expressing luciferase, Clontech), which are stably transfected with the TNFR-SF receptor of interest. Cross-linking of the receptor leads to promoter activation and, for example, secretion of luciferase protein in the culture medium. At the desired time point, luciferase activity can be measured by transferring a culture medium sample and adding a substrate. Luciferase activity can be measured in a luminometer, which is a measure of agonist activity. An example of OX40 is shown in Zhang et al. J Biol Chem. 2016 Dec 30; 291(53): 27134-27146.

[0402] In one embodiment of the invention, the polypeptide or antibody comprises increased agonist activity. That is, the polypeptide or antibody comprises increased agonist activity compared to the parent polypeptide or parent antibody.

[0403] In one embodiment of the invention, the polypeptide is an antibody, a monospecific antibody, a bispecific antibody or a multispecific antibody. In one embodiment, the polypeptide is a monospecific polypeptide, a bispecific polypeptide or a multispecific polypeptide.

[0404] The polypeptide of the present invention is not limited to antibodies with natural Fc domains, such as human Fc domain antibodies, but it can also be an antibody with mutations other than the mutations of the present invention, such as, for example, mutations that affect glycosylation or enable the antibody to become a bispecific antibody. The term "natural antibody" refers to any antibody that does not comprise any genetically introduced mutations. Antibodies comprising naturally occurring modifications, such as antibodies of different allotypes, are understood to be "natural antibodies" within the meaning of the present invention and are therefore understood to be parent antibodies. Such antibodies can serve as templates for at least two substitutions according to the present invention, thereby providing antibodies of the present invention. An example of a parent antibody comprising substitutions other than the substitutions of the present invention is a bispecific antibody as described in WO2011 / 131746 (Genmab), which utilizes reducing conditions to promote the exchange of half molecules of two antibodies comprising an IgG4-like CH3 region, thereby forming a bispecific antibody without the formation of aggregates. Other examples of parent antibodies include, but are not limited to, bispecific antibodies, such as heterodimeric bispecifics: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinatneurosciences Corporation); FcΔAdp (Regeneron); protrusion-into-cavity (Genentech); electrostatic manipulation (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof.

[0405] In one embodiment, the polypeptide or antibody comprises an Fc region comprising an R435H substitution.

[0406] In one embodiment, the invention provides a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) a substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, and c) an R435H substitution, wherein these positions correspond to human IgG1 according to EU numbering.

[0407] The polypeptide or antibody can be any human antibody of any isotype, e.g., IgGl, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA, optionally a fully human antibody, e.g., a fully human IgGl antibody.

[0408] In one embodiment of the invention, the polypeptide or antibody is a human IgG1 antibody, such as an IgG1m(za) or IgG1m(f) allotype.

[0409] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region that is a human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or mixed isotypes. That is, the Fc region of the polypeptide or antibody according to the invention comprises at least a first and a second mutation introduced into the Fc region corresponding to a human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA isotype, or mixed isotypes. In one embodiment of the invention, the Fc region is a mixed isotype selected from the group consisting of IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, and IgG3 / IgG4. In mixed isotypes, the Fc region is composed of amino acid sequences from more than one isotype.

[0410] In one embodiment of the invention, the Fc region is of human IgGl, IgG2, IgG3, IgG4 isotype or mixed isotypes.

[0411] In one embodiment of the invention, the Fc region is human IgG.

[0412] In a preferred embodiment of the invention, the polypeptide or antibody has an Fc region that is of the human IgG1 isotype.

[0413] In one embodiment of the invention, the polypeptide or antibody has an Fc region that is of the IgG1m(f), IgG1m(a), IgG1m(z), IgG1m(x) allotype or mixed allotypes.

[0414] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as shown in SEQ ID NO: 73, 74, 75, 76, 89, 168, 169 or 170, wherein the Fc region comprises a substitution in a position selected from the group corresponding to E430, E345 and S440, with the proviso that the substitution in S440 is S440Y or S440W, and a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324, K326, 1332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0415] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as shown in SEQ ID NO: 77, 78 or 90, wherein the Fc region comprises a substitution at one or more positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0416] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as shown in SEQ ID NO: 80, 82, 83, 84, 87 or 88.

[0417] In one embodiment of the invention, the polypeptide is a human antibody, a humanized antibody or a chimeric antibody.

[0418] Multispecific antibodies

[0419] The polypeptide of the present invention is not limited to antibodies with natural Fc domains, such as human Fc domain antibodies, but it can also be an antibody with mutations other than the mutations of the present invention, such as mutations that affect glycosylation or enable the antibody to become a multispecific antibody or a bispecific antibody. The term "natural antibody" refers to any antibody that does not comprise any genetically introduced mutations. Therefore, antibodies comprising naturally occurring modifications, such as antibodies of different allotypes are understood to be "natural antibodies" within the meaning of the present invention and are therefore understood to be parent antibodies. Such antibodies can serve as templates for at least two substitutions according to the present invention, thereby providing antibodies of the present invention. An example of a parent antibody comprising substitutions other than the substitutions of the present invention is a bispecific antibody as described in WO2011 / 131746 (Genmab), which utilizes reducing conditions to promote the exchange of half molecules of two antibodies comprising an IgG4-like CH3 region, thereby forming a bispecific antibody without the formation of aggregates. Other examples of parent antibodies include, but are not limited to, bispecific antibodies, such as heterodimeric bispecifics: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); protrusion-into-cavity (Genentech); electrostatic manipulation (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof.

[0420] It will be understood that any embodiment of the invention described herein may be used in conjunction with the multispecific antibodies described below.

[0421] Thus, in one embodiment, the variant of the invention is an antibody selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0422] In a specific embodiment, the bispecific antibody has the format described in WO 2011 / 131746.

[0423] The bispecific antibody of the present invention is not limited to a particular format, and it may be any of those described herein.

[0424] In another aspect, the invention relates to a polypeptide or antibody that is a bispecific polypeptide or antibody comprising a first heavy chain and a first antigen binding region of an immunoglobulin and a second polypeptide or antibody comprising a second heavy chain and a second antigen binding region of an immunoglobulin, wherein the first and second antigen binding regions bind to different epitopes on the same or different antigens, and wherein the first and / or second heavy chain comprises:

[0425] a) a substitution in a position selected from the group corresponding to: E430, E345 and S440, with the proviso that the substitution in S440 is S440Y or S440W,

[0426] b) one or more substitutions at positions selected from the group consisting of G236, S267, H268, S324, K326, I332, E333 and P396, and

[0427] c) wherein the first heavy chain comprises a further substitution in a position selected from the group consisting of K409, T366, L368, K370, D399, F405 and Y407; and

[0428] The second heavy chain comprises a further substitution in a position selected from the group consisting of: F405, T366, L368, K370, D399, Y407 and K409, and wherein the further mutation in the first polypeptide is different from the further mutation in the second polypeptide.

[0429] In one aspect, the invention provides a polypeptide or antibody comprising the Fc region of a human IgG, comprising a first heavy chain and a second antigen binding region, a second heavy chain and a second antigen binding region, wherein the first and second heavy chains comprise a) a substitution at a position selected from the group consisting of: E430, E345 and S440, with the proviso that the substitution in S440 is S440Y or S440W and b) a substitution at one or more positions selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333P396 and c) a further substitution in position F405 or K409; wherein the further substitution is different from the first heavy chain and the second heavy chain such that if the first heavy chain has a substitution in position F405, the second heavy chain has a substitution in K409 and vice versa.

[0430] Thus, embodiments are provided wherein the first heavy chain and the second heavy chain are not identical because the (c) further mutation is not located in the same position in the first and second heavy chain.

[0431] It will be understood that any embodiment of the invention described herein may be used in conjunction with the multispecific antibodies described below.

[0432] Thus, in one embodiment, the variant of the invention is an antibody selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0433] In a specific embodiment, the bispecific antibody has the format described in WO 2011 / 131746.

[0434] In a specific embodiment of the invention, the first heavy chain comprises a further substitution corresponding to K409, such as K409R; and

[0435] The second heavy chain contains further substitutions corresponding to F405, such as F405L.

[0436] In one embodiment of the invention, the first and / or second heavy chain comprises

[0437] a) corresponding to the substitution at position E430,

[0438] b) substitution at one or more positions selected from the group consisting of G236, S267, H268, S324, K326, I332, E333 and P396, and

[0439] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0440] In one embodiment of the invention, the first and / or second heavy chain comprises

[0441] a) corresponding to substitution at position E345,

[0442] b) substitution at one or more positions selected from the group consisting of G236, S267, H268, S324, K326, I332, E333 and P396, and

[0443] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0444] In one embodiment of the invention, the first and / or second Fc heavy chain comprises

[0445] a) a substitution corresponding to S440Y or S440W,

[0446] b) substitution at one or more positions selected from the group consisting of G236, S267, H268, S324, K326, I332, E333 and P396, and

[0447] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0448] In one embodiment of the invention, the first and / or second Fc heavy chain comprises

[0449] a) corresponding to the substitution of E430G,

[0450] b) two substitutions corresponding to K326W, E333S, and

[0451] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0452] In one embodiment of the invention, the first and / or second Fc heavy chain comprises

[0453] a) corresponding to the substitution of E430G,

[0454] b) two substitutions corresponding to K326W, E333T, and

[0455] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0456] In one embodiment of the invention, the first and / or second Fc heavy chain comprises

[0457] a) corresponding to the substitution of E430G,

[0458] b) two substitutions corresponding to K326A, E333A, and

[0459] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0460] In one embodiment of the invention, the first and / or second heavy chain comprises

[0461] a) corresponding to the substitution of E430G,

[0462] b) two substitutions corresponding to K333A, P396L, and

[0463] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0464] In one embodiment of the invention, the first and / or second heavy chain comprises

[0465] a) corresponding to the substitution of E430G,

[0466] b) three substitutions corresponding to K326A, E333A, P396L, and

[0467] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0468] In one embodiment of the invention, the first and / or second heavy chain comprises

[0469] a) corresponding to the substitution of E430G,

[0470] b) three substitutions corresponding to K326A, E333T, P396L, and

[0471] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0472] In one embodiment of the invention, the first and / or second heavy chain comprises

[0473] a) a substitution corresponding to E345K,

[0474] b) two substitutions corresponding to K326W, E333S, and

[0475] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0476] In one embodiment of the invention, the first and / or second heavy chain comprises

[0477] a) a substitution corresponding to E345K,

[0478] b) two substitutions corresponding to K326W, E333T, and

[0479] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0480] In one embodiment of the invention, the first and / or second heavy chain comprises

[0481] a) a substitution corresponding to E345K,

[0482] b) two substitutions corresponding to K326A, E333A, and

[0483] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0484] In one embodiment of the invention, the first and / or second heavy chain comprises

[0485] a) a substitution corresponding to E345K,

[0486] b) two substitutions corresponding to K333A, P396L, and

[0487] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0488] In one embodiment of the invention, the first and / or second heavy chain comprises

[0489] a) a substitution corresponding to E345K,

[0490] b) three substitutions corresponding to K326A, E333A, P396L, and

[0491] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0492] In one embodiment of the invention, the first and / or second heavy chain comprises

[0493] a) a substitution corresponding to E345K,

[0494] b) three substitutions corresponding to K326A, E333T, P396L, and

[0495] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0496] In one embodiment of the invention, the first and / or second heavy chain comprises

[0497] a) a substitution corresponding to E345R,

[0498] b) two substitutions corresponding to K326W, E333S, and

[0499] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0500] In one embodiment of the invention, the first and / or second heavy chain comprises

[0501] a) a substitution corresponding to E345R,

[0502] b) two substitutions corresponding to K326W, E333T, and

[0503] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0504] In one embodiment of the invention, the first and / or second heavy chain comprises

[0505] a) a substitution corresponding to E345R,

[0506] b) two substitutions corresponding to K326A, E333A, and

[0507] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0508] In one embodiment of the invention, the first and / or second heavy chain comprises

[0509] a) a substitution corresponding to E345R,

[0510] b) two substitutions corresponding to K333A, P396L, and

[0511] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0512] In one embodiment of the invention, the first and / or second heavy chain comprises

[0513] a) a substitution corresponding to E345R,

[0514] b) three substitutions corresponding to K326A, E333A, P396L, and

[0515] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0516] In one embodiment of the invention, the first and / or second heavy chain comprises

[0517] a) a substitution corresponding to S440Y,

[0518] b) two substitutions corresponding to K326W, E333S, and

[0519] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0520] In one embodiment of the invention, the first and / or second heavy chain comprises

[0521] a) a substitution corresponding to S440Y,

[0522] b) two substitutions corresponding to K326W, E333T, and

[0523] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0524] In one embodiment of the invention, the first and / or second heavy chain comprises

[0525] a) a substitution corresponding to S440Y,

[0526] b) two substitutions corresponding to K326A, E333A, and

[0527] c) wherein the first heavy chain comprises a further substitution corresponding to K409R, and the second heavy chain comprises a further substitution corresponding to F405L.

[0528] In one embodiment of the invention, the first and / or second heavy chain comprises

[0529] a) a substitution corresponding to S440Y,

[0530] b) two substitutions corresponding to K333A, P396L, and

[0531] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0532] In one embodiment of the invention, the first and / or second heavy chain comprises

[0533] a) a substitution corresponding to S440Y,

[0534] b) three substitutions corresponding to K326A, E333A, P396L, and

[0535] c) wherein the first heavy chain comprises a further substitution corresponding to: K409R; and the second heavy chain comprises a further substitution corresponding to: F405L.

[0536] Targets and methods of use

[0537] The polypeptide or antibody according to the present invention can bind to a target that activates a signal transduction pathway. In one embodiment, the target is a target that activates, inhibits, regulates and / or modulates a signal transduction pathway. Examples of targets that may be particularly suitable as targets according to the present invention are cell surface receptors and ligands.

[0538] For example, cell surface receptors include receptors belonging to receptor families such as 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-anchored receptor family, the tyrosine phosphatase receptor family, the adhesion factor family, and the hormone receptor family. Various references concerning receptors belonging to these receptor families and their characteristics are available, including, for example, Cooke BA, 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. Rev. Immunol., 10: 295-331; Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396; Fantl W I., et al. (1993) Annu. Rev. Biochem., 62: 453-481; Smith C A., et al. (1994) Cell, 76: 959-962; Flower D R. (1999) Biochim. Biophys. Acta, 1422: 207-234; and M. Miyasaka, ed., Cell Technology, Supplementary Volume, Handbook Series, "Handbook for Adhesion Factors" (1994) (Shujunsha, Tokyo, Japan).

[0539] In one embodiment of the invention, the polypeptide or antibody comprises an antigen binding region, wherein the antigen binding region binds to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) or the G protein coupled receptor (GPCR) superfamily.

[0540] In one embodiment of the present invention, the polypeptide or antibody binds to a cell surface receptor, including, for example, a hormone receptor and a cytokine receptor. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation control 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 (IFN-α) receptor, interferon-β (IFN-β) receptor, interferon-γ (IFN-γ) receptor, growth hormone (GH) receptor, insulin receptor, blood stem cell proliferation factor (SCF) receptor, vascular epidermal 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 (TGF-β) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M (OSM) receptor and Notch family receptors.

[0541] 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) through their cysteine-rich extracellular domains. TNF receptors form trimeric complexes in the plasma membrane. TNFRSF includes the following 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(UniprotQ9UBN6),DcR3(Uniprot O95407),OPG(Uniprot O00300),TROY(Uniprot Q9NS68),XEDAR(Uniprot Q9HAV5),LTbR(Uniprot P36941),HVEM(Uniprot Q92956),TWEAKR(UniprotQ9NP84),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).

[0542] Some TNFRSFs are involved in apoptosis and contain intracellular death domains, such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR. Other TNFRSFs are involved in other signal transduction pathways, such as proliferation, survival, and differentiation, such as DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT. TNF receptors are expressed in a wide variety of mammalian tissues, particularly in leukocytes.

[0543] In one embodiment of the invention, the antigen binding region binds to a TNFR-SF member selected from the group consisting of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, NGFR, OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR.

[0544] In one embodiment of the invention, the antigen binding region binds to a member of the TNFR-SF. In one embodiment of the invention, the antigen binding region binds to a member of the TNFR-SF that does not contain an intracellular death domain. In one embodiment of the invention, the TNFR-SF is selected from the group consisting of: OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. In one embodiment of the invention, the TNFR-SF is selected from the group consisting of: FAS, DR4, DR4, TNFR1, DR6, DR3, EDAR, and NGFR.

[0545] In one embodiment of the invention, the antibody comprises an antigen binding region that binds OX40, wherein the IgG1 Fc region comprises

[0546] a.E430G replaced, and

[0547] b.Replaced by K326W and E333S.

[0548] In one embodiment of the invention, the antibody comprises an antigen binding region that binds CD40, wherein the IgG1 Fc region comprises

[0549] a.E430G replaced, and

[0550] b.Replaced by K326W and E333S.

[0551] In one embodiment of the invention, the antibody comprises an antigen binding region that binds CD137, wherein the IgG1 Fc region comprises

[0552] a.E430G replaced, and

[0553] b.Replaced by K326W and E333S.

[0554] In one embodiment of the invention, the antibody comprises an antigen binding region that binds GITR, wherein the IgG Fc region comprises

[0555] a.E430G replaced, and

[0556] b.Replaced by K326W and E333S.

[0557] In one embodiment of the invention, the antibody comprises an antigen binding region that binds GITR, wherein the IgG1 Fc region comprises

[0558] a.E430G replaced, and

[0559] b.Replaced by K326W and E333S.

[0560] In one embodiment of the invention, the antibody comprises an antigen binding region that binds GITR, wherein the IgG2 Fc region comprises

[0561] a.E430G replaced, and

[0562] b.Replaced by K326W and E333S.

[0563] The polypeptides or antibodies according to the present invention may bind to any target, examples of such targets or antigens according to the present invention may be directed against: TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, EDAR, DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, RELT.

[0564] In one embodiment of the invention, the antibody comprises an antigen binding region that binds FAS, wherein the IgG1 Fc region comprises

[0565] a.E430G replaced, and

[0566] b.Replaced by K326W and E333S.

[0567] In one embodiment of the present invention, the antibody comprises an antigen binding region that binds to DR5, wherein the IgG1 Fc region comprises

[0568] a.E430G replaced, and

[0569] b. K326A and E333A substitutions.

[0570] In one embodiment of the present invention, the antibody comprises an antigen binding region that binds to DR5, wherein the IgG1 Fc region comprises

[0571] a.E430G replaced, and

[0572] b. K326A and E333T substitutions.

[0573] In one embodiment of the present invention, the antibody comprises an antigen binding region that binds to DR5, wherein the IgG1 Fc region comprises

[0574] a.E430G replaced, and

[0575] b.K326A substitution.

[0576] In one embodiment of the present invention, the antibody comprises an antigen binding region that binds to DR5, wherein the IgG1 Fc region comprises

[0577] a.E430G replaced, and

[0578] b.E333A substitution.

[0579] In one embodiment of the present invention, the antibody comprises an antigen binding region that binds to DR5, wherein the IgG1 Fc region comprises

[0580] a.E430G replaced, and

[0581] b.Replaced by K326W and E333S.

[0582] In one embodiment of the invention, the antibody comprises an antigen binding region that binds CD20, wherein the IgG1 Fc region comprises

[0583] a.E430G replaced, and

[0584] b.Replaced by K326W and E333S.

[0585] In one embodiment of the invention, the antigen binding region binds to a member of the tumor necrosis factor superfamily (TNF-SF):

[0586] In one embodiment of the invention, the antigen binding region binds to a TNF-SF member selected from the group consisting of lymphotoxin beta (TNF-C), OX40L, CD154, FasL, CD70, CD153, RANKL, APRIL and BAFF.

[0587] In one embodiment of the present invention, the polypeptide or antibody binds to a cell surface receptor, including, for example, a hormone receptor and a cytokine receptor. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation control 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 (IFN-α) receptor, interferon-β (IFN-β) receptor, interferon-γ (IFN-γ) receptor, growth hormone (GH) receptor, insulin receptor, blood stem cell proliferation factor (SCF) receptor, vascular epidermal 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 (TGF-β) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M (OSM) receptor and Notch family receptors.

[0588] In one embodiment of the invention, the antigen binding region binds to a cell surface receptor selected from the group consisting of CTLA-4, PD1, TIM-3, LAG-3, ICOS, CD28 and PDL-1.

[0589] The polypeptides or antibodies according to the invention may bind to any target, examples of such targets or antigens being described above.

[0590] Methods for increasing the agonist activity of polypeptides or antibodies

[0591] It should be understood that the embodiments described herein with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region and an antigen binding region of an immunoglobulin. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen binding region of an immunoglobulin, and a second polypeptide or antibody comprising a second Fc region and a second antigen binding region of an immunoglobulin.

[0592] In one aspect, the invention relates to methods of increasing the agonistic activity of a polypeptide or antibody by introducing Fc-Fc enhancing substitutions and C1q binding substitutions.

[0593] In one aspect, the invention relates to a method for increasing the agonist activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, the method comprising a) introducing at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitutions, and b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0594] The effect of introducing a) at least one substitution according to the invention in one of the following positions E430, E345 or S440 into a polypeptide or antibody on enhanced Fc-Fc interaction. The effect of introducing b) one or more substitutions according to the invention in one of the following positions G236, S239, S267, H268, S324, K326, I332, E333 and P396 into a polypeptide or antibody on increased agonist activity.

[0595] In another aspect, the invention relates to a method for increasing the agonist activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one substitution at a position selected from E430, E345: or a S440Y or S440W substitution, and the method comprises b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0596] In one embodiment, the invention relates to a method for increasing the agonist activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and the method comprises b) introducing at least two substitutions at positions selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0597] Increasing the agonist activity of a polypeptide or antibody according to the present invention should be understood as increasing the agonist activity of the polypeptide or antibody compared to the parent polypeptide or antibody, or increasing the agonist activity of the polypeptide or antibody can also refer to when the polypeptide or antibody is compared to a polypeptide or antibody comprising an Fc-Fc enhancing mutation rather than a C1q binding mutation. Thus, it is understood that the polypeptide or antibody can be compared to a parent polypeptide or parent antibody having the same antigen binding region and an Fc region without an Fc-Fc enhancing substitution and a C1q binding substitution, or the polypeptide or antibody can be compared to a polypeptide or antibody having the same antigen binding region and an Fc region with an Fc-Fc enhancing substitution rather than a C1q binding substitution.

[0598] In one embodiment of the present invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position G236 is not G236F, G236R, G236Y.

[0599] In one embodiment of the present invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position S267 is not S267H, S267I, S267K, S267G.

[0600] In one embodiment of the invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position H268 is not H268K, H268D, H268E.

[0601] In one embodiment of the invention, at least one substitution is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y. Thus, embodiments are provided wherein the substitutions enhance Fc-Fc interactions.

[0602] In one embodiment of the present invention, at least one substitution is selected from the group consisting of E430G, E430S, E430F, E430T.

[0603] In one embodiment of the invention, at least one substitution is selected from the group consisting of E345K, E345Q, E345R, E345Y.

[0604] In one embodiment of the invention, the polypeptide or antibody has at least an E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345R substitution. In one embodiment of the invention, the polypeptide or antibody has at least an S440Y substitution.

[0605] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0606] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T, the method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T and P396L.

[0607] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[0608] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0609] In one embodiment, the invention relates to a method of increasing the agonistic activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, said method comprising introducing a substitution selected from one of the following groups:

[0610] i) K326W and E333S,

[0611] ii) K326W and E333T,

[0612] iii) K326A and E333A,

[0613] iv) K326A, E333A and P396L,

[0614] v)K326W,

[0615] vi) E333S, and

[0616] vii)E333T.

[0617] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E345K, E345Q, E345R and E345Y, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0618] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E345K, E345Q, E345R and E345Y, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0619] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0620] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0621] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, said method comprising introducing a substitution from one of the following groups:

[0622] i) K326W and E333S,

[0623] ii) K326W and E333T,

[0624] iii) K326A and E333A,

[0625] iv) K326A, E333A and P396L,

[0626] v)K326W,

[0627] vi) E333S, and

[0628] vii)E333T.

[0629] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0630] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0631] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, said method comprising introducing a substitution selected from one of the following groups:

[0632] i) K326W and E333S,

[0633] ii) K326W and E333T,

[0634] iii) K326A and E333A,

[0635] iv) K326A, E333A and P396L,

[0636] v)K326W,

[0637] vi) E333S, and

[0638] vii)E333T.

[0639] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of S440Y and S440W, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0640] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of S440W and S440W, the method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0641] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0642] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0643] In one embodiment, the invention relates to a method of increasing the agonist activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising introducing a substitution from one of the following groups:

[0644] i) K326W and E333S,

[0645] ii) K326W and E333T,

[0646] iii) K326A and E333A,

[0647] iv) K326A, E333A and P396L,

[0648] v)K326W,

[0649] vi) E333S, and

[0650] vii)E333T.

[0651] Provided herein are embodiments that allow for increased agonistic properties of polypeptides or antibodies following cell surface antigen binding. In one embodiment, the polypeptide or antibody comprises increased agonistic properties. In one embodiment, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the substitutions mentioned above may be present in the first and / or second heavy chain.

[0652] In one embodiment of the invention, one or more substitutions are selected from the group consisting of K326, E333 and P396. In one embodiment of the invention, one or more substitutions, such as two or three substitutions, are selected from the group consisting of K326, E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions P396 and E333. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326, E333 and P396.

[0653] In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three, substitutions selected from the group consisting of K326A, K326W, E333S, E333A, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises a K326A substitution. In one embodiment of the invention, the polypeptide or antibody comprises a K326W substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises a P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and E333S. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and E333A. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and E333A. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and E333S. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions E333S and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326S, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333S and P396L.

[0654] In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, one or more substitutions, such as two or three substitutions, are selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267, H268, and S324.

[0655] In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three, substitutions selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises an S267E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an H268F substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S324T substitution. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and H268F. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions H268F and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E, H268F, and S324T.

[0656] In one embodiment, the invention relates to a method, wherein the Fc region comprises one or more further substitutions.

[0657] In one embodiment, the present invention relates to a method wherein the Fc region comprises a further substitution at one of the following positions in human IgG1 according to EU encoding: S440 or K439. In one embodiment of the invention, the Fc region comprises a further substitution corresponding to one of the following positions: S440 or K439, with the proviso that if the Fc-Fc enhancing substitution is in S440, the further substitution is not in S440. A polypeptide or antibody comprising an Fc-Fc enhancing substitution according to the present invention and a C1q binding substitution and a further substitution at position S440, such as S440K, does not form oligomers with a polypeptide or antibody comprising a mutation at position S440, such as S440K. A polypeptide or antibody comprising an Fc-Fc enhancing substitution according to the present invention and a C1q binding substitution and a further substitution at position K439, such as K439E, does not form oligomers with a polypeptide or antibody comprising a mutation at position K439, such as K439E. Provided herein are methods that allow oligomer formation between polypeptides or antibodies, wherein the first polypeptide or antibody comprises a K439E substitution and the second polypeptide or antibody comprises an S440K substitution. In this way, oligomers such as for example hexamers can be forced to form in certain patterns of the first and second polypeptides. This can be of interest in methods where the polypeptides bind to different targets or epitopes and oligomers should be formed in combinations of these different targets or epitopes.

[0658] In one embodiment, the invention relates to a method, wherein the further substitution is selected from S440K or K439E.

[0659] In one embodiment, the invention relates to a method for increasing agonistic activity, wherein the agonistic activity is increased by at least 20% compared to a parent polypeptide or parent antibody that is the same polypeptide or antibody, or a polypeptide or antibody having the same Fc-Fc enhancing substitutions but not the C1q binding substitutions. In another embodiment of the invention, the polypeptide or antibody has an increased agonistic activity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a parent polypeptide or parent antibody, or alternatively a polypeptide or antibody having the same Fc-Fc enhancing substitutions but not the C1q binding substitutions.

[0660] Methods for increasing CDC activity

[0661] In one aspect, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody by introducing Fc-Fc enhancing substitutions and C1q binding substitutions.

[0662] In one aspect, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, the method comprising a) introducing at least one substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0663] The effect of introducing a) at least one substitution according to the invention into a polypeptide or antibody on enhanced Fc-Fc interaction, said substitution being in one of the following positions: E430, E345 or S440. The effect of introducing b) one or more substitutions according to the invention into a polypeptide or antibody on increased CDC activity, said substitution being in one of the following positions: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[0664] In another aspect, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and the method comprises b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the position corresponds to human IgG1 according to EU numbering.

[0665] In one embodiment, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region and an antigen binding region of a human immunoglobulin, wherein the Fc region comprises a) at least one substitution at a position selected from E430, E345 or a S440Y or S440W substitution, and the method comprises b) introducing at least two substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396, wherein the positions correspond to human IgG1 according to EU numbering.

[0666] Increasing the agonist activity of a polypeptide or antibody according to the present invention should be understood as increasing the CDC activity of the polypeptide or antibody compared to the parent polypeptide or antibody, or increasing the agonist activity of a polypeptide or antibody can also refer to when the polypeptide or antibody is compared to a polypeptide or antibody comprising an Fc-Fc enhancing mutation but without a C1q binding mutation. Thus, it is understood that a polypeptide or antibody can be compared to a parent polypeptide or antibody having the same antigen binding region and an Fc region without an Fc-Fc enhancing substitution and without a C1q binding substitution, or a polypeptide or antibody can be compared to a polypeptide or antibody having the same antigen binding region and an Fc region with an Fc-Fc enhancing substitution but without a C1q binding substitution.

[0667] In one embodiment of the present invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position G236 is not G236F, G236R, G236Y.

[0668] In one embodiment of the present invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position S267 is not S267H, S267I, S267K, S267G.

[0669] In one embodiment of the invention, one or more substitutions are selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the proviso that the substitution at position H268 is not H268K, H268D, H268E.

[0670] In one embodiment of the invention, at least one substitution is selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y. Thus, embodiments are provided wherein the substitutions enhance Fc-Fc interactions.

[0671] In one embodiment of the present invention, at least one substitution is selected from the group consisting of E430G, E430S, E430F, E430T.

[0672] In one embodiment of the invention, at least one substitution is selected from the group consisting of E345K, E345Q, E345R, E345Y.

[0673] In one embodiment of the invention, the polypeptide or antibody has at least an E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least an E345R substitution. In one embodiment of the invention, the polypeptide or antibody has at least an S440Y substitution.

[0674] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0675] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E430G, E430S, E430F and E430T, the method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0676] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[0677] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0678] In one embodiment, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E430G substitution, said method comprising introducing a substitution from one of the following groups:

[0679] i) K326W and E333S,

[0680] ii) K326W and E333T,

[0681] iii) K326A and E333A,

[0682] iv) K326A, E333A and P396L,

[0683] v)K326W,

[0684] vi) E333S, and

[0685] vii)E333T.

[0686] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E345K, E345Q, E345R and E345Y, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0687] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of E345K, E345Q, E345R and E345Y, the method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0688] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0689] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0690] In one embodiment, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345K substitution, said method comprising introducing a substitution from one of the following groups:

[0691] i) K326W and E333S,

[0692] ii) K326W and E333T,

[0693] iii) K326A and E333A,

[0694] iv) K326A, E333A and P396L,

[0695] v)K326W,

[0696] vi) E333S, and

[0697] vii)E333T.

[0698] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0699] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0700] In one embodiment, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an E345R substitution, said method comprising introducing a substitution from one of the following groups:

[0701] i) K326W and E333S,

[0702] ii) K326W and E333T,

[0703] iii) K326A and E333A,

[0704] iv) K326A, E333A and P396L,

[0705] v)K326W,

[0706] vi) E333S, and

[0707] vii)E333T.

[0708] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of S440Y and S440W, the method comprising introducing one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0709] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises at least one substitution selected from the group consisting of S440W and S440W, the method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0710] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324K326, I332, E333 and P396.

[0711] In one embodiment, the invention relates to a method of increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising introducing one or more substitutions selected from the group consisting of K326A, K326W, E333A, E333S, E333T, and P396L.

[0712] In one embodiment, the invention relates to a method for increasing the CDC activity of a polypeptide or antibody, wherein the Fc region comprises an S440Y substitution, said method comprising introducing a substitution from one of the following groups:

[0713] i) K326W and E333S,

[0714] ii) K326W and E333T,

[0715] iii) K326A and E333A,

[0716] iv) K326A, E333A and P396L,

[0717] v)K326W,

[0718] vi) E333S, and

[0719] vii)E333T.

[0720] Provided herein are embodiments that allow for increased CDC activity of polypeptides or antibodies following cell surface antigen binding. In one embodiment, the polypeptide or antibody comprises increased CDC activity. In one embodiment, the polypeptide or antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the substitutions mentioned above may be present in the first and / or second heavy chain.

[0721] In one embodiment of the invention, one or more substitutions are selected from the group consisting of K326, E333 and P396. In one embodiment of the invention, one or more substitutions are selected from the group consisting of K326, E333 and P396, such as two or three substitutions. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions P396 and E333. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions K326, E333 and P396.

[0722] In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three, substitutions selected from the group consisting of K326A, K326W, E333S, E333A, and P396L. In one embodiment of the invention, the polypeptide or antibody comprises a K326A substitution. In one embodiment of the invention, the polypeptide or antibody comprises a K326W substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises a P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and E333S. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and E333A. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and E333A. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and E333S. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions E333S and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326A, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326S, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions K326W, E333S and P396L.

[0723] In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267, H268, and S324. In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267, H268, and S324, such as two or three substitutions. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions H268 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at positions S267, H268, and S324.

[0724] In one embodiment of the invention, one or more substitutions are selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises one or more, such as two or three, substitutions selected from the group consisting of S267E, H268F, and S324T. In one embodiment of the invention, the polypeptide or antibody comprises an S267E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an H268F substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S324T substitution. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and H268F. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions H268F and S324T. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions S267E, H268F, and S324T.

[0725] Composition

[0726] It should be understood that the embodiments described below with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an immunoglobulin Fc region and an antigen-binding region. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin and a second polypeptide or antibody comprising a second Fc region and a second antigen-binding region of an immunoglobulin.

[0727] The present invention also relates to compositions comprising the polypeptides or antibodies described herein and variations thereof. Specific aspects and embodiments are described below. In addition, such polypeptides or antibodies can be obtained according to any of the methods described herein.

[0728] In one aspect, the invention relates to a composition comprising at least one polypeptide or antibody described herein.

[0729] In one embodiment of the invention, a composition comprises one or more polypeptides or antibodies of any aspect or embodiment described herein.

[0730] In one embodiment of the invention, the composition comprises a first polypeptide or antibody and a second polypeptide or antibody, as described in any aspect or embodiment herein.

[0731] In one aspect of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and the second polypeptide or antibody comprise an Fc region comprising

[0732] (i) at least one substitution that is an Fc-Fc enhancing mutation; and

[0733] (ii) one or more substitutions that are C1q binding substitutions; and

[0734] (iii) a further mutation that prevents oligomerization between Fc regions comprising the same further mutation, wherein the first and second polypeptides or antibodies do not comprise the same further mutation.

[0735] In one embodiment of the invention, the composition comprises a first polypeptide or antibody and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies comprise i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions, and ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396, and iii) further mutations, wherein the first and second polypeptides or antibodies do not comprise the same further mutations. Thus, the composition comprises a first polypeptide or antibody comprising a first Fc region and a second polypeptide or antibody comprising a second Fc region.

[0736] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0737] (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions; and;

[0738] (ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0739] (iii) a further substitution at position K439 or S440, with the proviso that if the further substitution is at S440, the substitution according to (i) is not at S440, with the proviso that the first and second Fc regions do not comprise a further substitution according to (iii) at the same amino acid position,

[0740] (iv) wherein the substitution corresponds to an amino acid position in human IgG1 according to EU numbering.

[0741] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise (i) a first mutation, (ii) a second mutation, and (iii) a further mutation, wherein the mutations correspond to the following amino acid positions in human IgG1 according to EU numbering:

[0742] (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions; and;

[0743] (ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0744] (iii) a further substitution at position K439 in the first Fc region and a further substitution at position S440 in the second Fc region, or vice versa, provided that if the further substitution is at position S440, the first substitution is not at S440;

[0745] (iv) wherein the substitution corresponds to an amino acid position in human IgG1 according to EU numbering.

[0746] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0747] (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions; and

[0748] (ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0749] (iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, and

[0750] (iv) wherein the substitution corresponds to an amino acid position in human IgG1 according to EU numbering.

[0751] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0752] (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions; and

[0753] (ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0754] (iii) a further S440K substitution in the first Fc region and a further K439E substitution in the second Fc region;

[0755] (iv) wherein the substitution corresponds to an amino acid position in human IgG1 according to EU numbering.

[0756] Thus, embodiments are provided wherein both the first and second polypeptides or antibodies have increased agonist and / or CDC activity, or only the first or second polypeptide has increased agonist and / or CDC activity.

[0757] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0758] (i) a substitution in the amino acid position corresponding to E430, and

[0759] (i)(ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0760] (ii) (iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0761] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0762] i) a substitution in the amino acid position corresponding to E345, and

[0763] ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396; and

[0764] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0765] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0766] i) E430G, and

[0767] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A, P396L, S267E, H268F, S324T, G263A, S324E, I332E, and S239D; and

[0768] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0769] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0770] i) E430G, and

[0771] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A and P396L; and

[0772] (iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0773] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0774] i) E430G, and

[0775] ii) at least two substitutions selected from the group consisting of K326W, K326A, E333S, E333A, and P396L; and

[0776] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0777] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0778] i) E430G replaced, and

[0779] ii) K326W and E333S replacement; and

[0780] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0781] (ii).

[0782] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0783] i) E345K, and

[0784] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A, P396L, S267E, H268F, S324T, G263A, S324E, I332E, and S239D; and

[0785] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0786] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0787] i) E345K, and

[0788] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A and P396L; and

[0789] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0790] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0791] i) E345K, and

[0792] ii) at least two substitutions selected from the group consisting of K326W, K326A, E333S, E333A, and P396L; and

[0793] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0794] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0795] i) E345K replacement, and

[0796] ii) K326W and E333S replacement; and

[0797] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0798] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0799] i) E345R, and

[0800] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A, P396L, S267E, H268F, S324T, G263A, S324E, I332E, and S239D; and

[0801] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0802] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0803] i) E345R, and

[0804] ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A and P396L; and

[0805] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0806] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0807] i) E345R, and

[0808] ii) at least two substitutions selected from the group consisting of K326W, K326A, E333S, E333A, and P396L; and

[0809] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0810] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, and a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise:

[0811] i) E345R substitution, and

[0812] ii) K326W and E333S replacement; and

[0813] iii) a further K439E substitution in the first Fc region and a further S440K substitution in the second Fc region, or vice versa.

[0814] In another embodiment of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and the second polypeptide or antibody comprise an Fc region comprising:

[0815] (i) at least one substitution that is an Fc-Fc enhancing mutation;

[0816] (ii) a further mutation that prevents oligomerization of the Fc region with the same further mutation, wherein the first and second polypeptides or antibodies do not comprise the same further mutation,

[0817] (iii) and the first or second Fc region comprises one or more substitutions which are C1q binding substitutions. Thus, in some embodiments, only the first or second polypeptide or antibody comprises the second mutation that reduces Fc effector function.

[0818] In one embodiment of the present invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first and second Fc regions comprise

[0819] (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W,

[0820] (iii) a further K439E or S440K mutation, wherein the first and second Fc regions do not comprise the same further substitution, and wherein if at least one substitution is S440Y or S440W, the further mutation is not S440K;

[0821] (ii) and the first or second Fc region comprises one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396.

[0822] In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first Fc region comprises (i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions, and ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396, and iii) a further K439E mutation; and the second Fc region comprises i) at least one or more substitutions selected from E430, E345 or S440Y or S440W substitutions, and a further S440K mutation. Thus, embodiments are provided in which only the first polypeptide or antibody has increased agonist activity and / or increased CDC activity.

[0823] In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first Fc region comprises (i) at least one or more substitutions selected from the group consisting of E430, E345, and ii) one or more substitutions at positions selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333, and P396, and iii) further an S440K mutation; and the second Fc region comprises i) at least one or more substitutions selected from E430, E345 or an S440Y or S440W substitution, and further a K439E mutation. Thus, embodiments are provided in which only the first polypeptide or antibody has increased agonist activity and / or increased CDC activity.

[0824] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first Fc region comprises (i) an E430G substitution and ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A and P396L and iii) a further K439E substitution; and the second Fc region comprises i) an E430G substitution, and a further S440K substitution.

[0825] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first antigen binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen binding region and a second Fc region, wherein the first Fc region comprises (i) an E430G substitution and ii) one or more substitutions selected from the group consisting of K326W, K326A, E333S, E333A and P396L and iii) a further S440K substitution; and the second Fc region comprises i) an E430G substitution, and further a K439E substitution.

[0826] In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) an E430G substitution and ii) a K326W and E333S substitution, and iii) a further S440K substitution; and the second Fc region comprises i) an E430G substitution and a further K439E substitution. In one embodiment of the present invention, a composition comprises a first polypeptide or antibody comprising a first antigen-binding region and a first Fc region, a second polypeptide or antibody comprising a second antigen-binding region and a second Fc region, wherein the first Fc region comprises (i) an E430G substitution and ii) a K326W and E333S substitution, and iii) a further K439E substitution; and the second Fc region comprises i) an E430G substitution and a further S440K substitution.

[0827] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) or the G protein coupled receptor (GPCR) superfamily.

[0828] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member selected from the group consisting of TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, EDAR DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[0829] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member having an intracellular death domain selected from the group consisting of TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR, and EDAR.

[0830] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member without an intracellular death domain selected from the group consisting of DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0831] In one embodiment of the invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member belonging to the group of immune activators consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[0832] In one embodiment of the invention, the composition comprises polypeptides or antibodies, wherein the first polypeptide and the second polypeptide bind to different epitopes on one or more TNFR-SF members without an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, and RELT.

[0833] In one embodiment of the invention, the composition comprises a polypeptide or antibody, wherein a first polypeptide that binds to a member of the TNFR-SF without an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT does not block the binding of a second antibody that binds to a member of the TNFR-SF without an intracellular death domain selected from the group consisting of OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[0834] In one embodiment of the present invention, the composition comprising the first polypeptide or antibody and the second polypeptide and antibody is in a 1:49 to 49:1 molar ratio, such as a 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio. , 1:40 molar ratio, 1:45 molar ratio, 1:50 molar ratio, 50:1 molar ratio, 45:1 molar ratio, 40:1 molar ratio, 35:1 molar ratio, 30:1 molar ratio, 25:1 molar ratio, 20:1 molar ratio, 15:1 molar ratio, 10:1 molar ratio, 9:1 molar ratio, 8:1 molar ratio, 7:1 molar ratio, 6:1 molar ratio, 5:1 molar ratio, 4:1 molar ratio, 3:1 molar ratio, and 2:1 molar ratio are present in the composition.

[0835] In one embodiment of the invention, the composition comprises the first polypeptide and the second polypeptide and / or any additional polypeptides present in the composition in an equimolar ratio.

[0836] In one embodiment of the invention the composition according to any aspect or embodiment is a pharmaceutical composition.

[0837] Therapeutic applications

[0838] The polypeptide, antibody, bispecific antibody or composition according to any aspect or embodiment of the invention may be used as a medicament, ie for therapeutic applications.

[0839] In one aspect, the invention provides a polypeptide, antibody or composition according to any aspect or embodiment disclosed herein for use as a medicament.

[0840] In another aspect, the invention provides a polypeptide, antibody or composition according to any aspect or embodiment disclosed herein for use in treating cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

[0841] In another aspect, the invention relates to a method of treating an individual suffering from a disease, comprising administering to the individual an effective amount of a polypeptide, antibody or composition according to any aspect or embodiment disclosed herein.

[0842] In one embodiment of the present invention, the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

[0843] In one embodiment of the invention, the method according to any aspect or embodiment disclosed herein involves further administering an additional therapeutic agent.

[0844] In one embodiment of the invention, the additional therapeutic agent is one or more anticancer agents selected from the group consisting of chemotherapeutic agents (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), kinase inhibitors (including but not limited to sorafenib, sunitinib or everolimus), apoptosis regulators (including but not limited to recombinant human TRAIL or birinapant), RAS inhibitors, proteasome inhibitors (including but not limited to bortezomib), histone deacetylase inhibitors (including but not limited to vorinostat), nutritional supplements, cytokines (including but not limited to IFN-γ), antibodies or antibody mimetics (including but not limited to anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR antibodies and antibody mimetics), antibody-drug conjugates.

[0845] Reagent test kit

[0846] It should be understood that the embodiments described below with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin, and a second polypeptide or antibody comprising a second Fc region and a second antigen-binding region of an immunoglobulin.

[0847] The present invention also relates to kits for simultaneous, separate or sequential use in therapy comprising a polypeptide or antibody as described herein. In addition, such variants may be obtained according to any of the methods described herein.

[0848] In one aspect, the invention relates to a kit comprising a polypeptide, antibody or composition according to any aspect or embodiment described herein, wherein the polypeptide, antibody or composition is in one or more containers such as vials.

[0849] In one embodiment of the invention, a kit comprises a polypeptide, an antibody or a composition according to any aspect or embodiment described herein for simultaneous, separate or sequential use in therapy.

[0850] In another aspect, the present invention relates to the use of a polypeptide, an antibody, a composition or a kit according to any embodiment described herein for use in a diagnostic method.

[0851] In another aspect, the present invention relates to a method of diagnosis comprising administering a polypeptide, antibody, composition or kit according to any embodiment described herein to at least a part of the body of a human or other mammal.

[0852] In another aspect, the present invention relates to the use of a polypeptide, an antibody, a composition or a kit according to any embodiment described herein for imaging at least a part of the body of a human or other mammal.

[0853] In another aspect, the invention relates to a method for imaging at least a portion of the body of a human or other mammal comprising administering a variant, composition or kit according to any embodiment described herein.

[0854] Further Application

[0855] It should be understood that the embodiments described below with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin, and a second polypeptide or antibody comprising a second Fc region and a second antigen-binding region of an immunoglobulin.

[0856] In another aspect, the present invention relates to a polypeptide or antibody of the present invention as described above for use as a medicament, in particular for use as a medicament for treating a disease or condition. Examples of such diseases and conditions include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, bacterial, viral or fungal infections.

[0857] In another aspect, the present invention relates to the polypeptides, antibodies, bispecific antibodies, compositions and kits described herein for use in treating a disease, such as cancer.

[0858] In another aspect, the invention relates to a method of treating a human disease comprising administering a variant, composition or kit as described herein.

[0859] In another aspect, the invention relates to a method of treating cancer in a human comprising administering a variant, composition, or kit.

[0860] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of alleviating, ameliorating, preventing or eradicating (curing) symptoms or disease states.

[0861] An "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount of an antibody can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0862] dose

[0863] It should be understood that the embodiments described below with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin, and a second polypeptide or antibody comprising a second Fc region and a second antigen-binding region of an immunoglobulin.

[0864] The effective dose and dosage regimen of the antibody depends on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary non-limiting range for a therapeutically effective amount of an antibody of the invention is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, such as about 0.1 to 20 mg / kg, such as about 0.1 to 10 mg / kg, such as about 0.5, about 0.3, about 1, about 3, about 5 or about 8 mg / kg.

[0865] The polypeptides or antibodies of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, a medicament comprising an antibody is used in combination with one or more other therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, or an anti-angiogenic agent. Such combination administration may be simultaneous, separate, or sequential.

[0866] In another embodiment, the present invention provides a method of treating or preventing a disease such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a variant or pharmaceutical composition of the present invention in combination with radiation therapy and / or surgery.

[0867] Preparation method

[0868] It should be understood that the embodiments described below with reference to polypeptides or antibodies refer to polypeptides or antibodies comprising an Fc region and an antigen-binding region of an immunoglobulin. The polypeptides or antibodies may also be multispecific polypeptides or antibodies comprising a first Fc region and a first antigen-binding region of an immunoglobulin, and a second polypeptide or antibody comprising a second Fc region and a second antigen-binding region of an immunoglobulin.

[0869] The present invention also provides nucleic acid and carrier encoding the separation of variants according to any of the above aspects, and carrier and expression system encoding the variant. Suitable nucleic acid constructs, carriers and expression systems for antibodies and variants thereof are known in the art and are described in the Examples. In embodiments where variants not only comprise heavy chain (or its fragment containing Fc) but also comprise light chain, the nucleotide sequence encoding heavy chain and light chain moieties may be present in identical or different nucleic acids or carriers.

[0870] The present invention also provides a method for producing the polypeptide or antibody according to any of the above aspects in a host cell, wherein the polypeptide or antibody comprises at least the Fc region of the heavy chain, the method comprising the following steps:

[0871] a) providing a nucleotide construct encoding said Fc region of said variant,

[0872] b) expressing the nucleotide construct in a host cell, and

[0873] c) recovering the antibody variant from the cell culture of the host cell.

[0874] In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody also contains a light chain, and thus the host cell further expresses a light chain encoding construct on the same or a different vector.

[0875] Host cells suitable for recombinant expression of antibodies are well known in the art, including CHO, HEK-293, Expi293, PER-C6, NS / 0 and Sp2 / 0 cells. In one embodiment, the host cell is a cell capable of Asn-linked glycosylation of a protein, 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 that has been genetically engineered to produce a glycoprotein with a human-like or human glycosylation. Examples of such cells are genetically modified Pichia pastoris (Pichia pastoris) ((Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325)) and genetically modified Lemna minor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

[0876] In one embodiment, the host cell is one that is incapable of efficiently removing 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 a number of such antibody production systems, e.g., Sp2 / 0, NS / 0, or transgenic mammary gland (goat), in which only partial removal of the C-terminal lysine is achieved. In one embodiment, the host cell is one that has altered glycosylation machinery. Such cells have been described in the art and can be used as host cells in which variants of the present invention are expressed, thereby producing antibodies with altered glycosylation. See, e.g., Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1 and EP1176195; WO03 / 035835; and WO99 / 54342. Other methods for generating engineered glycoforms are known in the art and include, but are not limited to, Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al, 2003, J Biol Chem 278:3466-3473), US 6602684, WO 00 / 61739 A1; WO 01 / 292246 A1; WO 02 / 311140 A1; WO 02 / 30954 A1; Potelligent TM Technology (Biowa, Inc. Princeton, NJ); GlycoMAb TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; those described in Okazaki et al., 2004, JMB, 336: 1239-49.

[0877] The present invention also relates to antibodies obtained or obtainable by the above-described method of the present invention.

[0878] In another aspect, the present invention relates to a host cell capable of producing a polypeptide or antibody of the present invention. In one embodiment, the host cell has been transformed or transfected with a nucleotide construct of the present invention.

[0879] The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0880] Table 1

[0881]

[0882]

[0883]

[0884]

[0885]

[0886]

[0887]

[0888]

[0889]

[0890]

[0891]

[0892]

[0893]

[0894]

[0895]

[0896]

[0897] Example

[0898] Example 1: Antibody generation, production and purification.

[0899] Antibody expression constructs

[0900] For antibody expression, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into the pcDNA3.3 expression vector (ThermoFisher Scientific, US) containing the IgG1 heavy chain (HC) and light chain (LC) constant regions. Desired mutations were introduced by gene synthesis or site-directed mutagenesis. The antibodies mentioned in the present application are derived from the previously described DR5 antibodies hDR5-01, hDR5-05 (WO2014 / 009358) and Conatumumab (US7521048B2 and WO2010 / 138725), DR4 antibody chCTB007 (US 2009 / 0136503), FAS antibody E09 (Chodorge Cell Death Differ. 2012 Jul; 19(7): 1187–1195), APO1 (WO 2014 / 076292) and HFE7A (US6972323), OX40 antibody SF2 (US2014 / 0377284), CD40 antibodies SGN 40 (US6838261) and CP870893 (US7338660), 4-1BB antibody MOR7480 (WO 2012 / 032433) and BMS-663513 (US8475790), CD20 antibodies HuMab-7D8 and 11B8 (WO2004 / 035607), CD52 antibody alemtuzumab (Crowe et al., Clin Exp Immunol. 1992;87(1):105–10), and EGFR antibody 2F8 (WO2002 / 100348) VH and VL sequences. In some instances, human IgG1 antibody b12, a gp120-specific antibody, was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-23).

[0901] transient expression

[0902] Antibodies were expressed as IgG1, K. A mixture of plasmid DNA encoding both antibody heavy and light chains was transiently transfected in Expi293 cells (Life / Thermo Scientific, USA) using Expifectamine (Invitrogen, US) essentially as described by the manufacturer.

[0903] Protein purification and analysis

[0904] The antibody was purified by protein A affinity chromatography. The culture supernatant was filtered with a 0.20 μM dead-end filter and loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), cleaned 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 buffer was exchanged into 12.6 mM NaH PO , 140 mM NaCl, pH 7.4 buffer (B.Braun or Thermo Fisher). After buffer exchange, the sample was sterile filtered with a 0.20 μM dead-end filter. The purified protein was analyzed by many bioanalytical assays, including capillary electrophoresis and high performance size exclusion chromatography (HP-SEC) on sodium dodecyl sulfate-polyacrylamide gel (CE-SDS). The concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2-8 ° C.

[0905] Generation of bispecific antibodies

[0906] Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reduction conditions. The basis of this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011 / 131746. F405L and K409R (EU numbering) mutations were introduced into anti-DR5 IgG1 antibodies to generate antibody pairs with complementary CH3 domains. The F405L mutation was introduced into IgG1-b12-K326A / E333A / P396L / E430G and IgG1-CONA-C49W-K326W / E333S / E430G; the K409R mutation was introduced into IgG1-b12-K326W / E333S / E430G and IgG1-hDR5-01-G56T-K326A / E333A / P396L / E430G. To produce bispecific antibodies, two parent complementary antibodies (each antibody final concentration is 0.5 mg / mL) are incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL PBS at 31°C for 5 hours. The reduction reaction is terminated by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol. The antibody buffer is exchanged into 12.6 mM NaH PO , 140 mM NaCl, pH 7.4 buffer (B.Braun or Thermo). In this way, the bispecific antibodies IgG1-hDR5-01-G56T-K326A / E333A / P396L / K409R / E430G x IgG1-b12-K326A / E333A / P396L / F405L / E430G, termed BsAb(hDR5-01-G56T-K409Rxb12-F405L)-K326A / E333A / P396L / E430G and IgG1-CONA-C49W-F405L-K326W / E333S / E430G x IgG1-b12-K409R-K326W / E333S / E430G, termed BsAb(hDR5-01-G56T-K409Rxb12-F405L)-K326A / E333A / P396L / E430G xIgG1-b12-K409R)-K326W / E333S / E430G.

[0907] Example 2: Effect of the combination E430G and K326A / E333A / P396L on the efficacy of agonistic anti-DR5 antibodies.

[0908] A viability assay was performed to evaluate the effects of the combination of Fc-Fc enhancing substitutions E430G (WO2013 / 004842; WO2014 / 108198; WO2014 / 006217; de Jong et al., 2016) and K326A / E333A / P396L (WO2016 / 116635) on the agonistic activity of anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1-hDR-05 on DR5-positive BxPC-3 cells (ATCC, CRL-1687). Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation at 1,200 rpm for 5 minutes and concentrated at a concentration of 0.5×10 5 Cells were resuspended at 5000 cells / mL in culture medium (RPMI 1640, containing 25 mM Hepes and L-glutamine (Lonza Cat nr BE12-115F) + 10% heat-inactivated donor bovine serum with iron (DBSI; Life Technologies Cat nr 10371-029) + 50 U / mL penicillin / streptomycin (Pen / Strep; Lonza; Cat nr DE17-603E). 100 μL of the single-cell suspension (5,000 cells per well) was seeded into a polystyrene 96-well flat-bottom plate (Greiner Bio-One, Cat nr 655182) and adhered overnight at 37°C. Next, 50 μL of a serially diluted antibody preparation series (ranging from 0.0003 to 20,000 ng / mL final concentration, diluted 4-fold) was added and cultured at 37°C for 3 days. As negative and positive controls, cells were incubated in the absence of antibody or 5 μM staurosporine (Sigma Aldrich, Cat nr S6942), respectively. The viability of the cell culture was determined in the CellTiter-Glo luminescent cell viability assay (Promega, Catnr G7571), which quantifies the presence of ATP, an indicator of metabolically active cells. From the kit, 20 μL of luciferin solution reagent was added to each well and mixed by shaking the plate at 500 rpm for 2 minutes. Next, the plate was incubated at 37°C for 1.5 hours. 100 μL of supernatant was transferred to a white OptiPlate-96 (Perkin Elmer, Cat nr 6005299) and luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using GraphPad Prism software using nonlinear regression (sigmoidal dose response with variable slope). Figure 1The percentage of viable cells as calculated using the following formula: % viable cells = [(luminescence of antibody sample - luminescence of staurosporine sample) / (luminescence of no antibody sample - luminescence of staurosporine sample)] * 100 is shown.

[0909] Figure 1 showed that combining the Fc-Fc enhancing substitution E430G and the triple substitutions K326A / E333A / P396L resulted in an anti-DR5 antibody IgG1-hDR5-01-G56T ( Figure 1 A) and IgG1-hDR5-05 ( Figure 1 B) Induction of killing efficacy when tested as single agents in an in vitro viability assay against adherent human BxPC-3 pancreatic cancer cells. In contrast, when only E430G or K326A / E333A / P396L were present, these antibodies did not show effective killing of these pre-adherent BxPC-3 cells. Likewise for the combination of non-cross-blocking antibodies IgG1-hDR5-01-G56T + IgG1-hDR5-05, introduction of the combined substitutions K326A / E333A / P396L / E430G resulted in the most effective killing of pre-adherent BxPC-3 cells ( Figure 1 C).

[0910] These data show that the K326A / E333A / P396L / E430G substitutions induce strong agonistic activity of anti-DR5 antibodies on adherent BxPC-3 cells.

[0911] Example 3: Efficacy of a monovalent anti-DR5 antibody containing K326A / E333A / P396L / E430G.

[0912] Viability assays were performed on human BxPC-3 pancreatic cancer and COLO205 colon cancer cells to investigate the efficacy of monovalent anti-DR5 antibodies containing K326A / E333A / P396L / E430G. Monovalent DR5 antibodies were generated by controlled Fab arm exchange between IgG1-hDR5-01-G56T-K326A / E333A / P396L / K409R / E430G and IgG1-b12-K326A / E333A / P396L / F405L / E430G as described in Example 1. The resulting bispecific antibody, designated BsAb (hDR5-01-G56T-K409Rxb12-F405L)-K326A / E333A / P396L / E430G, contains one arm specific for DR5 and one nonspecific arm directed against HIV glycoprotein gp120, resulting in monovalent DR5 binding on DR5-positive human cancer cells. BxPC-3 cells were harvested as described in Example 2. COLO205 cells (ATCC, CCL-222) were harvested by combining culture supernatants containing non-adherent cells and trypsin-treated adherent COLO205 cells. Cells were pelleted by centrifugation at 1,200 rpm for 5 minutes and plated at a concentration of 0.5 x 10 5 100 μL of single cell suspension (5,000 cells per well) was seeded into a polystyrene 96-well flat-bottom plate and allowed to adhere overnight at 37°C. Next, 50 μL of a serially diluted antibody preparation series (ranging from 0.0024 to 10,000 ng / mL final concentration, diluted 4-fold) was added and incubated at 37°C for 3 days. As negative and positive controls, cells were incubated in the absence of antibody or with 5 μM staurosporine, respectively. The viability of the cultured cells was determined in the CellTiter-Glo luminescent cell viability assay, as described in Example 2.

[0913] Figure 2 It was shown that in the presence of K326A / E333A / P396L / E430G mutations, a monovalent variant of IgG1-hDR5-01-G56T could still induce killing of human BxPC-3 pancreatic cancer and COLO205 colon cancer cells.

[0914] Example 4: Effect of combining E430G and K326A / E333A, K326A / P396L or E333A / P396L on C1q binding and efficacy of agonistic anti-DR5 antibodies.

[0915] A viability assay was performed to investigate the effects of the Fc-Fc enhancing substitution E430G in combination with two of the three substitutions in K326A / E333A / P396L on the agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T against DR5-positive BxPC-3 and COLO 205 cells. For reference, the experiment included combinations of E430G with all three substitutions, K326A / E333A / P396L, as described in Example 2. The viability assay was performed as described in Example 3. The viability of the cultured cells was determined in the CellTiter-Glo luminescent cell viability assay as described in Example 2.

[0916] Figure 3 We show that combining the Fc-Fc enhancing substitution E430G and two substitutions from K326A / E333A / P396L (E333A / P396L, K326A / E333A or K326A / P396L) results in an anti-DR5 antibody IgG1-hDR5-01-G56T that is highly resistant to Fc-Fc binding when targeting human BxPC-3 pancreatic cancer cells ( Figure 3 A) and COLO 205 colon cancer ( Figure 3 B) Induction of killing efficacy when tested as a single agent in an in vitro cell viability assay. In contrast, no killing of these pre-adherent cancer cells was observed when E430G alone was present. The most potent killing was observed when E430G was combined with all three mutations, K326A / E333A / P396L.

[0917] Binding ELISAs were performed to assess the effect of different substitutions on C1q binding. Purified antibody samples of IgG-hDR5-01-G56T variants containing the E430G substitution in combination with K326A / E333A, K326A / P396L, E333A / P396L, or K326A / E333A / P396L substitutions were tested and compared to WT IgG-hDR5-01-G56T and IgG-hDR5-01-G56T-E430G. IgG-2F8-I253D / K322A was used as a negative control for C1q binding. 96-well Microlon ELISA plates (Greiner Cat#655092) were coated by incubation overnight at 4°C with 1 μg / mL antibody sample in 100 μL PBS. Plates were washed and blocked with 200 μL / well of 0.5x PBS supplemented with 0.025% Tween 20 and 0.1% gelatin for 1 hour at room temperature with shaking. After washing between incubations, the plate was incubated with 100 μL / well of a serial dilution series of purified C1q (Quidel Cat#A400; final C1q concentration range 30-0.010 μg / mL, 3-fold dilution) at 37°C for 1 hour, 100 μL / well of rabbit anti-human C1q (DAKO, product#A0136, 1 / 4.000) at room temperature for 1 hour, and 100 μL / well of pig anti-rabbit IgG-HRP (DAKO, P0399, 1:10.000) as detection antibody at room temperature for 1 hour, and finally with 100 μL / well of substrate containing 1 mg / mL 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche Cat#11112 597001) at room temperature for approximately 15 minutes. The reaction was stopped by adding 100 μL of 2% oxalic acid. Absorbance was measured at 405 nm in a BioTek EL808 microplate reader (BioSPX).Log-transformed data were analyzed by fitting sigmoidal dose-response curves with variable slope using GraphPad Prism software.

[0918] Figure 3 C shows that the introduction of the E430G Fc-Fc enhancing substitution did not affect the apparent C1q binding affinity for 1 μg / mL coated IgG1-hDR5-01-G56T antibody, while antibody variants containing the E430G substitution in combination with K326A / E333A, K326A / P396L, E333A / P396L, or K326A / E333A / P396L substitutions showed enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G (Table 2).

[0919] Table 2: EC50 values ​​of C1q binding to IgG1-hDR5-01-G56T antibody variants (ELISA)

[0920]

[0921]

[0922] 1 One-way ANOVA p value = 0.0022; Bonferroni post hoc test Ab vs. WT: p < 0.05 as indicated.

[0923] Together, these data show that combining the E430G Fc-Fc enhancing substitution with K326A / E333A, K326A / P396L, E333A / P396L, or K326A / E333A / P396L substitutions results in increased C1q binding and increased agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T-E430G that possesses only the E430G Fc-Fc enhancing mutation.

[0924] Example 5: Effect of the combination E430G and K326W / E333S on CIq binding and efficacy of agonistic anti-DR5 antibodies.

[0925] Binding ELISAs were performed to assess the effects of K326A / E333A and K326W / E333S on C1q binding to antibodies containing the E430G Fc-Fc enhancing mutation. Purified antibody samples of IgG1-CONA-C49W variants containing the E430G substitution in combination with either the K326A / E333A or K326W / E333S mutations were tested and compared to WT IgG1-CONA-C49W and IgG1-CONA-C49W-E430G. IgG1-CONA-C49W-K326W / E333S, which does not contain the E430G substitution, was also tested. IgG1-2F8-I253D / K322A was used as a negative control for C1q binding. C1q binding ELISAs were performed on ELISA plates coated with 1 μg / mL of the antibody as described in Example 4.

[0926] When compared to the WT antibody, a strong enhancement of C1q binding achieved by introducing the K326W / E333S substitution was demonstrated ( Figure 4A). In contrast, introduction of the E430G Fc-Fc enhancer mutation did not affect the apparent C1q binding affinity for 1 μg / mL coated IgG1-CONA-C49W antibody. Antibody variants containing the E430G substitution and combinations of K326A / E333A or K326W / E333S substitutions showed strongly enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G (Table 3).

[0927] Table 3: EC50 values ​​for binding of C1q to IgG1-CONA-C49W antibody variants (ELISA)

[0928]

[0929]

[0930] 1 One-way ANOVA p value = 0.0013; Bonferroni post hoc test Ab vs. WT: p < 0.05 as indicated.

[0931] A viability assay was performed to investigate the effect of the combination of the Fc-Fc enhancing mutation E430G and the C1q binding substitutions K326A / E333A or K326W / E333S on the agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T on DR5-positive BxPC-3 and COLO 205 cells. The viability assay was performed as described in Example 3. The viability of the cultured cells was determined in the CellTiter-Glo luminescent cell viability assay as described in Example 2.

[0932] Figure 4 B / C shows that the combination of the Fc-Fc enhancing substitution E430G and the two mutations K326W / E333S resulted in the anti-DR5 antibody IgG1-hDR5-01-G56T when expressed against adherent human BxPC-3 pancreatic Figure 4 B) and COLO 205 colon ( Figure 4 C) Induction of strong killing efficacy when tested as a single agent in an in vitro viability assay of cancer cells. In contrast, WT antibody and IgG1-hDR5-01-G56T-E430G showed no efficacy. The killing efficacy of IgG1-hDR5-01-G56T-K326W / E333S / E430G was superior to that of IgG1-hDR5-01-G56T-K326A / E333A / E430G on both BxPC-3 and COLO205 cancer cells.

[0933] Together, these data show that combining the E430G Fc-Fc enhancing substitution with either the K326A / E333A or K326W / E333S substitutions results in increased CIq binding and increased agonistic activity of the anti-DR5 antibody IgGl-CONA-C49W-E430G that possesses only the E430G hexamerization enhancing mutation.

[0934] Example 6: Effect of combining E430G with other Fc variants on CIq binding and efficacy of agonistic anti-DR5 antibodies.

[0935] A C1q binding ELISA was performed to investigate the effect of C1q binding substitutions S267E / H268F / S324T or the IgG1 / IgG3 chimeric isotype IgG1 variant 113F on C1q binding to antibodies containing the E430G Fc-Fc enhancing substitution (Tammen et al., J Immunol. 2017). Purified antibody samples of IgG1-hDR5-01-G56T variants with and without these substitutions were tested and compared to WT IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G. IgG1-2F8-I253D / K322A was used as a negative control for C1q binding. The C1q binding ELISA was performed on ELISA plates coated with 1 μg / mL antibody as described in Example 4.

[0936] Figure 5 A shows that the introduction of the E430G Fc-Fc enhancing substitution did not affect the apparent C1q binding affinity for 1 μg / mL coated IgG1-hDR5-01-G56T antibody. In contrast, antibody variants containing the E430G substitution and the combination of the S267E / H268F / S324T substitutions showed strongly enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G, while the introduction of the E430G substitution in the IgG113F-hDR5-01-G56T format variant resulted in a slight enhancement of C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G (Table 4).

[0937] Table 4: EC50 values ​​for binding of C1q to IgG1-hDR5-01-G56T antibody variants (ELISA)

[0938]

[0939] 1 One-way ANOVA p value = 0.0013; Bonferroni post hoc test Ab vs. WT: p < 0.05 as indicated.

[0940] Viability assays were performed to investigate the effects of the combination of the Fc-Fc enhancing substitution E430G with the C1q binding substitutions S267E / H268F / S324T (Moore et al., MAbs 2010) or the IgG1 / IgG3 chimeric isotype variant 113F (Natsume et al., Cancer Res. 2008) on the agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T on DR5-positive BxPC-3 and COLO205 cells. Viability assays were performed as described in Example 3. The viability of cultured cells was determined in the CellTiter-Glo luminescent cell viability assay as described in Example 2.

[0941] Figure 5 B / C shows that the combination of the Fc-Fc enhancing substitution E430G and the C1q binding substitutions S267E / H268F / S324T resulted in the anti-DR5 antibody IgG1-hDR5-01-G56T to adhere to human BxPC-3 pancreatic Figure 5 B) and COLO 205 colon ( Figure 5 C) Induction of killing efficacy when tested as a single agent in an in vitro viability assay of cancer cells. When E430G was incorporated into the IgG1 / IgG3 chimeric isotype IgG1 variant 113F of IgG1-hDR5-01-G56T, induction of killing efficacy was observed on COLO 205 ( Figure 5 C), and slight induction of killing efficacy was observed on BxPC-3, where agonistic activity was only observed at the highest antibody concentration tested ( Figure 5 B). However, on both cell lines, the efficacy of these variants, IgG1-hDR5-01-G56T-S267E / H268F / S324T / E430G and IgG113F-hDR5-01-G56T-E430G, was significantly lower than that of IgG1-hDR5-01-G56T-K326W / E333S / E430G and IgG1-hDR5-01-G56T-K326A / E333A / P396L / E430G. As in the previous example, the WT antibody and IgG1-hDR5-01-G56T-E430G showed no efficacy.

[0942] Together, these data show that combining the E430G Fc-Fc enhancing substitution with the S267E / H268F / S324T substitutions resulted in strongly increased C1q binding and agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T-E430G, which possesses only the E430G Fc-Fc enhancing substitution. Introduction of the E430G substitution into the IgG113F-hDR5-01-G56T format variant resulted in slightly enhanced C1q binding and agonistic activity of the antibody.

[0943] Example 7: Summary of the effects of E430G in combination with other Fc mutations and variants on the efficacy of agonistic anti-DR5 antibodies.

[0944] In the previous examples, a viability assay was described in which the effect of the Fc-Fc enhancing substitution E430G on the agonistic activity of the anti-DR5 antibody IgG1-hDR5-01-G56T was tested when combined with other Fc region substitutions or variants described as affecting DR5 agonism or C1q binding. In this example, a summary of all viability assays on adherent human pancreatic BxPC-3 cancer cells is presented by expressing and ranking the percentage of viable cells after three days of incubation with 10 μg / mL of the indicated antibodies relative to WT IgG1-hDR5-01-G56T (which showed no effect in Examples 2, 4, 5, and 6). Details of the viability assay and CellTiter-Glo luminescence assay for adherent BxPC-3 cells are described in Example 2.

[0945] Figure 6 The results show that the combination of the Fc-Fc enhancing substitution E430G and the C1q binding double substitution K326W / E333S showed the most significant effect when compared to the WT IgG1-hDR5-01-G56T antibody after a 3-day incubation period of adherent human BxPC-3 pancreatic cancer cells in complete medium containing heat-inactivated fetal bovine serum at 10 μg / mL of antibody. In addition, the combination of E430G with K326A / E333A / P396L, E333A / P396L, and K326A / E333A resulted in a significantly lower percentage of viable cells than the WT antibody. Other Fc variants that have been shown to enhance C1q binding, such as S267E / H268F / S324T and IgG1 / IgG3 chimeric IgG-113F, did not show significant induction of killing efficacy against adherent BxPC-3 cells when combined with E430G in IgG1-hDR5-01-G56T in the experimental setting herein with 10 μg / mL of antibody, where IgG1-hDR5-01-G56T-E430G also did not result in induction of killing efficacy when tested as a single agent.

[0946] Example 8: Effect of CIq on the in vitro activity of agonistic anti-DR5 antibodies with Fc-Fc enhancing substitutions in combination with CIq binding substitutions.

[0947] Previous examples have shown that enhanced C1q binding contributes to better agonistic activity of the tested anti-DR5 antibodies containing the E430G Fc-Fc enhancer mutation. To test the effect of C1q, a viability assay was performed on WIL2-S SF cells in serum-free medium with IgG1-CONA-K326A / E333A / P396L / E430G and IgG1-hDR5-01-G56T-K326W / E333S / E430G in the presence or absence of purified human C1q. WIL2-S SF cells are derived from WIL2-S (ATCC, CRL-8885) B lymphoblastoid cells and adapted for growth in serum-free conditions in medium containing 50 U / mL Pen / Strep and 1 mM sodium pyruvate in the presence of HyQ-ADCF-Mab (Perbio, Cat# SH30349). The WIL2-S SF suspension cells were passed through a cell strainer, pelleted by centrifugation at 300 x g for 5 minutes, and plated at 0.5 x 10 6 The cells were resuspended in serum-free medium at a concentration of 50,000 cells / mL. 100 μL of single-cell suspension (50,000 cells per well) was seeded in a polystyrene 96-well flat-bottom plate (Greiner Bio-One, Cat nr 655182). 25 μL of a serially diluted antibody preparation (ranging from 0.0003 to 20,000 ng / mL final concentration, 4-fold dilution) and 25 μL of purified C1q (Quidel, Cat#A400; 2.5 μg / mL final concentration) were added and incubated at 37°C for 1 day. As negative and positive controls, cells were cultured in medium without antibody or containing 5 μM staurosporine (Sigma Aldrich, Cat nr S6942), respectively. Cell viability was determined by TO-PRO-3 staining. TO-PRO-3 is a cell-impermeable carbonyl cyanine monomer stain that binds to double-stranded DNA. Therefore, TO-PRO-3 can be used as a dead cell indicator. All samples were transferred to a polystyrene 96-well U-bottom plate (Greiner Bio-One, Cat nr 650261) and centrifuged at 300 x g for 3 minutes, after which 70 μL of supernatant was removed. 10 μL of TO-PRO-3 mixture (Invitrogen, Cat # T3605) (20 μL TO-PRO-3 + 1980 μL PBS) was added, and the cells were resuspended by pipetting. The number of TO-PRO-3-positive cells was determined by flow cytometry on a BD LSRFortessa X-20 cell analyzer (BD Biosciences).

[0948] Figure 7 It was shown that the addition of purified C1q to serum-free medium greatly enhanced the expression of IgG1-CONA-K326A / E333A / P396L / E430G ( Figure 7 A) and IgG1-hDR5-01-G56T-K326W / E333S / E430G ( Figure 7 B) Potency on WIL2-S SF cells. These data indicate that C1q binding contributes to better agonistic activity of agonistic anti-DR5 antibodies containing the E430G Fc-Fc enhancing substitution in combination with either the K326A / E333A / P396L or C1q binding K326W / E333S substitutions.

[0949] Example 9: Effect of CIq on the in vitro agonistic activity of agonistic anti-DR5 antibodies with Fc-Fc enhancing substitutions in combination with CIq binding substitutions.

[0950] In Example 8, the effect of C1q on the efficacy of agonistic anti-DR5 antibodies was tested in a viability assay of WIL2-SSF cells in serum-free medium with an antibody concentration series and a fixed C1q concentration. In this example, a C1q concentration series was tested for the effect of the efficacy of agonistic IgG1-hDR5-01-G56T antibody variants with a combination of Fc-Fc enhancing substitutions (E430G) and C1q binding substitutions (K326A / E333S / P396L, K326W / E333S, or K326A / E333A) in a viability assay of WIL2-SSF cells in serum-free medium. Viability was performed essentially as described in Example 8, with a fixed antibody concentration of 2.5 μg / mL and a concentration series of purified C1q at a final concentration ranging from 0.0002 to 2.5 μg / mL in 4-fold dilutions.

[0951] Figure 8 The addition of purified C1q to serum-free medium was shown to enhance the potency of anti-DR5 antibodies containing E430GFc-Fc enhancing substitutions. All tested IgG1-hDR5-01-G56T-E430G antibody variants containing C1q binding enhancing substitutions (K326A / E333S / P396L, K326W / E333S or K326A / E333A) showed efficacy against WIL2-SSF cells in a C1q dose-dependent manner ( Figure 8A). IgG1-hDR5-01-G56T-K326W / E333S / E430G showed the highest efficacy of all antibodies tested, and maximal killing was achieved across a range of C1q concentrations starting at 0.16 μg / mL. These data indicate that C1q binding contributes to the better activity of agonistic anti-DR5 antibodies containing the E430G Fc-Fc enhancing substitution in combination with the K326A / E333A / P396L, K326W / E333S, or K326A / E333A C1q binding substitutions. The dual-epitope targeting antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G also showed a dose-dependent increase in C1q killing of WIL2S-SF cells in serum-free medium, reaching maximal killing at approximately 0.16 μg / mL C1q ( Figure 8 B).

[0952] Example 10: Effect of C1q neutralization on the in vitro agonistic activity of agonistic anti-DR5 antibodies with Fc-Fc enhancing substitutions in combination with C1q binding substitutions.

[0953] In a viability assay of WIL2-S SF cells in serum-free medium containing purified C1q, the efficacy of the agonistic anti-DR5 IgG1-hDR5-01-G56T-K326W / E333S / E430G combination containing the E430G Fc-Fc enhancing substitutions for binding to C1q was tested using an anti-C1q neutralizing antibody directed against the C1q globular head region. Similarly, the effect of neutralizing C1q was also tested in the same setting with the dual epitope targeting antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G. The viability assay was performed essentially as described in Example 8. Briefly, WIL2-S SF cells were plated at 0.67×10 6 The cells were resuspended in serum-free medium at a concentration of 50,000 cells / mL. 75 μL of single cell suspension (50,000 cells per well) was inoculated in serum-free medium in a polystyrene 96-well flat-bottom plate. Next, 25 μL of anti-DR5 antibody sample (2.5 μg / mL final concentration), 25 μL of purified C1q (0.01 μg / mL final concentration) and 25 μL of anti-C1q antibody sample (Sanquin, CLB / C1q-85CAT#MW1828; 10 μg / mL final concentration) were added and incubated at 37°C for 1 day. Cell viability was determined by TO-PRO-3 staining as described in Example 8.

[0954] In this experiment, the effect of adding purified C1q to serum-free medium to enhance the potency of the anti-DR5 antibody IgG1-hDR5-01-G56T-K326W / E333S / E430G, as described in Example 9 ( Figure 9 A). Furthermore, this efficacy was reduced when the binding of supplemented C1q to anti-DR5 antibodies was neutralized by the presence of excess anti-C1q antibodies ( Figure 9 A). These data suggest that C1q binding is required for optimal activity of agonistic anti-DR5 antibodies containing the E430G Fc-Fc enhancing substitution in combination with the K326W / E333S C1q binding substitutions. C1q-dependent efficacy in killing WIL2S-SF cells was also demonstrated for the dual-epitope targeting antibody combination IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G, which showed enhanced efficacy when C1q was added to serum-free medium and this effect was neutralized by the presence of excess anti-C1q antibody ( Figure 9 B).

[0955] Example 11: Solution-phase complement activation assay of antibodies with combinations of Fc-Fc enhancing and CIq binding substitutions.

[0956] Target binding-independent complement activation of the antibody variants was determined by quantification of C4d (a marker of classical complement pathway activation) after incubation of the antibodies in normal human serum (NHS). A three-step ELISA procedure was performed using the MicroVue C4d enzyme immunoassay (Quidel, Cat# A0008), which comprises (1) a microplate coated with a mouse monoclonal antibody that specifically binds to the C4d-containing activation fragment of human C4, (2) a goat anti-human C4d antibody conjugated to HRP, and (3) a chromogenic substrate. The kit was supplemented with internal controls and standards and used as described in the manufacturer's instructions. As a positive control, heat-aggregated gamma globulin was prepared as follows. A 1 mL aliquot of a 1.5 mL vial of IVIG solution (60 mg / mL; Sanquin, Cat# 04H04H443A) was heated at 63°C for 20 minutes. The vials were combined and diluted to 20 mg / mL with PBS and filtered through a cellulose acetate (SFCA) membrane syringe filter (Corning, Cat#431219) without surfactant in 0.22 μm pores. Approximately 0.2 mL aliquots were stored at 4 ° C. For antibody samples, 50 μ L 100 μ g / mL antibody preparation samples in 90% normal human serum (NHS, Sanquin M0008AC) were incubated for 1 hour in polypropylene 96-well U-shaped bottom plates (Greiner Bio-One; Cat#650261) at 37 ° C. Then, 5 μ L of these samples were diluted 90 times with sample diluent, and 100 μ L of diluted samples were incubated 30 minutes per well at room temperature in the coating strip under shaking conditions, and the coating strip was pre-cleaned three times with 250 μ L cleaning solutions. Then, the wells were cleaned five times with 250 μ L cleaning solutions, and then 50 μ L C4d conjugates were incubated 30 minutes per well at room temperature under shaking conditions. The wells were washed five times with 250 μL of wash solution and then incubated with 100 μL of substrate per well for 30 minutes at room temperature with shaking. The reaction was terminated by adding 50 μL of stop solution per well and the color intensity was measured spectrophotometrically at 405 nm on a BioTek EL808 microplate reader (BioSPX).

[0957] Compared with the negative control samples, the positive control samples showed significantly enhanced C4d levels ( Figure 10 In contrast, no clear enhancement of C4d levels was observed for all tested IgG1-hDR5-01-G56T antibody variants containing the E430G Fc-Fc enhancing substitution in combination with the C1q binding substitutions K326W / E333S, K326A / E333A or K326A / E333A / P396L when incubated in NHS in the absence of target cells ( Figure 10), whereas C4d was produced when the positive control HAGG, representing a random immune complex, and IgG1-CONA-RGY, representing a solution-phase IgG1 hexamer, were incubated in NHS. These data indicate that the IgG1-hDR5-01-G56T antibody variant containing the E430G Fc-Fc enhancing substitution in combination with the C1q binding substitutions K326W / E333S, K326A / E333A, or K326A / E333A / P396L did not exhibit target-independent hexamerization and complement activation in solution phase.

[0958] Example 12: Effect of combining E430G and K326W, E333S or K326W / E333S on C1q binding and efficacy of agonistic anti-DR5 antibodies.

[0959] C1q binding ELISA was performed to evaluate the effect of introducing K326W, E333S, or K326W / E333S substitutions on C1q binding of IgG-CONA-C49W variants with or without the E430G substitution. IgG-2F8-I253D / K322A was used as a negative control for C1q binding. ELISA experiments were performed in 96-well plates coated with 1 μg / mL antibody to test the binding of various concentrations of purified C1q (range 0.010-30 μg / mL, 3-fold dilutions) as described in Example 4. Absorbance was measured at 405 nm, and log-transformed data were analyzed by fitting a sigmoidal dose-response curve with a variable slope using GraphPad Prism software. Figure 11 A, B show that for both the anti-DR5 antibody IgG1-CONA-C49W and its variant IgG1-CONA-C49W-E430G with the E430G Fc-Fc interaction-enhancing and hexamerization-enhancing mutation, introduction of K326W, E333S, or K326W / E333S substitutions resulted in increased C1q binding to randomly immobilized antibody. IgG1-CONA-C49W-K326W / E333S / E430G showed the highest apparent C1q binding affinity of all tested antibody variants.

[0960] Binding of the purified antibody variants to WIL2-S SF suspension cells was analyzed by flow cytometry. Cells were harvested, counted, washed in PBS, and plated at 3.33 × 10 6 Resuspend in culture medium at 1×10 cells / mL. 5cells) were pipetted into a 96-well plate. 50 μL of an antibody titration series sample (range 0.001-2.5 μg / mL final antibody concentration, 3-fold dilution) was added and incubated at 37°C for 15 minutes. Subsequently, 20 μL of purified C1q (2.5 μg / mL final concentration) was added and incubated at 4°C for 45 minutes. Next, 100 μL of FACS buffer (PBS + 0.1% (w / v) bovine serum albumin (BSA) + 0.02% (w / v) sodium azide) was added, and the cells were washed twice with 150 μL of FACS buffer. The washed cells were incubated at 4°C with 50 μL of FITC-labeled rabbit anti-human C1q antibody (20 μg / mL final concentration; DAKO Cat. No. F0254) for 30 minutes. 100 μL of FACS buffer was added, and the cells were washed twice with FACS buffer. Cells were resuspended in 30 μL FACS buffer and fluorescence was measured by flow cytometry using iQueScreener (IntelliCyt).Binding curves with a log-transformed C1q concentration axis were analyzed using nonlinear regression analysis (sigmoidal dose response with variable slope) using GraphPad Prism software. Figure 11 C, D show that the introduction of E333S or E430G substitutions into the anti-DR5 antibody IgG1-CONA-C49W alone had no effect on C1q binding to the antibody bound to DR5-positive WIL2-S SF cells ( Figure 11 C). Introduction of the K326W mutation into the anti-DR5 antibodies IgG1-CONA-C49W or IgG1-CONA-C49W-E430G resulted in increased C1q binding to WIL2-S SF cells opsonized with anti-DR5 antibodies, consistent with the increased C1q binding observed for cells opsonized with IgG1-CONA-C49W-K326W / E333S / E430G ( Figure 11 C, D). Introduction of E333S into the anti-DR5 antibody IgG1-CONA-C49W-E430G resulted in a modest increase in C1q binding to antibody-opsonized WIL2-SSF cells ( Figure 11 D) These flow cytometry data indicate that cell-bound IgG1-CONA-C49W-K326W / E333S / E430G showed the highest affinity binding to C1q.

[0961] A viability assay was performed to evaluate the effect of introducing K326W, E333S, or K326W / E333S into anti-DR5 IgG1-CONA-C49W variants with or without the E430G substitution on DR5 agonist activity on WIL2-S SF cells. A 1-day viability assay was performed essentially as described in Example 8. Briefly, 100 μL of cells (50.000 cells / well) in serum-free medium were pipetted into a 96-well plate. 25 μL of purified C1q (final concentration 2.5 μg / mL) and 25 μL of a concentration dilution series of antibody samples (range 0.0003-20 μg / mL final concentration, 5-fold dilution) were added and incubated at 37°C for 1 day. Cell viability was determined using the CellTiterGlo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Log-transformed CIq concentration data were analyzed using nonlinear regression (sigmoidal dose response with variable slope) and plotted using GraphPad Prism software. Figure 11 EG showed that introduction of only K326W, E333S or E430G mutations into the anti-DR5 antibody IgG1-CONA-C49W did not result in the induction of DR5 agonist activity in WIL2-S SF cells, whereas the K326W / E333S double mutation in IgG1-CONA-C49W resulted in the induction of DR5 agonist activity and partial killing of WIL2-S SF cells ( Figure 11 E). Combining the mutations K326W, E333S or the double mutation K362W / E333S with the Fc-Fc enhancing mutation E430G in the anti-DR5 antibody IgG1-CONA-C49W resulted in the induction of DR5 agonist activity, with K362W / E333S / E430G resulting in the highest maximal killing in WIL2-S SF cells ( Figure 11 F).

[0962] Example 13: Effect of combining E430G and K326W, E333S or K326W / E333S on the C1q-dependent efficacy of agonistic anti-DR5 antibodies.

[0963] The effect of introducing K326W, E333S or K326W / E333S substitutions on C1q-dependent agonist activity in anti-DR5IgG-CONA-C49W antibody variants with or without the mutation E430G was tested. A 1-day viability assay was performed in vitro using WIL2-S SF cells in serum-free medium with a C1q concentration dilution series, essentially as described in Example 8. Briefly, 100 μL of cells (50.000 cells / well) in serum-free medium were pipetted in a 96-well plate. 25 μL of antibody sample (2.5 μg / mL final concentration) and 25 μL of a concentration dilution series of purified C1q (range 42 pg / mL–2.5 μg / mL final concentration, 3-fold dilution) were added and incubated at 37°C for 1 day. Cell viability was determined using the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Log-transformed data of CIq concentrations were analyzed using nonlinear regression (sigmoidal dose response with variable slope) and plotted using GraphPad Prism software. Figure 12 It was shown that introduction of the Fc-Fc enhancing substitution E430G or the C1q binding enhancing substitutions K326W or E333S as single mutations in the anti-DR5 antibody IgG1-CONA-C49W resulted in the induction of C1q dose-dependent killing of WIL2-S SF cells, and that introduction of the K326W / E333S double mutation resulted in more potent induction of C1q dose-dependent killing of WIL2-S SF cells. Combining the E430G Fc-Fc enhancing substitution with the tested C1q binding substitutions resulted in more potent killing, with IgG-CONA-C49W-K326W / E333S / E430G inducing the most potent C1q dose-dependent killing of WIL2-S SF cells ( Figure 12 ).

[0964] Example 14: Effect of C1q neutralization on the in vitro agonistic activity of anti-DR5 antibodies having the E430G mutation in combination with K326W, E333S or K326W / E333S mutations.

[0965] To test the contribution of C1q to the efficacy of agonist anti-DR5 antibodies containing the E430G Fc-Fc enhancer mutation, C1q neutralizing antibodies were added to the viability assay of WIL2-S SF cells opsonized with the IgG1-CONA-C49W variant in serum-free medium containing purified human C1q. The experiment was performed essentially as described in Example 8. Briefly, 75 μL of cell suspension was seeded in a polystyrene 96-well flat-bottom plate in serum-free medium (50,000 cells per well). 25 μ L IgG1-CONA-C49W antibody variant (2.5 μ g / mL final concentration), 25 μ L purified C1q (at a final C1q concentration close to the EC90 concentration of each different antibody according to Table 5) and 25 μ L (10 μ g / mL final concentration) C1q neutralizing antibody (CLB-C1q-85; Sanquin, Article No. MW1828) or isotype control antibody (purified mouse IgG1, kappa clone MOPC-21; BD Biosciences Cat. No. 555746) were added to WIL2-S SF cells and cultured at 37 ° C for 1 day. Cell viability was determined using the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). GraphPad Prism software was used to analyze and plot data. Figure 13 The results showed that in the presence of C1q neutralizing antibody, the DR5 agonist activity of IgG1-CONA-C49W variants with K326W / E430G, E333S / E430G or K326W / E333S substitutions was completely inhibited. For IgG1-CONA-C49W-K326W / E333S / E430G, C1q neutralization resulted in partial inhibition of DR5 agonist activity.

[0966] Table 5: CIq EC90 values ​​for 2.5 μg / mL of the indicated antibodies in a viability assay on WIL2-S SF cells in serum-free medium supplemented with a concentration series of purified CIq as described in Example 13 (data not shown).

[0967]

[0968] Example 15: Effect of Fc-Fc interaction inhibition on the in vitro agonistic activity of anti-DR5 antibodies having the E430G mutation with K326W, E333S or the combination of K326W / E333S.

[0969] To test the involvement of Fc-Fc-mediated antibody hexamerization in IgG1-CONA antibody variant-induced cell death, we used the 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science 2000 Feb 18; 287(5456): 1279-83), which binds to Fc (Diebolder et al., Science. 2014 Mar 14; 343(6176): 1260-3) in a region containing core amino acids in the hydrophobic knot region involved in Fc-Fc interactions. The viability of WIL2-S SF cells was determined in the presence or absence of the DCAWHLGELVWCT peptide, essentially as described in Example 14. Briefly, 75 μL of WIL2-S SF cell suspension was seeded in serum-free medium in a polystyrene 96-well flat-bottom plate (50,000 cells per well). 25 μL of antibody (2.5 μg / mL final concentration) was added and incubated at room temperature for 10 minutes. Next, 25 μL of Fc-Fc inhibitory peptide DCAWHLGELVWCT or scrambled control peptide WCDLEGVTWHACL (80 μg / mL) was added and incubated at room temperature for 10 minutes. Then, 25 μL of purified C1q (at a final C1q concentration close to the EC90 of each different antibody, as listed in Example 14, Table 1) was added and the reaction mixture was incubated at 37°C for 1 day. Cell viability was determined using the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). GraphPadPrism software was used to analyze and plot data. Figure 14 The results showed that the DR5 agonist activity of IgG1-CONA-C49W variants with K326W / E430G, E333S / E430G, K326W / E333S, or K326W / E333S / E430G substitutions was partially inhibited in the presence of the Fc-Fc inhibitory peptide DCAWHLGELVWCT. The Fc-Fc inhibitory peptide inhibited the agonist activity of IgG1-CONA-C49W-K326W / E333S / E430G with the Fc-Fc enhancing mutation E430G more strongly than that of IgG1-CONA-C49W-K326W / E333S without the mutation E430G.

[0970] Example 16: Effect of combining K326W / E333S with the Fc-Fc enhancing mutations E345K, E345R or S440Y on the agonist activity of anti-DR5 antibodies

[0971] A viability assay was performed to investigate the effect of combining the C1q binding substitutions K326W / E333S with the Fc-Fc enhancing mutations E345K, E345R, or S440Y on the agonist activity of the anti-DR5 antibody IgG1-CONA-C49W opsonized against BxPC-3 cells. The viability assay was performed essentially as described in Example 2. Briefly, 100 μL of a BxPC-3 single cell suspension was seeded in a polystyrene 96-well flat-bottom plate (5,000 cells per well) in complete medium (RPMI with 10% DBSI) and allowed to adhere overnight at 37°C. Next, 50 μL of a serially diluted antibody preparation (ranging from 0.0003 to 20 μg / mL final concentration, 5-fold dilutions) was added and incubated at 37°C for 3 days. Cell viability was determined using the CellTiter-Glo assay. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed using GraphPad Prism software with log-transformed concentration axes and plotted using nonlinear regression (sigmoidal dose response with variable slope). Figure 15 A shows that killing of BxPC-3 cells by IgG1-CONA-C49W was strongly induced by introduction of the E345R mutation, slightly induced by the E430G or E345K mutation, while S440Y had no effect. Figure 15 B shows that the killing of BxPC-3 cells by the IgG1-CONA-C49W-K326W / E333S variant was increased by the introduction of the E430G, E345K, or E345R mutations, but was not further enhanced by the introduction of the S440Y mutation. These data collectively indicate that the C1q binding-enhancing K326W / E333S mutation can enhance the efficacy of anti-DR5 agonist IgG1 antibodies with different Fc-Fc enhancing mutations such as E430G, E345K, or E345R.

[0972] Example 17: Effect of combining E430G with other Fc modifications on the efficacy of agonistic anti-DR5 antibodies

[0973] A viability assay was performed to investigate the effects of combining the Fc-Fc enhancing substitution E430G with the C1q binding substitutions S267E / H268F / S324T or the IgG1 / IgG3 chimeric isotype variant 113F on the agonistic activity of the anti-DR5 antibody IgG1-CONA-C49W, opsonized against DR5-positive BxPC-3 cells. The viability assay was performed essentially as described in Example 3. Briefly, 100 μL of a BxPC-3 single cell suspension was seeded in a polystyrene 96-well flat-bottom plate (5,000 cells per well) in culture medium (RPMI containing 10% heat-inactivated DBSI) and allowed to adhere overnight at 37°C. 25 μL of purified C1q (2.5 μg / mL final concentration) and 25 μL of antibody sample from a concentration dilution series (ranging from 0.0003 to 20 μg / mL final concentration, in 5-fold dilutions) were added and incubated at 37°C for 3 days. The viability of cultured cells was determined in the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data with logarithmic concentration axes were analyzed using nonlinear regression (sigmoidal dose response with variable slope) and plotted using GraphPad Prism software. Figure 16 showed that the E430G Fc-Fc enhancing substitutions were combined with the C1q binding enhancing form S267E / H268F / S324T ( Figure 16 A) or IgG113F( Figure 16 B) Combination results in induction of agonist activity of the anti-DR5 antibody IgG1-CONA-C49W against adherent human BXPC-3 pancreatic cancer cells. Combining the E430G substitution with the K326W / E333S C1q binding enhancing mutations results in stronger DR5 agonist activity of IgG1-CONA-C49W-K326W / E333S / E430G compared to IgG1-CONA-C49W-S267E / H268F / S324T / E430G and IgG113F-CONA-C49W-E430G.

[0974] Example 18: Efficacy of a monovalent anti-DR5 antibody containing K326W / E333S / E430G substitutions

[0975] A viability assay was performed to investigate the effect of the agonist activity of a monovalent anti-DR5 antibody containing K326W / E333S / E430G substitutions on BxPC-3 pancreatic cancer cells. As described in Example 1, monovalent DR5 antibodies were generated by controlled Fab arm exchange between IgG1-CONA-C49W-F405L-K326W / E333S / E430G and IgG1-b12-K409R-K326W / E333S / E430G. The resulting bispecific antibody, designated BsAb (IgG1-CONA-C49W-F405L x IgG1-b12-K409R)-K326W / E333S / E430G), contains one arm specific for DR5 and one nonspecific arm against HIV glycoprotein gp120, resulting in monovalent DR5 binding on DR5-positive human cells. Essentially as described in Example 8, WIL2-S SF cells were subjected to a 1-day viability assay. Briefly, 100 μL WIL2-S SF cells in serum-free medium were pipetted into a 96-well plate (50.000 cells / well). 25 μL purified C1q (2.5 μg / mL final concentration) and 25 μL antibody samples of a concentration dilution series (ranging from 0.0003 to 20 μg / mL final concentration, 5-fold dilution) were added to the cells and incubated at 37°C for 1 day. Cell viability was determined using the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data with logarithmic concentration axes were analyzed using nonlinear regression (sigmoidal dose response with variable slope) and plotted using GraphPad Prism software. Figure 17 It was shown that in the presence of K326W / E333S / E430G mutations, the IgG1-CONA-C49W monovalent variant could still induce killing of WIL2-S SF cells.

[0976] Example 19: Effect of the combination E430G and K326W / E333S on the agonist activity of different isotype variants of anti-DR5 antibodies.

[0977] To test whether the introduction of the K326W / E333S / E430G substitutions could induce agonist activity in anti-DR5 antibodies in non-IgG1 antibody backbones, an IgG3 isotype variant of IgG1-CONA-C49W with a human IgG3 constant domain was generated by methods known in the art, generating IgG3-CONA-C49W. The IgG3 backbone also contained the R345H mutation to enhance FcRn binding (Stapleton et al., 2011 Nat Commun). The K326W / E333S / E430G substitutions were introduced in both the IgG1 and IgG3 isotype variants, and the agonist activity of the different antibodies was tested in an in vitro viability assay using different cell lines: human WIL2-S SF B lymphoblastoid cells, BxPC-3 and HPAF-II (ATCC, CRL-1997) pancreatic cancer cells, and HT-29 (ATCC, HTB-38) colon cancer cells. Essentially as described in Example 8, a viability assay was performed using WIL2-S SF suspension cells. Briefly, 100 μL of WIL2-S SF cells were pipetted into serum-free medium in a 96-well plate (50,000 cells / well). Next, 25 μL of purified C1q sample (2.5 μg / mL final concentration) and 25 μL of a concentration dilution series of antibody samples (range 0.0003-20 μg / mL final concentration, 5-fold dilution) were added to the cells and incubated at 37°C for 1 day. For adherent cells BxPC-3, HPAF-II, and HT-29, a 3-day viability assay was performed essentially as described in Example 3. In brief, 100 μL of cells in culture medium (RPMI1640 with 25 mM Hepes and L-glutamine + 10% heat-inactivated DBSI + 50 U / mL Pen / Strep) were pipetted into 96-well plates (5.000 cells / well) and allowed to adhere by incubation overnight at 37 ° C. Next, 25 μL of purified C1q sample (2.5 μg / mL final concentration) and 25 μL of a concentration dilution series of antibody samples (range 0.0003-20 μg / mL final concentration, 5-fold dilution) were added to the cells and incubated at 37 ° C for 3 days. Cell viability was determined using the CellTiter-Glo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data with logarithmic concentration axes were analyzed using nonlinear regression (sigmoidal dose response with variable slope) and plotted using GraphPad Prism software. Figure 18The introduction of K326W / E333S / E430G substitutions in an IgG3 variant of an anti-DR5 antibody (IgG3-DR5-CONA-C49W-R435H-K326W / E333S / E430G) was shown to induce agonist activity in all tested cell lines: WIL2S-SF ( Figure 18 A), BxPC-3( Figure 18 B), HPAF-II( Figure 18 C) and HT29( Figure 18 D) In ​​all cell lines tested, the IgG1 variant IgG1-DR5-CONA-C49W-K326W / E333S / E430G was more potent than the IgG3 variant IgG3-DR5-CONA-C49W-R435H-K326W / E333S / E430G.

[0978] Example 20: Effect of Combining E430G with K326W / E333T on the Agonist Activity of Anti-DR5 Antibodies A viability assay was performed to evaluate the effect of the combination of the Fc-Fc enhancing substitutions E430G and K326W / E333T on the agonist activity of the anti-DR5 antibody IgG1-CONA-C49W compared to K326W / E333S on DR5-positive WIL2-S cells. The in vitro viability assay was performed essentially as described in Example 8. Briefly, 100 μL of WIL2-S cells were pipetted into a 96-well plate (50,000 cells / well) in culture medium (RPMI 1640 containing 25 mM Hepes and L-glutamine (Lonza, Cat. No. BE12-115F) + 10% heat-inactivated DBSI + 1 mM sodium pyruvate (Lonza, Cat. No. BE13-115E) + 50 U / mL Pen / Strep). Next, 50 μL of a concentration dilution series of antibody samples (range 0.001-20 μg / mL final concentration, 5-fold dilution) and 10 μL of purified C1q sample (2.5 μg / mL final concentration) were added to the cells and incubated at 37°C for 1 day. Cell viability was determined using the cellTiterGlo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using GraphPad Prism software using nonlinear regression (sigmoidal dose response with variable slope). Figure 19 It was shown that introduction of K326W / E333T / E430G in IgG1-CONA-C49W resulted in induction of DR5 agonist activity of the single agent with similar killing potency in an in vitro viability assay on WIL2-S cells as by introduction of K326W / E333S / E430G.

[0979] Example 21: Pharmacokinetic (PK) analysis of IgG1-CONA-C49W antibody variants containing Fc-Fc enhancing mutations and / or Fc mutations affecting C1q binding.

[0980] The effects of the E430G Fc-Fc enhancing mutation and the C1q binding enhancing mutation on the clearance of IgG1-CONA-C49W were studied in a PK experiment in SCID mice. All tested antibody variants are listed in Table 6. Animal experiments were performed in accordance with the Dutch Animal Protection Act (WoD) converted from the Directive (2010 / 63 / EU) and the Code of Practice "Animal Experiments in Cancer Research" (Inspection V&W, Zutphen, The Netherlands, 1999), if applicable, and approved by the Utrecht Ethics Committee. Animals were raised and handled in an animal house (GDL) accredited by AAALAC and ISO 9001:2000 according to the good animal practice defined by FELASA. Female SCID mice (CB- Hsd-Prkdc scid; Envigo SCID mice) were injected intravenously with 450 μg of antibody (22.5 mg / kg) in a 200 μL injection volume (3 mice per group). 50 μL blood samples were collected from the saphenous vein 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 20 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged at 14,000g for 10 minutes. 20 μL plasma samples were diluted with 380 μL PBS (1:20) and stored at -20°C until the antibody concentration was determined. Total human IgG concentration was determined using a sandwich ELISA. Mouse anti-human IgG-kappa mAb clone MH16 (CLBSanquin, catalog number M1268) was used as the capture antibody and coated overnight in 100 μL at 4°C at a concentration of 2 μg / mL in PBS to a 96-well Microlon ELISA plate (Greiner, Germany). The plates were blocked by incubating them with PBS supplemented with 0.2% bovine serum albumin (BSA) at room temperature on a plate shaker. After washing, 100 μL of diluted plasma samples were added and incubated on a plate shaker at room temperature for 1 hour. The plates were washed three times with 300 μL PBST (PBS supplemented with 0.05% Tween 20) and then incubated on a plate shaker at room temperature with 100 μL peroxidase-labeled goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA; 1:10.000 in PBST supplemented with 0.2% BSA) for 1 hour. The plate was washed three more times with 300 μL PBST and then incubated with 100 μL substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS; Roche, catalog number 11112422001; 1 tablet (Roche, catalog number 11112597001) in 50 mL ABTS buffer] for 15 minutes at room temperature, protected from light. The reaction was terminated by adding 100 μL 2% oxalic acid and incubating at room temperature for 10 minutes. The absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT). Concentrations were calculated by using the injected material as a reference curve. As a plate control, human myeloma protein containing IgG (binding site, catalog number BP078) was included. The human IgG concentration (in μg / mL) was plotted ( Figure 20 A) and the area under the curve (AUC) was calculated using Graphpad prism. The clearance until the last day of blood sampling (day 21) was determined by the formula D*1.000 / AUC, where D is the injected dose of 22.5 mg / kg ( Figure 20B). All tested IgG1-CONA-C-49W variants containing the E430G Fc-Fc enhancing mutation and / or the C1q binding enhancing mutation showed comparable clearance to WT IgG1 ( Figure 20 A, B). In summary, the introduction of C1q binding-enhancing mutations such as K326W / E333S or K326A / E333A did not significantly affect the clearance of IgG1 antibodies containing the E430G Fc-Fc-enhancing mutation, such as in IgG1-CONA-C49W-K326W / E333S / E430G and IgG1-CONA-C49W-K326A / E333A / E430G.

[0981] Table 6: IgG1-CONA-C49W antibody variants tested in PK analysis in scid mice

[0982]

[0983]

[0984] Example 22: Effect of combining the E430G Fc-Fc enhancing mutation and the C1q binding enhancing mutations K326A / E333A or K326W / E333S on FcRn binding of IgG1 antibodies.

[0985] Neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting IgG from degradation. After antibody internalization, FcRn binds to the antibody Fc region in the endosome, where the interaction is stable in a mild acidic environment (pH 6.0). Upon recycling to the plasma membrane (wherein the environment is neutral (pH 7.4)), the interaction is lost and the antibody is released back into circulation. This affects the plasma half-life of IgG.

[0986] FcRn binding ELISA was performed to evaluate the effect of the combination of the introduction of Fc-Fc enhancing mutations and the C1q binding enhancing mutations K326A / E333A or K326W / E333S on the binding of human FcRn to IgG1-7D8 antibody variants. IgG1-7D8-I235A / H310A / H435A was used as a negative control for FcRn binding (FcRn knockout; Shields et al., J. Biol. Chem. 2001; 276: 6591); IgG1-7D8-M252Y / S254T / T256E was used as a control for enhanced FcRn binding (Dall'Acqua et al., J Biol Chem. 2006 Aug 18; 281(33): 23514-24). All incubations were performed at room temperature. 96 streptawell plates (Roche, catalog number 1734776001) were coated with 5 μg / mL (100 μL / well) of the recombinantly produced biotinylated extracellular domain of human FcRn (FcRnECDHis-B2M-BIO, i.e., the extracellular domain of human FcRn with a C-terminal His and BAP tag, as a dimer with β2 microglobulin) diluted in PBST and 0.2% BSA for 1 hour. The plates were washed three times with PBST. Serially diluted antibody samples (range 0.003-10 μg / mL final concentration, 3-fold dilution in PBST / 0.2% BSA, pH 6.0) were added and incubated for 1 hour. The plates were washed with PBST / 0.2% BSA, pH 6.0. Polyclonal goat-anti-human IgG (1:10,000; Jackson ImmunoResearch, catalog number 109-035-097) conjugated with horseradish peroxidase (HRP) diluted in PBST / 0.2% BSA, pH 6.0 or 7.4 was added and the plate was incubated for 1 hour. After washing, 100 μL ABTS (1 mg / mL) was added as a substrate and the plate was incubated in the dark for 30 minutes. The reaction was terminated with 100 μL 2% oxalic acid and the absorbance was measured at 405 nm using an ELx808 absorbance microplate reader (BioTek). Logarithmic transformed data were analyzed by fitting a sigmoidal dose-response curve with a variable slope using GraphPad Prism software. The negative control (IgG1-7D8-I235A / H310A / H435A) showed complete loss of human FcRn binding at pH 6.0 ( Figure 21 A), while the positive control (IgG1-7D8-M252Y / S254T / T256E) showed enhanced binding to human FcRn compared to WT IgG1-7D8 at pH 6.0 and loss of binding at pH 7.4 ( Figure 21B). All tested IgG1-7D8 variants with Fc-Fc enhancing mutations, with or without C1q binding enhancing mutations, showed efficient binding to human FcRn at pH 6.0 and loss of binding at pH 7.4. However, introduction of the Fc-Fc enhancing mutation E430G alone resulted in a slight decrease in binding to human FcRn at pH 6.0 compared to WT IgG1-7D8, which was slightly further reduced when combined with the C1q binding enhancing mutations K326A / E333A or K326W / E333S.

[0987] Example 23: Effect of C1q on ADCC activity of anti-CD20 IgG1-7D8 antibody variants containing K326W / E333S / E430G substitutions.

[0988] The effect of C1q on the ADCC activity of anti-CD20 IgG1-7D8 antibody variants containing both the E430GFc-Fc enhancing mutation and the C1q binding enhancing substitutions K326W / E333S was tested in a chromium release assay using WIL2-S SF cells in serum-free medium. 6 cells / mL), washed (twice in PBS, 1,200 rpm, 5 minutes) and collected in 1 mL of serum-free medium (HyQ ADCF-Mab medium supplemented with 10% sodium pyruvate). 200 μCi 51 Cr (chromium-51; Amersham Biosciences Europe GmbH) was added and incubated at 37°C in a shaking water bath for 1 hour. After washing the cells (twice in PBS, 1,200 rpm, 5 minutes), the cells were resuspended in serum-free medium. Chromium-labeled cells were counted by trypan blue exclusion and diluted to 1×10 5 Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats of healthy donors (Sanquin) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza). After resuspending PBMCs in serum-free medium, they were counted by trypan blue exclusion and concentrated to 1 × 10 7Cells / mL. For ADCC experiments, 50 μL of chromium-labeled WIL2-S SF cells were pipetted into a 96-well plate (5,000 cells / well). 25 μL of antibody sample from a dilution series (ranging from 0.003-10 μg / mL final concentration, 3-fold dilution) and 25 μL of purified human C1q (2.5 μg / mL final concentration) or culture medium were added and pre-incubated at room temperature for 10 minutes. Next, 50 μL of PBMC (500.000 cells / well) was added, resulting in an effector to target ratio of 100: 1, and incubated at 37°C for 4 hours. Maximum cell lysis was determined by incubating 50 μL of chromium-labeled WIL2-S SF cells (5,000 cells / well) with 100 μL of 5% Triton-X100 (Sigma-Aldrich). Spontaneous lysis was determined by incubating chromium-labeled WIL2-S SF cells (5,000 cells / well) in 150 μL medium without antibodies and effector cells. Antibody-independent cell lysis was determined by incubating chromium-labeled WIL2-S SF cells (5,000 cells / well) with PBMC (500.000 cells / well) in a total volume of 150 μL in the absence of antibodies. The amount of cell lysis was determined using a scintillation counter. The cells were centrifuged (1,200 rpm; 3 minutes) and 25 μL of the supernatant was transferred to 96-well white optiplates filled with 100 μL microscint-40 solution. The released proteins in the supernatant were counted using a scintillation counter. 51 Cr. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis according to the following formula: (cpm sample - cpm antibody-independent lysis) / (cpm maximum lysis - cpm spontaneous lysis) × 100%. As negative controls, nonspecific IgG1-b12 antibody and IgG1-7D8 variant with L234A / L235A / P329G substitutions were tested, which are known to abolish complement binding and activation as well as FcγR binding and ADCC induction (Lo et al. JBC 2017). As expected, no ADCC activity of the nonspecific antibody IgG1-b12 was observed in the absence or presence of C1q ( Figure 22 For the IgG1-7D8-F405L-K326W / E333S / E430G antibody, the addition of 2.5 μg / mL purified human C1q resulted in inhibition of ADCC activity in WIL2-S SF cells in serum-free medium. In contrast, C1q did not affect the ADCC activity of WT IgG1-7D8 and IgG1-7D8-E430G.

[0989] Example 24: Effect of K326W / E333S / E430G on the agonist activity of anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05.

[0990] The effect of introducing the combination of the Fc-Fc enhancing mutation E430G and the C1q binding enhancing mutation K326W / E333S on the agonist activity of the anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 was tested in an in vitro viability assay using WIL2-S SF cells. A 1-day viability assay was performed essentially as described in Example 8. Briefly, cells in 100 μL of serum-free medium were pipetted into a 96-well plate (50,000 cells / well). 25 μL of a concentration dilution series of antibody samples (ranging from 0.0003 to 20 μg / mL final concentration, 5-fold dilution) and 25 μL of purified C1q (2.5 μg / mL final concentration) were added and incubated at 37°C for 1 day. Cell viability was determined using the CellTiterGlo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism software.

[0991] Introduction of both the E430G Fc-Fc enhancement and the K326W / E333S C1q binding enhancement mutations resulted in the two tested antibodies, IgG1-hDR5-01-G56T and IgG1-hDR5-05, inducing DR5 agonist activity, whereas these antibodies did not induce cell killing when only the E430GFc-Fc enhancement mutation was introduced ( Figure 23 ).

[0992] Example 25: Compatibility of K326W / E333S / E430G with the complementary Fc mutation pair K439E; S440K in agonist anti-DR5 antibody combinations.

[0993] The compatibility of the K326W / E333S / E430G mutation with other Fc engineered mutations, such as the complementary Fc mutation pair K439E; S440K, which can control intermolecular Fc-Fc interactions between antibodies binding to different cell surface targets, was tested in an in vitro viability assay on WIL2-S SF cells using the anti-DR5 agonist antibody combination IgG1-hDR5-01-K326W / E333S / E430G + IgG1-hDR5-05-K326W / E333S / E430G. A 1-day viability assay was performed essentially as described in Example 8. Briefly, cells in 100 μL of serum-free medium were pipetted into a 96-well plate (50,000 cells / well). 25 μL of a concentration dilution series of antibody samples (ranging from 0.0003 to 20 μg / mL final concentration, 5-fold dilution) and 25 μL of purified C1q (2.5 μg / mL final concentration) were added and incubated at 37°C for 1 day. Cell viability was determined using the CellTiterGlo assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using GraphPad Prism software using nonlinear regression (sigmoidal dose response with variable slope). Figure 24 The results showed that a single antibody, IgG1-hDR5-01-K326W / E333S / E430G-K439E, containing the Fc-Fc inhibitory mutation K439E, induced almost no cell killing. IgG1-hDR5-05-K326W / E333S / E430G-S440K, containing the Fc-Fc inhibitory mutation S440K, induced some cell killing, although the maximum killing was not 100%. In contrast, the combination of two complementary Fc-Fc control mutations, K439E and S440K (IgG1-hDR5-01-K326W / E333S / E430G-K439E + IgG1-hDR5-05-K326W / E333S / E430G-S440K), showed potent cell killing, which was similar to the combination without the complementary Fc-Fc control mutations K439E and S440K. The cell killing by the antibody combination containing the K326W / E333S / E430G mutations (with and without the complementary mutations K439E; S440K) was much more effective than the antibody combination containing only the E430G Fc-Fc enhancing mutation (IgG1-hDR5-01-K326W / E333S / E430G-K439E + IgG1-hDR5-05-K326W / E333S / E430G-S440K).

[0994] Example 26: Effect of combining the C1q binding enhancing substitutions K326W / E333S with K248E / T437R on the CDC efficacy of anti-CD52 antibodies

[0995] The effect of combining the K326W / E333S C1q binding enhancing mutation with the K248E / T437R substitution (Zhang et al., 2017 MAbs(9)7:1129-42), which promotes antibody multimerization on the cell surface, on CDC efficacy was tested using the anti-CD52 IgG1-Campath variant (based on alemtuzumab) against CD52-sensitive Wien 133 B-cell lymphoma cells. Wien 133 cells (provided by Dr. Geoff Hale, BioAnaLab Limited, Oxford, UK) were harvested and resuspended in culture medium [RPMI (Lonza, Cat No. BE12-115F) containing 0.2% bovine serum albumin (BSA; Roche Cat No. 10735086001). 40 μL of cells were pipetted into a round-bottom 96-well plate (0.1×10 6 Cells / well). Add 40 μL serially diluted antibody samples (ranging from 0.002 to 40 μg / mL final concentration, 4-fold dilution) and incubate at room temperature for 15 minutes. Then, add 20 μL NHS (20% final concentration) as a source of human complement and incubate at 37°C for 45 minutes. The reaction is terminated by placing the sample on ice. The cooled cells are pelleted and resuspended in 30 μL 2 μg / mL propidium iodide (PI; Sigma Aldrich). Samples are analyzed by flow cytometry on Intellicyt iQue Screener PLUS, and the percentage of lysis is determined according to the following formula: % lysis = (number of PI positive cells / total number of cells) x 100%. Figure 25 The results showed that the introduction of the hexamerization-enhancing single mutation E430G or the multimerization-enhancing double mutation K248E / T437R into WT IgG1-Campath resulted in increased CDC efficacy against Wien 133 cells. CDC efficacy was further enhanced by combining the K248E / T437R mutation, which promotes multimerization on the cell surface, with the K326W / E333S mutation, which enhances C1q binding, in IgG1-Campath-K248E / K326W / E333S / T437R.

[0996] Example 27: Effect of combining E430G and K326W / E333S on the efficacy of agonistic anti-DR4 antibodies in the presence of CIq.

[0997] A viability assay was performed to investigate the effect of the combination of hexamerization-enhancing mutations E430G and K326W / E333S on the agonistic activity of the anti-DR4 antibody chCTB007 on DR4-positive BxPC-3 cells. Viability was performed essentially as described in Example 2. Briefly, 100 μL of a BxPC-3 single cell suspension was seeded in culture medium (RPMI containing 10% heat-inactivated DBSI) in a polystyrene 96-well flat-bottom plate (5,000 cells per well) and allowed to adhere overnight at 37°C. 25 μL of purified C1q sample (2.5 μg / mL final concentration) and 25 μL of a concentration dilution series of antibody samples (range 0.00001-20 μg / mL final concentration, 5-fold dilution) were added and incubated at 37°C for 3 days. The viability of the cultured cells was determined in the CellTiter-Glo luminescent cell viability assay as described in Example 2. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer), and data were analyzed and plotted using GraphPad Prism software using nonlinear regression (sigmoidal dose response with variable slope). Figure 26 The results show that when tested as single agents in an in vitro viability assay against adherent human BxPC-3 pancreatic cancer cells in the presence of heat-inactivated fetal bovine serum supplemented with 2.5 μg / mL purified C1q, the combination of the hexamerization-enhancing mutation E430G and the two mutations K326W / E333S resulted in the induction of potent killing efficacy similar to that of the triple mutant E345R / E430G / S440Y anti-DR4 antibody IgG1-DR4-chCTB007. In contrast, when tested as wild-type antibody IgG1-DR4-chCTB007 or in the presence of only the E430G mutation, these anti...

Claims

1. A polypeptide comprising an Fc region and an antigen-binding region of an immunoglobulin, wherein the substitutions at positions E430, K326, and E333 in the Fc region are E430G, K326W, and E333S, respectively; Therein the positions correspond to human IgG1 according to EU numbering, and there are no further mutations in the Fc region.

2. The polypeptide according to claim 1, wherein the polypeptide is an antibody, a monospecific antibody, a bispecific antibody or a multispecific antibody.

3. The polypeptide according to claim 1, wherein the Fc region is of human IgG1, IgG2, IgG3, IgG4 isotype or mixed isotypes.

4. The polypeptide according to claim 1, wherein the Fc region is of the human IgG1 isotype.

5. The polypeptide according to claim 1, wherein the polypeptide is a human antibody, a humanized antibody or a chimeric antibody.

6. The polypeptide according to claim 1, wherein the antigen binding region binds to a member of the tumor necrosis factor receptor superfamily (TNFR-SF) or the G protein coupled receptor (GPCR) superfamily.

7. The polypeptide of claim 1, wherein the antigen binding region binds to a TNFR-SF member selected from the group consisting of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, NGFR, OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR.

8. A composition comprising at least one polypeptide according to any one of claims 1 to 7.

9. The composition according to claim 8, comprising a first polypeptide and a second polypeptide.

10. The composition according to any one of claims 8-9, wherein the composition is a pharmaceutical composition.

11. Use of a polypeptide according to any one of claims 1 to 7 or a composition according to any one of claims 8 to 10 for the preparation of a medicament for treating a disease, wherein the disease is cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

12. A kit comprising a polypeptide or composition according to any one of the preceding claims, wherein the polypeptide or composition is in one or more containers.

13. The kit according to claim 12, wherein the polypeptide or composition according to any one of the preceding claims is for simultaneous, separate or sequential use in therapy.

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