Polypeptide, methods for increasing the in vitro agonistic activity of a polypeptide and for increasing the in vitro cdc activity of a polypeptide, composition, parts kit, and use of a polypeptide or a composition

Abstract

Polypeptides and antibodies having an fc region and an antigen-binding region are described here, wherein the fc region has an enhancing fc-fc mutation and an enhancing c1q binding mutation, providing polypeptides or antibodies with increased cdc activity and / or agonist activity.

Description

POLYPEPTIDE, METHODS FOR INCREASING THE IN VITRO AGONISTIC ACTIVITY OF A POLYPEPTIDE AND FOR INCREASING THE IN VITRO CDC ACTIVITY OF A POLYPEPTIDE, COMPOSITION, PARTS KIT, AND USE OF A POLYPEPTIDE OR COMPOSITION FIELD OF THE INVENTION

[001] The present invention relates to polypeptides containing a region Fc comprising a binding region, such as antibodies, which have at least two amino acid substitutions in the Fc region compared to a parental polypeptide or antibody. FUNDAMENTALS OF THE INVENTION

[002] 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 the therapeutic window defined by efficacy and toxicity. CDC is initiated by C1q binding to the Fc regions of antibodies. C1q is a multimeric protein consisting of six globular binding heads attached to a stem.

[003] The hexamerization of IgG upon binding to the target on the cell surface has been shown to be enhanced by point mutations in the Fc region. Hexamerization is mediated through non-covalent intermolecular Fc-Fc interactions, and Fc-Fc interactions can be enhanced by point mutations in the CH3 domain, including E345R and E430G.

[004] WO2013 / 004842 describes antibodies or polypeptides comprising variant Fc regions having one or more amino acid modifications resulting in modified effector functions such as complement-dependent cytotoxicity (CDC).

[005] WO2014 / 108198 describes polypeptides as antibodies Petition 870260062878, dated 06 / 26 / 2026, page 11 / 410 / 194 comprising variant Fc regions having one or more amino acid modifications resulting in increased complement-dependent cytotoxicity (CDC).

[006] WO2016 / 164480 describes antigen-binding complexes having agonistic activity.

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

[008] Consequently, it is an object of the present invention to provide a polypeptide or antibody comprising an Fc region of a human IgG and an antigen-binding region which polypeptide has increased Fc-Fc interactions and agonistic activity such as increased activation of a target receptor upon binding, when compared with a parental polypeptide, where the parental polypeptide is a human IgG of the same isotype and having the same antigen-binding region, but without any mutations in the Fc region, i.e., a parental polypeptide or parental antibody.

[009] 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, for example antibody, is bound to the corresponding antigen compared with a parental polypeptide, wherein the parental polypeptide has no mutations in the Fc region.

[0010] It is yet another object of the present invention to provide a polypeptide with enhanced Fc-Fc interaction properties and enhanced effector functions such as CDC. It is a further object of the present invention to provide a polypeptide with enhanced Fc-Fc interactions and enhanced C1q binding properties, when compared with a parental polypeptide without any mutations in the Fc region. Petition 870260062878, dated 06 / 26 / 2026, page 12 / 410 / 194 SUMMARY OF THE INVENTION

[0011] 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 enhancing Fc-Fc mutation and a C1q-binding mutation providing polypeptides or antibodies with enhanced CDC activity and / or agonist activity.

[0012] Without being limited to theory, it is believed that the polypeptides or antibodies of the invention are capable of a stable binding interaction between the Fc regions of two polypeptide or antibody molecules when bound to the target on a cell surface, which leads to enhanced oligomerization, such as hexamer formation, thus providing an avid surface. The polypeptides or antibodies of the invention additionally have an enhanced Fc effector response compared to their parent polypeptide or parent antibody without any mutations in the Fc region, i.e., a parent polypeptide or antibody of the same isotype.

[0013] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) at least one Fc-Fc enhancing substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) at least one C1q binding substitution, wherein the positions correspond 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).

[0014] In one aspect, the invention provides a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region, wherein the Fc region comprises, a) a substitution at a position selected from the group consisting of: Petition 870260062878, dated 06 / 26 / 2026, page 13 / 410 / 194 replacement E430, E345 or S440Y or S440W, and b) a replacement in one or more position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, where the positions correspond to human IgG1, according to EU numbering.

[0015] A substitution in a position corresponding to the E430, E345 or S440Y or S440W substitution is considered an intensifying Fc-Fc substitution, according to the present invention.

[0016] A substitution at one or more position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, is considered a C1q linkage substitution according to the present invention.

[0017] That is, the inventors of the present invention in a first aspect of the invention have found that introducing a first mutation that enhances Fc-Fc interaction together with a second mutation that enhances binding to C1q provides a polypeptide or antibody with enhanced agonist and / or CDC activity.

[0018] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) a substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) a substitution at one or more position(s) 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.

[0019] That is, the inventors found that an Fc-Fc enhancing mutation together with one or more C1q linking substitution(s) at one or more selected position(s) from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396 can provide agonist activity. Petition 870260062878, dated 06 / 26 / 2026, page 14 / 410 / 194

[0020] The inventors further verified that an enhancing Fc-Fc mutation together with one or more C1q-linking substitution(s) at one or more selected position(s) from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396 can provide enhanced Fc-mediated effector functions, such as enhanced CDC.

[0021] The combination of an Fc-Fc enhancing mutation and the C1q linkage substitution in a polypeptide or antibody has the surprising effect of generating a polypeptide or antibody with agonistic properties when compared to a parental polypeptide or antibody.

[0022] In one embodiment of the present invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y.

[0023] In one embodiment of the present invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of: E430G, E430S, E430F and E430T.

[0024] In one embodiment of the present invention, the polypeptide or antibody comprises at least one substitution selected from the group consisting of: E345K, E345Q, E345R and E345Y.

[0025] In one embodiment of the present invention, the polypeptide or antibody comprises at least one E430G substitution. In one embodiment of the present invention, the polypeptide or antibody comprises at least one E345K substitution. In one embodiment of the present invention, the polypeptide or antibody comprises at least one S440Y substitution.

[0026] In one embodiment of the present invention, the polypeptide or antibody comprises a substitution at one or more position(s) Petition 870260062878, dated 06 / 26 / 2026, page 15 / 410 / 194 selected from the group consisting of: K326, E333 and P396.

[0027] In one embodiment of the present invention, the polypeptide or antibody comprises a substitution at one or more positions, such as two or three positions selected from the group consisting of: K326A, K326W, E333S, E333A and P396L.

[0028] In one embodiment of the present invention, the polypeptide or antibody comprises a substitution at one or more positions, such as two or three positions selected from the group consisting of: K326A, K326W, E333S, E333A, E333T and P396L.

[0029] In one embodiment of the present invention, the polypeptide or antibody comprises the K326W and E333S substitutions.

[0030] In a further aspect, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody comprising an Fc region of a human IgG and an antigen-binding region, the method comprising a) introducing a substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) introducing substitutions at one or more position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the position corresponds to human IgG1, according to EU numbering.

[0031] In a further aspect, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody comprising an Fc region of a human IgG and an antigen-binding region, the method comprising a) introducing a substitution at a position selected from the group consisting of: substitution E430, E345 or S440Y or S440W, and b) introducing substitutions at one or more position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, where the position corresponds to human IgG1, according to EU numbering. Petition 870260062878, dated 06 / 26 / 2026, p. 16 / 410 / 194

[0032] In another aspect, the present invention relates to a composition comprising at least one polypeptide or antibody as described herein.

[0033] In another aspect, the present invention relates to a polypeptide, antibody or a composition, as described herein, for use as a medicament.

[0034] In another aspect, the present invention relates to a polypeptide, antibody or a composition, as described herein, for use in the treatment of cancer, autoimmune disease, inflammatory disease or infectious disease.

[0035] In another aspect, the present invention relates to a method of treating an individual having a disease comprising administering to said individual an effective amount of a polypeptide, an antibody or composition, as described herein.

[0036] These and other aspects of the invention, particularly various therapeutic uses and applications for the polypeptide or antibody, are described in further detail below. Brief Description of the Drawings

[0037] Figure 1 shows the effect of E430G, K326A / E333A / P396L and K326A / E333A / P396L / E430G on the efficacy of anti-DR5 IgG1hDR5-01-G56T antibodies (A), IgG1-hDR-05 (B) and the antibody combination (C) on adherent human pancreatic cancer cells BxPC-3 as determined in a 3-day viability assay (CellTiter-Glo). Representative examples from two experiments are shown.

[0038] Figure 2 shows the efficacy of a monovalent anti-DR5 antibody with K326A / E333A / P396L / E430G on adherent human pancreatic cancer cells BxPC-3 (A) and colon cells COLO 205 as determined in a 3-day viability assay (CellTiter-Glo). As a monovalent anti-DR5 antibody, a bispecific antibody with an arm Petition 870260062878, dated 06 / 26 / 2026, page 17 / 410 / 194: A specific DR5 arm derived from IgG1-hDR5-01-G56T and a non-specific arm against HIV protein gp120 derived from IgG1-b12 were generated by controlled Fab arm exchange. Representative examples of these experiments are shown.

[0039] Figure 3 shows the effect of E430G combined with K326A / E333A / P396L or two of these substitutions E333A / P396L, K326A / E333A or K326A / P396L, on the efficacy of anti-DR5 IgG1-hDR5-01-G56T antibody on adherent human pancreatic cancer cells BxPC-3 (A) and colon cancer cells COLO 205 (B), as determined in a 3-day viability test (CellTiter-Glo), and on binding to C1q as determined in an ELISA test (C). Representative examples of these experiments are shown.

[0040] Figure 4 shows the effect of E430G combined with K326A / E333A or K326W / E333S on C1q binding anti-DR5 IgG1-CONA-C49W antibody as determined in an ELISA test (A) and on the efficacy of anti-DR5 IgG1-hDR5-01-G56T antibody on adherent human pancreatic cancer cells BxPC-3 (B) and colon cancer cells COLO 205 (C) as determined in a 3-day viability test (CellTiter-Glo). Representative examples of these experiments are shown.

[0041] Figure 5 shows the effect of E430G combined with C1q-binding substitutions S267E / H268F / S324T or the 113F variant of chimeric IgG1 / IgG3 isotype IgG1 in C1q-binding anti-DR5 antibody IgG1-hDR5-01-G56T as determined in an ELISA test (A) and on the efficacy of anti-DR5 antibody IgG1-hDR5-01-G56T on adherent human pancreatic cancer cells BxPC-3 (B) and colon cells COLO 205 (C) as determined in a 3-day viability test (CellTiter-Glo). Representative examples of these experiments are shown.

[0042] Figure 6 shows a summary of 3-day viability tests (CellTiter-Glo) on human BxPC-3 pancreatic cancer cells. Petition 870260062878, dated 06 / 26 / 2026, page 18 / 410 / 194 adherents with 10 pg / mL of IgG1-hDR5-01-G56T variants containing the indicated mutations. The effect is represented and classified by the percentage of viable cells in relation to WT IgG1-hDR5-01-G56T, which was defined as 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 comparisons test).

[0043] Figure 7 shows the agonistic effect of anti-DR5 antibody IgG1-CONA-C49W-K326A / E333A / P396L / E430G (A) and IgG1-hDR5-01G56T-K326W / E333S / E430G (B) on WIL2-S SF cell suspension in serum-free medium in the presence or absence of 2.5 μg / mL purified human C1q as determined in a 24-hour viability test. The percentage of viable cells is represented by the percentage of TO-PRO-3 negative cells.

[0044] Figure 8 shows the agonistic effect of 2.5 μg / mL anti-DR5 antibody variants IgG1-hDR5-01-G56T with E430G, an enhancing Fc-Fc substitution in combination with C1q-binding substitutions (A), and the combination of IgG1-hDR5-01-G56T-E430G + IgG1-hDR505-E430G antibodies (B) on cells suspended in WIL2-S SF in serum-free medium in the presence or absence of a range of purified human C1q concentrations as determined in a 24-hour viability test. Percent viable cells are represented by the percentage of TO-PRO-3 negative cells. Data from four different experiments are represented with error bars indicating the standard deviation.

[0045] Figure 9 shows the efficacy of 2.5 μg / mL of anti-DR5 IgG1hDR5-01-G56T-K326W / E333S / E430G agonistic (A) and the combination of IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G antibodies (B) in cells suspended in WIL2-S SF in serum-free medium with or without purified human C1q and anti-C1q neutralizing antibody as determined in a 24-hour viability test. The percent viable cells are Petition 870260062878, dated 06 / 26 / 2026, page 19 / 410 / 194 represented by the percentage of negative cells for TO-PRO-3.

[0046] Figure 10 shows solution-phase complement activation as measured by quantification of C4d deposition when antibody samples were incubated in NHS for IgG1hDR5-01-G56T antibody variants containing the 430G Fc-Fc enhancing substitutions in combination with C1q-binding substitutions K326W / E333S, K326A / E333A, or K326A / E333A / P396L. HAGG (heat-aggregated gamma globulin) and IgG1-CONA-RGY were tested as positive controls for solution-phase complement activation.

[0047] Figure 11 shows the effect of introducing K326W, E333S, or K326W / E333S substitutions in IgG-CONA-C49W and IgG1-CONAC49W-E430G on C1q binding, as determined by an ELISA test (A,B), 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 cells in suspension as determined by a 3-day viability test (CellTiter-Glo) (E,F,G). Standard deviations were calculated from two independent experiments.

[0048] Figure 12 shows the effect of introducing K326W, E333S, or K326W / E333S substitutions into IgG-CONA-C49W and IgG1-CONAC49W-E430G (2.5 pg / mL) on the viability of WIL2-S SF cell suspensions in serum-free medium in the presence of a range of purified human C1q concentrations, as determined by a 24-hour viability test. Percent viable cells were determined by a CellTiter-Glo assay.

[0049] Figure 13 shows the effect of adding an anti-C1q antibody on the efficacy of anti-DR5 IgG1-CONA-C49W antibody variants with mutations enhancing C1q binding and / or enhancing Fc-Fc interaction in a 24-hour viability assay in cells suspended in WIL2-S SF in serum-free medium supplemented with purified human C1q. As Petition 870260062878, dated 06 / 26 / 2026, page 20 / 410 / 194 viable percentage cells were determined in a CellTiter-Glo test.

[0050] Figure 14 shows the effect of adding a peptide that inhibits Fc-Fc interactions between variants of anti-DR5 IgG1-CONA-C49W antibodies opsonized in WIL2-S SF cell suspensions, incubated in serum-free medium supplemented with purified human C1q, in a 24-hour viability test. Percent viable cells were determined in a CellTiter-Glo assay. Scrambled peptide WCDLEGVTWHACL was used as a non-specific control peptide.

[0051] Figure 15 shows the effect of combining C1q linking substitutions K326W / E333S with Fc-Fc enhancing mutations E345K, E345R, or S440Y on the agonist activity of anti-DR5 IgG1CONA-C49W antibody on adherent human pancreatic cancer cells BxPC-3, as determined in a 3-day viability assay (CellTiterGlo).

[0052] Figure 16 shows the effect of the E430G mutation, combined with mutations enhancing C1q binding S267E / H268F / S324T or an 113F variant of the chimeric IgG1 isotype IgG1 / IgG3, introduced into anti-DR5 IgG1-CONA-C49W antibody, on the viability of adherent human pancreatic cancer cells BxPC-3, as determined in a 3-day viability assay (CellTiter-Glo).

[0053] Figure 17 shows the effect of a functionally monovalent anti-DR5 antibody with K326W / E333S / E430G mutations on the viability of WIL2-S SF cells in suspension as determined in a one-day viability assay (CellTiter-Glo). The functionally monovalent anti-DR5 antibody was generated as a bispecific antibody by controlled Fab-arm exchange of IgG1-CONA-C49W-F405LK326W / E333S / E430G (DR5-specific arm) and IgG1-b12-K409RK326W / E333S / E430G (non-specific arm, directed against HIV gp120 protein). RLU: relative luminescence units. Petition 870260062878, dated 06 / 26 / 2026, page 21 / 410 / 194

[0054] Figure 18 shows the effect of introducing K326W / E333S / E430G substitutions on the agonist activity of IgG1 and IgG3 isotype variants of an anti-DR5 antibody (IgG1-CONA-C49W and IgG3-CONAC49W-R345H) as determined in a 1-day viability test on WIL2-S SF cells (A) and 3-day viability tests on BxPC-3 (B), HPAF-II (C), and HT-29 (D) cells. Viability was determined using the CellTiter-Glo kit.

[0055] Figure 19 shows the effect of introducing both the E430G hexamerization-enhancing mutation and the K326W / E333T or K326W / E333S C1q binding-enhancing mutations in anti-DR5 IgG1-CONA-C49W antibody in WIL2-S cell suspensions, as determined in a 24-hour viability assay (CellTiterGlo).

[0056] Figure 20 shows the clearance rate of 450 pg of antibody administered intravenously in SCID mice. (A) Total human IgG in serum samples was determined by ELISA and plotted on a concentration-versus-time curve. Each data point represents the mean standard deviation + / - of samples in triplicate. (B) Clearance after day 21 following antibody administration was determined 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. A representative example of the two independent ELISA experiments is shown.

[0057] Figure 21 shows the effect of the Fc-Fc E430G enhancing mutation in combination with C1q binding enhancing mutations (K326A / E333A or K326W / E333S) on the binding of IgG1-7D8 antibody variants to human FcRn, as determined by an FcRnECDHis-B2M-BIO coated ELISA at pH 6.0 and 7.4. IgG1-7D8I235A / H310A / H435A was used as a negative control for FcRn binding at pH 6.0 (FcRn knockout); IgG1-7D8-M252Y / S254T / T256E was used as a control for enhanced FcRn binding at pH 7.4. Petition 870260062878, dated 06 / 26 / 2026, page 22 / 410 / 194

[0058] Figure 22 shows an ADCC chromium release assay using WIL2-S SF as target cells and human PBMCs (3 donors) as effector cells (E:T ratio 100:1) in serum-free medium with and without the addition of purified human C1q. In the absence and presence of C1q, chromium-labeled WIL2-S SF cells were incubated with a series of antibody concentrations to compare the ADCC activity of IgG1-7D8-F405LK326W / E333S / E430G with that of IgG1-7D8-E430G and WT IgG1-7D8. The non-specific IgG1-b12 antibody was used as a negative control.

[0059] Figure 23 shows the effect of K326W / E333S / E430G on the agonistic activity of anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1hDR5-05 in cells suspended in WIL2-S SF as determined in a 24-hour viability test (CellTiterGlo).

[0060] Figure 24 shows the effect of the complementary Fc mutation pair K439E; S440K on the agonistic activity of a dual anti-DR5 epitope targeting the IgG1-hDR5-01-G56TK326W / E333S / E430G + IgG1-hDR5-05-K326W / E333S / E430G antibody combination in cells suspended in WIL2-S SF as determined in a 24-hour viability assay (CellTiterGlo).

[0061] Figure 25 shows the results of a CDC test in Wien 133 cells testing IgG1-Campath antibody variants with the E430G or K248E / T437R hexamerization-enhancing mutations, and the K248E / T437R hexamerization-enhancing mutations combined with the K326W / E333S C1q-enhancing mutations. Wien 133 cells were incubated with a series of concentrations of the antibody variants in the presence of 20% pooled human serum (NHS).

[0062] Figure 26 shows the effect of combining the E430G hexamerization-enhancing mutation and the K326W / E333S C1q-enhancing mutations on the efficacy of anti-DR4 IgG1-DR4chCTB007 antibody on adherent human BxPC-3 pancreatic cancer cells. Petition 870260062878, dated 06 / 26 / 2026, page 23 / 410 / 194 as determined in a 3-day feasibility test (CellTiter-Glo).

[0063] Figure 27 shows a CDC assay using human WIL2S SF B lymphocytes with anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1CD95-APO1 (B), and IgG1-CD95-HFE7A (C) in the presence of 20% pooled human serum. WIL2-S SF cells were incubated for 45 minutes with a series of concentrations of the antibody variants in the presence of 20% pooled human serum (NHS). IgG1-b12 was used as a non-binding control antibody.

[0064] Figure 28 shows the 45-minute viability test (CellTiter-Glo) using WIL2-S SF cells incubated with anti-FAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B) and IgG1CD95-HFE7A (C) in serum-free medium without C1q.

[0065] Figure 29 shows a 24-hour viability test (CellTiterGlo) using WIL2-S SF cells with anti-FAS antibody variants IgG1FAS-E09 (A), IgG1-CD95-APO1 (B) and IgG1-CD95-HFE7A (C) in serum-free medium with C1q as a crosslinker.

[0066] Figure 30 shows a 24-hour viability test (CellTiterGlo) using WIL2-S SF cells incubated with antiFAS antibody variants IgG1-FAS-E09 (A), IgG1-CD95-APO1 (B) and IgG1-CD95-HFE7A (C) in serum-free medium without C1q.

[0067] Figure 31 shows the antibody activity of IgG1-CD134SF2 (WT), IgG1-CD134-SF2-E345R, IgG1-CD134-SF2-E430G, and IgG1CD134-SF2-K326W / E333S / E430G in an OX40 Jurkat reporter assay. Thaw-and-Use GloResponse NFkB-1uc2 / OX40 Jurkat cells were incubated for 5 hours with a range of antibody concentrations in the presence of 8% fetal bovine serum. OX40 assay responses were recorded by OX40 detected after stimulation by anti-OX40 antibodies inducing the expression of a luciferase reporter gene. RLU: Relative luminescence units. Petition 870260062878, dated 06 / 26 / 2026, page 24 / 410 / 194

[0068] Figure 32 shows the effect of the Fc-Fc E430G mutation enhancing combined with C1q K326W / E333S binding-enhancing mutations on the CD40 response of IgG1-CD40-SGN40 and IgG1-CD40CP870893. Thaw-and-Use GloResponse NFkB-1uc2 / CD40 Jurkat cells were incubated for 5 hours with a range of antibody concentrations in the presence of 8% fetal bovine serum. CD40 assay responses were recorded by luminescence detected after CD40 stimulation by anti-CD40 antibodies or CD40 ligand, which induce the expression of a luciferase reporter gene. RLU: Relative luminescence units.

[0069] Figure 33 shows the effect of the Fc-Fc E430G mutation enhancing combined with C1q K326W / E333S binding-enhancing mutations on the IgG1-CD137-MOR7480 and IgG1BMS-663513 4-1BB response. Thaw-and-Use GloResponse™ NFkB-1uc2 / 41BB Jurkat cells were incubated for 5 hours with a range of antibody concentrations in the presence of 1% fetal bovine serum. 41BB assay responses were recorded by luminescence detected after stimulation of 41BB by anti-4-1BB antibodies or by ligating 4-1BB with anti-His antibody, which induces the expression of a luciferase reporter gene. IgG-b12K326W / E333S / E430G was used as a negative control. RLU: Relative luminescence units.

[0070] Figure 34 shows the effect of the Fc-Fc E430G mutation combined with C1q K326W / E333S binding-enhancing mutations on the IgG1-GITR-INCAGN01876 GITR response. Thaw-and-Use GloResponse NFkB-1uc2 / GITR Jurkat cells were incubated for 6 hours with a range of antibody concentrations in the presence of 1% fetal bovine serum. GITR assay responses were recorded by luminescence detected after GITR stimulation with anti-GITR antibodies, which induce the expression of a luciferase reporter gene. RLU: Relative luminescence units Petition 870260062878, dated 06 / 26 / 2026, page 25 / 410 / 194

[0071] Figure 35 shows the effect of the Fc-Fc E430G mutation enhancing combined with the C1q K326W / E333S binding-enhancing mutations on the GITR antibody response to GITR-36E5 in IgG1, IgG2, IgG3, and IgG4 subclasses. Thaw-and-Use GloResponse NFkB-1uc2 / GITR Jurkat cells were incubated for 6 hours with a final antibody concentration of 111 ng / mL in the presence of 1% fetal bovine serum. GITR assay responses were recorded by luminescence detected after GITR stimulation with anti-GITR antibodies, which induce the expression of a luciferase reporter gene. IgG1b12 antibody was used as a non-binding control. RLU: Relative luminescence units. DETAILED DESCRIPTION OF THE INVENTION

[0072] In describing embodiments of the invention, specific terminology will be introduced for the sake of clarity. However, the invention is not intended to be limited by the specific terms thus 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. Definitions

[0073] The term “parental polypeptide” or “parental antibody” should be understood as a polypeptide or antibody that is identical to a polypeptide or antibody according to the invention, but where the parental polypeptide or parental antibody does not have an enhancing Fc-Fc mutation and a C1q-binding mutation according to the present invention.

[0074] The term “polypeptide comprising an Fc region of an immunoglobulin and a binding region” refers, in the context of the present invention, to a polypeptide comprising an Fc region of an immunoglobulin and a binding region that is capable of binding to any molecule, such as a polypeptide, for example present in a Petition 870260062878, dated 06 / 26 / 2026, page 26 / 410 / 194 cell, bacterium, or virion. The Fc region of an immunoglobulin is defined as the fragment of an antibody that would typically be generated after the digestion of an antibody with papain (which is known to some experts in the technique) that includes the two CH2-CH3 regions of an immunoglobulin and a connecting region, for example the hinge region. The constant domain of an antibody heavy chain defines the antibody isotype, for example IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates the effector functions of antibodies with cell surface receptors called Fc receptors and complement system proteins. The AO binding region can be a polypeptide sequence, such as a protein, protein ligand, receptor, antigen-binding region, or a ligand-binding region capable of binding to a cell, bacterium, or virion.If the binding region is, for example, a receptor, the “polypeptide comprising an Fc region of an immunoglobulin and a binding region” may have been prepared as a protein fusion of the Fc region of an immunoglobulin and said binding region. If the binding region is an antigen-binding region, the “polypeptide comprising an Fc region of an immunoglobulin and a binding region” may be an antibody, such as a chimeric, humanized, or human antibody, or a heavy chain-only antibody, or an ScFv-Fc fusion. The polypeptide comprising an Fc region of an immunoglobulin and a binding region may typically comprise a connecting region, for example a hinge region, and two CH2-CH3 regions of the heavy chain of an immunoglobulin; thus, the “polypeptide comprising an Fc region of an immunoglobulin and a binding region” may be a “polypeptide comprising at least one Fc region of an immunoglobulin and a binding region”.The term "Fc region of an immunoglobulin" means, in the context of the present invention, a connecting region, for example a hinge depending on the antibody subtype, e.g. Petition 870260062878, dated 06 / 26 / 2026, page 27 / 410 / 194, a CH2 and CH3 region of an immunoglobulin is present, for example, in human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM, or IgE. The polypeptide is not limited to human origin but may have any origin, such as, for example, mouse or cynomolgus origin. The term "wild-type Fc region" means, in the context of the present invention, an Fc region of immunoglobulin with an amino acid sequence as it occurs in nature.

[0075] The term “hinge region” as used here is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

[0076] The term “CH2 region” or “CH2 domain” as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be any of the other subtypes, as described herein.

[0077] The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be any of the other subtypes, as described herein.

[0078] The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four potentially interconnected by disulfide bonds. The structure of immunoglobulins was Petition 870260062878, dated 06 / 26 / 2026, page 28 / 410 / 194, is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a variable heavy chain region (abbreviated here as VH) and a constant heavy chain region. The constant heavy chain region typically consists of three domains, CH1, CH2, and CH3. The heavy chains are interconnected via disulfide bonds in the so-called "hinge region". Each light chain typically consists of a variable light chain region (abbreviated here as VL) and a constant light chain region. The constant light chain region typically consists of one domain, CL.The VH and VL regions can be further subdivided into hypervariability regions (or hypervariable regions that may be hypervariable sequentially and / or formed of structurally defined loops), also called complementarity-determining regions (CDRs), interdispersed with regions that are more conserved, called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the aminotherm to the carboxytherm in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradicted by the context, CDR sequences are identified herein according to IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet address www.imgt.org / ).Unless otherwise specified or contradicted by the context, reference to amino acid positions in the Fc / Fc region domain in the present invention is in accordance with 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. 913242). Petition 870260062878, dated 06 / 26 / 2026, p. 29 / 410 / 194

[0079] 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 thereof, which has the ability to specifically bind to an antigen. The antibody of the present invention comprises an Fc domain of an immunoglobulin and an antigen-binding region. An antibody generally contains two CH2-CH3 regions and a connecting region, for example, the hinge region, for example, at least one Fc domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen.The constant or “Fc” regions of antibodies can mediate the binding of an immunoglobulin to host tissues or factors, including various immune system cells (such as effector cells) and components of the complement system, such as C1q, the first component in the classical complement activation pathway. An antibody can also be multispecific, such as a bispecific antibody or similar molecule. The term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same or different cells or cell types.As indicated above, unless otherwise described or clearly contradicted by the term, the term antibody herein includes fragments of an antibody comprising at least a portion of an Fc region and retaining the ability to specifically bind to antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of an antibody. Petition 870260062878, dated 06 / 26 / 2026, p. 30 / 410 / 194 full length. Examples of link fragments encompassed within the term “Ab” or “antibody” include, without limitation, monovalent antibodies (described in WO2007059782 by Genmab); heavy chain antibodies, consisting of only two heavy chains and naturally occurring in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), strand-exchange engineered domains (SEED or Seed-body) that are asymmetric and bispecific antibody-type 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, US Patent No.7,612,181); dual-domain double-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone / Eli Lilly), node-in-hole antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), electrostatically directed antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 antibodies (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 recognizing two targets (Genentech, NovImmune, Adimab), crosslinked mAbs (Karmanos Cancer Center), covalently fused mAbs (AIMM), CovX-body (CovX / Pfizer), FynomAbs (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), bispecific IgG type (ImClone / Eli Lilly, Shen, J., et al.).J Immunol Methods, 2007. 318(1-2): p. 6574), TIG-body, DIG-body and PIG-body (Pharmabcine), reactive molecules. Petition 870260062878, dated 06 / 26 / 2026, p. 31 / 410 / 194 Dual affinity targeting (Fc-DART or Ig-DART, by Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybodies (Zyngenia), common light chain (Crucell / Merus, US7262028) or common heavy chain (κλBodies by NovImmune, WO2012023053) approaches, as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc region such as scFv- fusions, such as BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ) (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Genetech / Roche, scFv fusion by Novartis, scFv fusion by Immunomedics, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008 / 003116), and dual scFv fusions.It should also be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal antibodies), for example, those generated by technologies exploited by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, fusion proteins as described in WO2014 / 031646, and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated may potentially possess any isotype.

[0080] The term “full-length antibody” when used herein refers to an antibody (e.g., a parental antibody) that contains all the variable and constant heavy and light chain domains corresponding to those that are normally found in a wild-type antibody of this isotype.

[0081] The term “human antibody”, as used herein, is intended for Petition 870260062878, dated 06 / 26 / 2026, page 32 / 410 / 194, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which germline-derived CDR sequences from another mammalian species, such as a mouse, have been grafted onto the human scaffold sequences.

[0082] The term “chimeric antibody,” as used herein, refers to an antibody in which both chain types, i.e., heavy chain and light chain, are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (originating from a non-human species, or synthetic or genetically engineered from any species, including humans) linked to a constant region of human origin.

[0083] The term “humanized antibody,” as used herein, refers to an antibody in which both chain types are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the complementarity-determining regions (CDRs) of the variable domains are foreign (originating from a species other than human, or synthetic) while the remainder of the chain is of human origin. The assessment of humanization is based on the resulting amino acid sequence, not the methodology itself, which allows the use of protocols other than grafting.

[0084] The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb” or similar terms, as used herein, refer to a preparation of Ab molecules of composition Petition 870260062878, dated 06 / 26 / 2026, page 33 / 410 / 194 unique molecular structure. A monoclonal antibody composition exhibits a unique specificity and binding affinity for a particular epitope. Therefore, the term “human monoclonal antibody” refers to Abs exhibiting a unique binding specificity that possesses variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be generated by a hybridoma that includes a B cell obtained from a non-human transgenic or transchromosomal animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell.

[0085] The term “isotype” as used herein refers to a class of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM or any allotypes derived therefrom, such as IgG1m(za) and IgG1m(f)) that is encoded by genes in the constant region of the heavy chain. In addition, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain. The term “mixed isotype” used herein refers to the Fc region of an immunoglobulin generated by combining structural features of one isotype with the analogous region of another isotype, thus generating a hybrid isotype. A mixed isotype may comprise an Fc region having a sequence composed of two or more isotypes selected from the following: IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, thus generating combinations such as, for example, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0086] The term “antigen-binding region,” “antigen-binding domain,” or “binding region,” as used herein, refers to a region of an antibody that is capable of binding to antigen. This binding region is typically defined by the VH and VL domains of the antibody, which may be further subdivided into regions of Petition 870260062878, dated 06 / 26 / 2026, page 34 / 410 / 194 hypervariability (or hypervariable regions that can be hypervariable in sequence and / or form structurally defined loops), also called complementarity-determining regions (CDRs), interdispersed with regions that are more conserved, called framework regions (FRs). The antigen can be any molecule, such as a polypeptide, for example, present in a cell, bacterium, or virion.

[0087] The term “target,” as used herein, refers to a molecule to which the antigen-binding region of the antibody binds. The target includes any antigen to which the created antibody is directed. The terms “antigen” and “target” may be used interchangeably with respect to an antibody and have the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0088] The term “epitope” means a protein determinant capable of specific binding to a variable antibody domino. Epitopes generally consist of surface clusters of molecules, such as amino acids, sugar side chains, or a combination thereof, and generally have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former but not the latter may be lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding.

[0089] An “antibody” or “antibody variant” or “variant of a parental antibody” of the present invention is an antibody molecule comprising one or more mutations as compared to a “parental antibody.” The different terms may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary antibody forms Petition 870260062878, dated 06 / 26 / 2026, page 35 / 410 / 194. Parental antibodies include, without limitation, a wild-type antibody, a full-length antibody or antibody fragment containing Fc, a bispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, or any combination thereof. Similarly, a “polypeptide” or “a variant of a polypeptide comprising an Fc region of an immunoglobulin and a binding region” or “a variant of a parental polypeptide comprising an Fc region of an immunoglobulin and a binding region” of the present invention is a “polypeptide comprising an Fc region of an immunoglobulin and a binding region” that comprises one or more mutations as compared to a “parental polypeptide comprising an Fc region of an immunoglobulin and a binding region”.The different terms may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Amino acid substitutions may exchange a native amino acid for another naturally occurring amino acid, or for a derivative of a non-naturally occurring amino acid. Amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined as substitutions within the amino acid classes reflected in one or more of the following three tables: Amino acid residue classes for conservative substitutions. Acidic residues Asp (D) and Glu (E) Basic residues Lys (K), Arg (R) and His (H) Hydrophilic uncharged residues Ser (S), Thr (T), Asn (N) and Gln (Q) Aliphatic uncharged residues Gly (G), Ala (A), Val (V), Leu (L) and Ile (I) Nonpolar uncharged residues Cys (C), Met (M) and Pro (P) Aromatic residues Phe (F), Tyr (Y) and Trp (W) Alternative conservative amino acid residue substitution classes 1 AST 2 OF 3 NQ 4 RK 5 ILM 6 FYW Petition 870260062878, dated 06 / 26 / 2026, page 36 / 410 / 194 Alternative physical and functional classifications of amino acid residues Waste containing alcohol group Aliphatic waste Waste associated with cycloalkenyl Hydrophobic waste Negatively charged waste Polar waste Positively charged waste Small waste Very small waste Waste involved in shift formation Flexible waste S and TI, L, V and MF, H, W and YA, C, F, G, H, I, L, M, R, T, V, W and YD and EC, D, E, H, K, N, Q, R, S and TH, K and RA, C, D, G, N, P, S, T and VA, G and SA, C, D, E, G, H, K, N, Q, R, S, P and TQ, T, K, S, G, N, D, E and R

[0090] In the context of the present invention, a substitution in a variant is indicated as: original amino acid - position - substituted amino acid; The three-letter code, or one-letter code, is used, including the codes Xaa and X to indicate the amino acid residue. Therefore, the notation “E345R” or “Glu345Arg” means that the variant comprises a glutamic acid substitution for arginine at the variant amino acid position corresponding to the amino acid at position 345 in the parental antibody.

[0091] When such a position is not present in an antibody, but the variant comprises an amino acid insertion, for example: Position - substituted amino acid; the notation, for example, “448E” is used.

[0092] This notation is particularly relevant in connection with modification(s) in a series of homologous polypeptides or antibodies.

[0093] Similarly, when the identity of the replacement amino acid residue(s) is immaterial: original amino acid - position; or “E345”.

[0094] For a modification where the original amino acid(s) and / or substituted amino acid(s) may comprise more than one, but not all, amino acid(s), the substitution of glutamic acid by arginine, lysine, or tryptophan at position 345: Petition 870260062878, dated 06 / 26 / 2026, p. 37 / 410 / 194 “Glu345Arg, Lys,Trp” or “E345R,K,W” or “E345R / K / W” or “E345 a R, K or W” may be used interchangeably in the context of the invention.

[0095] Furthermore, the term “a substitution” encompasses a substitution in any of the other nineteen natural amino acids, or in other amino acids, such as non-natural amino acids. For example, an E amino acid substitution 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 designates any amino acid. These substitutions may also be designated E345A, E345C, etc., or E345A,C, etc., or E345A / C / etc. The same applies to the analogy for each and every position mentioned here, to specifically include any of such substitutions.

[0096] As used herein, the term “effector cell” refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Examples of immune cells include a cell of myeloid or lymphoid origin, for example lymphocytes (such as B cells and T cells including cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and perform specific immune functions. In some embodiments, an effector cell, such as, for example, a natural killer cell, is capable of inducing ADCC.For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcR are involved in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells that present antigens. In some cases... Petition 870260062878, dated 06 / 26 / 2026, page 38 / 410 / 194 modalities, ADCC can be further increased by antibody-directed classical complement activation, resulting in the deposition of activated C3 fragments on the target cell. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote an enhanced Fc receptor-mediated ADCC. In some modalities, antibody-directed classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some modalities, an effector cell can phagocytose a target antigen, a target particle, or a target cell. The expression of a particular FcR or complement receptor on an effector cell can be regulated by humoral factors, such as cytokines.For example, FcyRI expression has been found to be upregulated by interferon γ (IFN γ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcyRI-bearing cells against targets. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. In some modes, antibody-directed classical complement activation leads to C3 fragments in the target cell. These C3 cleavage products can promote direct phagocytosis by effector cells or indirectly enhance antibody-mediated phagocytosis.

[0097] The term “Fc effector functions,” as used herein, is intended to refer to functions that are a consequence of polypeptide or antibody binding to its target, such as an antigen, on a cell membrane where the Fc effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) binding to C1q, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) cell-mediated cytotoxicity dependent on Petition 870260062878, dated 06 / 26 / 2026, p. 39 / 410 / 194 antibody (ADCC), (v) Fc-gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated binding to the complement receptor of an opsonized antibody, (x) opsonization, and (xi) a combination of any one of (i) to (x).

[0098] The term “cluster-dependent functions” as used herein is intended to refer to functions that are a consequence of the formation of antigen complexes after the oligomerization of polypeptides or antibodies bound to their antigens, optionally on a cell, a cell membrane, on a virion, or on another particle. Examples of cluster-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 modulation, such as reduction, inhibition or stimulation of proliferation, and (vii) a combination of any of (i) to (vi).

[0099] The term “agonistic,” as used herein, is understood as stimulation or activation of a receptor on a cell membrane, resulting in a biological response, such as intracellular signaling. Such an agonistic effect could result in the induction of apoptosis (programmed cell death) or activation of immune cells, or activation of an intracellular pathway.

[00100] Agonistic activity or enhanced agonistic activity can be determined in a viability test for antibodies directed against targets expressing an intracellular death domain, as described in Example 2, using the following steps: i) Inoculate a cell line expressing a target corresponding to an antibody, for example DR5, in a 96-well flat-bottom polystyrene plate overnight at 37°C. Petition 870260062878, dated 06 / 26 / 2026, page 40 / 410 / 194 ii) Add a serial dilution of the antibody, for example an anti-DR5 antibody in a range (0.0003 to 20.000 ng / mL) and incubate for 3 days at 37°C, iii) Determine cell viability by quantifying the presence of ATP, for example by using the CellTiler-Glo luminescent cell viability test, iv) Calculate viable cells using the following formula: % viable cells = [(luminescence in antibody sample - luminescence in staurosporine sample) / (luminescence in antibody sample - luminescence in staurosporine sample)]*100.

[00101] Agonistic activity or increased agonist activity can be determined in a reporter assay for antibodies directed to targets activating the intracellular signaling pathway, as described in Examples 29, 30, 31 and 32 using the following steps: i) Stably transfected Jurkat cells with the target, for example OX40, 4-1BB, CD40 or GITR, and a luciferase reporter gene downstream of an NFAT response element expressing the cells, incubate them in a 96-well flat-bottom plate overnight at 37°C, ii) Add a serial dilution of the antibody, for example an anti-OX49, anti-4-1BB, anti-CD40 or anti-GITR antibody in a range of, for example, 19.5 to 5,000 ng / mL and incubate for 5 hours, iii) Add a firefly luciferase substrate (5'fluoroluciferin) to the cells and incubate for 5-10 minutes, iv) Determine the luminescence using an Envision MultiLable Plate reader.

[00102] The term “vector,” as used herein, refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment bound to the vector. One type of vector is a “plasmid,” which is in the form of a circular loop of double-stranded DNA. Petition 870260062878, dated 06 / 26 / 2026, p. 41 / 410 / 194 Another type of vector is a viral vector, in which the nucleic acid segment can be ligated to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomic mammalian vectors). Other vectors (such as non-episomic mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell and, in this way, are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to here as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids.In this report, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention aims to include other forms of expression vectors, such as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[00103] The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the cell in question, but also to the offspring of such a cell. As certain modifications may occur in successive generations due to either mutation or environmental influences, this progeny may not, in fact, be identical to the parent cell, but is still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomes such as CHO cells, HEK 293 cells, PER.C6 cells, NS0 cells and lymphocytes, and prokaryotic cells such as E. coli and other hosts. Petition 870260062878, dated 06 / 26 / 2026, page 42 / 410 / 194 eukaryotes, such as plant cells and fungi.

[00104] The term “transfectome”, as used herein, includes recombinant eukaryotic host cells expressing Ab or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK 293 cells, plant cells or fungi, including yeast cells.

[00105] The term “preparation” refers to preparations of antibody variants and mixtures of different antibody variants that may have an increased ability to form oligomers when interacting with antigen associated with a cell (e.g., an antigen expressed on the cell surface), a cell membrane, a virion, or other structure, which may result in antigen-enhanced signaling and / or activation.

[00106] As used herein, the term “affinity” is the binding strength of one molecule, for example, an antibody, to another, for example, a target or antigen, at a single site, such as the monovalent binding of an antibody’s single antigen-binding site to an antigen.

[00107] As used herein, the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen-binding sites of antibodies simultaneously interacting with a target or, for example, between the antibody and C1q. When more than one binding interaction is present, the two structures will only dissociate when all binding sites dissociate and thus the rate of dissociation will be slower than for individual binding sites, thus providing greater total effective binding strength (avidity) compared to the binding strength of individual binding sites (affinity).

[00108] As used herein, the term “oligomer” refers to a molecule consisting of more than one, but a limited number, of monomeric units (e.g., antibodies) in contrast to a polymer which, at least in principle, consists of an unlimited number of Petition 870260062878, dated 06 / 26 / 2026, page 43 / 410 / 194 monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Greek prefixes are frequently used to designate the number of monomeric units in the oligomer, for example, a tetramer being composed of four units and a hexamer of six units.

[00109] The term “oligomerization”, as used herein, is intended to refer to a process that converts monomers to a finite degree of polymerization. Here, it is observed that polypeptides, antibodies and / or other dimeric proteins comprising target-binding regions according to the invention can form oligomers, such as hexamers, through non-covalent association of Fc regions after binding to the target, for example, to a cell surface.

[00110] The term “clustering”, as used herein, is intended to refer to the oligomerization of antibodies, polypeptides, antigens, or other proteins through non-covalent interactions.

[00111] The term “Fc-Fc strengthening,” as used herein, is intended to refer to increasing the binding strength between, or stabilizing the interaction between, the Fc regions of two antibodies containing Fc regions or polypeptides so that the polypeptides form oligomers upon binding to the target.

[00112] Fc-Fc enhancing substitutions, as used herein, refer to substitutions at the following positions corresponding to human IgG1 according to EU numbering: E430, E345, or S440, provided that the substitutions at position S440 are S440Y or S440W. Thus, Fc-Fc enhancing substitutions, as used herein, 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.

[00113] The term “C1q binding”, as used here, is intended to make Petition 870260062878, dated 06 / 26 / 2026, page 44 / 410 / 194 refers to the direct interaction between C1q and a polypeptide or antibody. Direct binding to C1q can be evaluated, for example, using antibody immobilized on an artificial surface (as described in Examples 4, 5 and 6). The multivalent interaction resulting in high-avidity binding of C1q to an oligomeric antibody can be evaluated when bound to a predetermined antigen on a cell surface or virion.

[00114] C1q binding to a polypeptide or antibody can be determined in an ELISA test using the following steps: i) coat a 96-well Microlon ELISA plate with 1 pg / mL of polypeptide or antibody in 100 pL PBS at 4°C overnight; ii) incubate the plate with 100 pL / well of serial dilutions of C1q, from a final C1q concentration range of 30-0.01 pg / mL in 3-fold dilutions for 1 h at 37°C; iii) incubate the plate with 100 pL / well of rabbit anti-human C1q for 1 h at RT; iv) incubate the plate with 100 pL / well of pig anti-rabbit IgG-HRP for 1 h at RT; v) incubate the plate with 100 pL / well of substrate with 1 mg / mL of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is added for 15 min at RT, vi) the reaction is stopped by adding 100 µL of 2% oxalic acid per cavity. Absorbance is measured at 405 nm using a BioTek EL808 microplate reader.

[00115] The term C1q-binding substitution, as used herein, is intended to refer to a substitution in a polypeptide comprising an Fc region of an immunoglobulin and an antigen-binding region, which enhances the direct interaction with C1q. Enhanced C1q binding may, for example, result in a decreased EC50 of the interaction between C1q and the polypeptide comprising an Fc region of an immunoglobulin and an antigen-binding region, measured according to the method for determining C1q binding described above.

[00116] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is initiated by a large Petition 870260062878, dated 06 / 26 / 2026, page 45 / 410 / 194 macromolecular complex called C1 binding for antibody-antigen complexes on a surface. C1 is a complex consisting of 6 C1q recognition proteins and a hetero-tetramer of serine proteases, C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions that begins with the cleavage of C4 into C4a and C4b and C2 into C2a and C2b. C4b is deposited and forms, together with C2a, an active enzymatic convertase called C3 convertase, which cleaves the complement component C3 into C3b and C3a, which forms a C5 convertase. This C5 convertase splits C5 into C5a and C5b, and the latter component is deposited on the membrane, which in turn triggers the late activation events of complement in which 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 that cause cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be assessed using C1q efficacy, CDC kinetics, CDC assays (as described in WO2013 / 004842, WO2014 / 108198) or by C3b and C4b cell deposition methods described in Beurskens et al. April 1, 2012, vol. 188, no. 7, 3532-3541.

[00117] The term “complement-dependent cytotoxicity” (“CDC”), as used herein, is intended to refer to the antibody-mediated complement activation process leading to cell or virion lysis when the antibody is bound to its target on a cell or virion as a result of pores in the membrane, which are created by MAC assembly.

[00118] The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein, is intended to refer to a mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of Petition 870260062878, dated 06 / 26 / 2026, p. 46 / 410 / 194 antibody-bound. The term “antibody-dependent cellular phagocytosis” (“ADCP”) as used herein is intended to refer to a mechanism of elimination of antibody-coated target cells or virions by internalization by phagocytes. The internalized antibody-coated target cells or virions are contained within a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP can be assessed using an in vitro cytotoxicity assay with macrophages as effector cells and video microscopy, as described by van Bij et al. in the Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685.

[00119] The term “complement-dependent cellular cytotoxicity” (“CDCC”) as used herein is intended to refer to the mechanism of killing of target cells or virions by cells expressing complement receptors that recognize complement 3 (C3) cleavage products that are covalently bound to target cells or virions as a result of antibody-mediated complement activation. CDCC can be assessed in a similar manner as described for ADCC.

[00120] The term “plasma half-life,” as used herein, indicates the time required to reduce the concentration of a polypeptide in blood plasma to half its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase will typically be 1 to 3 days, during which there is a decrease of approximately 50% in blood plasma concentration due to redistribution between plasma and tissues. Plasma half-life can be measured by well-known methods in the art.

[00121] The term “plasma clearance rate”, as used herein, is a quantitative measurement of the rate at which a polypeptide is removed from the blood after administration to a living organism. The plasma clearance rate can be calculated as the dose / AUC (mL / day / kg), where the value Petition 870260062878, dated 06 / 26 / 2026, page 47 / 410 / 194 of AUC (area under the curve) is determined from a concentration-time curve.

[00122] The term “antibody-drug conjugate”, as used herein, refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a ligand coupling the drug to, for example, an antibody. The ligand is either cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell.

[00123] The term “antibody-drug conjugate uptake,” as used herein, refers to the process in which antibody-drug conjugates are bound to a target on a cell followed by uptake / engulfment by the cell membrane and are thus introduced into the cell. Antibody-drug conjugate uptake can be assessed as “antibody-mediated internalization and cell death by anti-TF ADC in an in vitro killing assay” as described in WO 2011 / 157741.

[00124] The term “apoptosis,” as used herein, refers to the process of programmed cell death (PCD) that can occur in a cell. Biochemical events lead to characteristic cellular changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. The binding of an antibody to a certain receptor can induce apoptosis.

[00125] The term “programmed cell death” or “PCD,” as used herein, refers to the death of a cell in any form mediated by an intracellular program. Different forms of PCD exist, the various types of PCD having in common the fact that they are executed by active cellular processes that can be intercepted by interference with intracellular signaling. In one particular embodiment, the occurrence of any form of PCD in a cell or tissue can be determined by staining of Petition 870260062878, dated 06 / 26 / 2026, page 48 / 410 / 194 cell or tissue with conjugated Annexin V, correlating with exposure to phosphatidylserine.

[00126] The term “Annexin V”, as used herein, refers to an annexin group protein that binds phosphatidylserine (PS) on the cell surface.

[00127] The term “FcRn”, as used herein, is intended to refer to the neonatal Fc receptor, which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from breast milk across the intestinal epithelium of the newborn rodent into the newborn's bloodstream. Further studies have revealed a similar receptor in humans. In humans, however, it is found in the placenta to help facilitate the transport of IgG from the mother to the growing fetus and has also been shown to play a role in monitoring IgG turnover. FcRn binds to IgG at an acidic pH of 6.0 to 6.5, but not at neutral or higher pH. Therefore, FcRn can bind to IgG from the intestinal lumen (the inside of the intestine) at a slightly acidic pH and ensure efficient unidirectional transport to the basolateral side (inside the body), where the pH is neutral to basic (pH 7.0-7.5).This receptor also plays a role in the adult salvage of IgG through its occurrence in the endocytosis pathway in endothelial cells. FcRn receptors in acidic endosomes bind to IgG internalized by pinocytosis, recycling it to the cell surface and releasing it in the basic pH of the blood, thus preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the longer half-life of IgG in the blood compared to other isotypes.

[00128] The term “Protein A”, as used herein, is intended to refer to a 56 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology. Petition 870260062878, dated 06 / 26 / 2026, page 49 / 410 / 194 cellular osmolarity and a two-component system called ArlS-ArlR. It has found use in biochemical research due to its ability to bind to immunoglobulins. It is composed of five homologous Ig-binding domains that duplicate in a three-helix bundle. Each domain is capable of binding to proteins of many mammalian species, most notably IgGs. It binds to the Fc region of the heavy chain of most immunoglobulins (overlapping the conserved binding site of FcRn receptors) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind to bacteria via their Fc region rather than just via their Fab regions, through which the bacteria break through opsonization, complement activation, and phagocytosis.

[00129] The term “G protein”, as used herein, refers to an immunoglobulin-binding protein expressed in group C and G Streptococcus bacteria, very similar to Protein A, but with different specificities. It is a 65 kDa (G148 G protein) and 58 kDa (C40 G protein) cell surface protein that has found application in antibody purification through its binding to the Fc region. Specific embodiments of the invention

[00130] As described herein, surprisingly, amino acid substitutions in the Fc region of a polypeptide or antibody provide polypeptides or antibodies with enhanced effector functions, for example, CDC and / or agonist activity. The inventors found that by introducing a mutation that enhances Fc-Fc interactions, such as a substitution at a selected position from the group consisting of: E430, E345 and S440 along with the C1q-binding substitution, the Fc effector functions of the polypeptide or antibody can be enhanced. Furthermore, the inventors also found that the combination of an Fc-Fc-enhancing mutation and the C1q-binding substitution can result in polypeptides, such as antibodies, with agonist or agonist properties. Petition 870260062878, dated 06 / 26 / 2026, page 50 / 410 / 194 intensified agonistic properties.

[00131] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, 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, provided that the mutation in S440 is S440Y or S440W, and b) at least one C1q binding substitution, wherein the positions correspond 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., Protein Sequences of Immunological Interest, 5th ed., 1991 NIH Publication No. 91-3242)

[00132] In one aspect, the invention provides a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region, wherein the Fc region comprises, a) a substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) a substitution at one or more position(s) 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.

[00133] A substitution in a position corresponding to the E430, E345 or an S440Y or S440W substitution is considered an intensifying Fc-Fc substitution according to the present invention.

[00134] A substitution at one or more position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, is considered a C1q linking substitution according to the present invention.

[00135] A mutation at one of the following positions E430, E345, or S440, provided that the mutation at S440 is S440Y or S440W, introduces the effect of enhanced Fc-Fc interactions and oligomerization in the polypeptide or Petition 870260062878, dated 06 / 26 / 2026, page 51 / 410 / 194 antibody. Enhanced oligomerization occurs when the antigen-binding region of the polypeptide or antibody is linked to the corresponding target antigen. Enhanced oligomerization generates oligomers such as, for example, hexamers. The generation of oligomeric structures, such as hexamers, has the effect of increasing the effector functions of Fc, for example CDC, by increasing the avidity of C1q binding of the polypeptide. The combination of the Fc-Fc enhancing mutation with a C1q-binding substitution, such as one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333, and P396, generates a polypeptide or antibody with enhanced effector functions. The combination of an Fc-Fc enhancing substitution and a C1q binding substitution additionally has the effect of generating a polypeptide or antibody with agonist activity.In one embodiment, the polypeptide or antibody may have increased agonistic activity when compared to a parental polypeptide or parental antibody.

[00136] Polypeptides or antibodies according to the present invention are of particular interest when activating an intracellular signaling pathway by ligand-binding to the cell surface receptor.

[00137] In one embodiment, according to the invention, an enhanced or intensified Fc effector function or activity of a polypeptide or antibody having an enhancing Fc-Fc substitution and a C1q-binding substitution shall be understood to mean when the polypeptide or antibody is compared with a parental polypeptide or parental antibody, which is the parental polypeptide or parental antibody without the substitutions, according to the invention, but otherwise identical.

[00138] The present invention enables novel therapies based on polypeptides or antibodies with enhanced properties, such as CDC agonist activity. That is, the polypeptides or antibodies according to the invention have enhanced properties depending on the Fc region such as Petition 870260062878, dated 06 / 26 / 2026, page 52 / 410 / 194 as CDC and they may also have enhanced properties depending on the antigen-binding region, such as agonistic activity.

[00139] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) a substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, 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 the group consisting of: E430, E345 or S440Y or S440W substitution. In one embodiment of the invention, the polypeptide or antibody comprises substitution(s) at one or more positions selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00140] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at two or three position(s) selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00141] In one embodiment of the invention, the polypeptide or antibody comprises C1q-binding substitutions selected from one of the groups consisting of; i) two C1q bonding substitutions at positions K326, E333 ii) three C1q bonding substitutions at positions f: K326, E333 and P396, and iii) three C1q bonding substitutions at positions S267, H268 and S324.

[00142] In one embodiment of the invention, one or more Petition 870260062878, dated 06 / 26 / 2026, page 53 / 410 / 194 C1q linkage substitution(s) are at a position selected from the group consisting of G236, S239, S267, H268, S324, K326, I332, E333 and P396, with the condition that the substitution at position G236 is not G236F, G236R, G236Y.

[00143] In one embodiment of the invention, one or more linking substitution(s) to C1q 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.

[00144] In one embodiment of the invention, one or more linking substitution(s) to C1q 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 H268 is not H268K, H268D, or H268E.

[00145] In one embodiment of the invention, at least one Fc-Fc intensifying substitution is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y.

[00146] In one embodiment of the invention, at least one Fc-Fc intensifying substitution is selected from the group consisting of: E430G, E430S, E430F and E430T.

[00147] In one embodiment of the invention, at least one Fc-Fc intensifying substitution is selected from the group consisting of: E345K, E345Q, E345R and E345Y.

[00148] In one embodiment of the invention, the polypeptide or antibody has at least one E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345R substitution. In one embodiment of the invention, the polypeptide or Petition 870260062878, dated 06 / 26 / 2026, page 54 / 410 / 194: the antibody has at least one S440Y substitution.

[00149] 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 position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00150] 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 of: K326, E333 and P396.

[00151] In one embodiment of the invention, the Fc region comprises an E430G substitution and a substitution at one or more positions selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00152] 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, and a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00153] 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, and a substitution at one or more position(s) selected from the group of: K326, E333 and P396.

[00154] 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 of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00155] In one embodiment of the invention, the Fc region comprises the E345R substitution and a substitution at one or more position(s) Petition 870260062878, dated 06 / 26 / 2026, page 55 / 410 / 194 selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00156] In one embodiment of the invention, the Fc region comprises at least one substitution selected from the group consisting of: S440Y or S440W and a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00157] In one embodiment of the invention, the Fc region comprises at least one substitution selected from the group consisting of: S440Y or S440W and a substitution at one or more position(s) selected from the group of: K326, E333 and P396.

[00158] In one embodiment of the invention, the Fc region comprises an S440Y substitution and a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00159] Thus, embodiments are provided here that allow for C1q binding and / or enhanced agonistic properties of polypeptides or antibodies upon antigen binding on the cell surface. In one embodiment, the polypeptides or antibodies comprise enhanced agonistic properties. In another embodiment, the polypeptides or antibodies comprise an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the aforementioned substitutions may be present in the first and / or second heavy chain.

[00160] In one embodiment of the invention, the polypeptide or antibody comprises substitutions at one or more position(s) selected from the group consisting of: K326, E333 and P396. In another embodiment of the invention, the polypeptide or antibody comprises a substitution, such as two or three substitutions at one or more position(s) selected from the group consisting of: K326, E333 and P396. In one embodiment of the Petition 870260062878, dated 06 / 26 / 2026, page 56 / 410 / 194 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.

[00161] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at one or more position(s) 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, such as two or three position(s) 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 substitutions at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions E333 and P396.In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions K326, E333, and P396.

[00162] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at one or more position(s) 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, such as two or three position(s) selected from the group consisting of: K326, E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution in Petition 870260062878, dated 06 / 26 / 2026, page 57 / 410 / 194 position E345 and substitutions at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at positions E333 and P396. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at positions K326, E333 and P396.

[00163] 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 an S440Y or S440W substitution and a substitution at one or more positions, such as two or three 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 substitutions at positions K326 and E333. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at S440Y or S440W and substitutions at positions K326 and P396. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution at positions E333 and P396.In one embodiment of the invention, the polypeptide or antibody comprises an S440Y substitution or S440W substitutions at positions K326, E333, and P396.

[00164] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at the K326 position selected from the group consisting of: K326W, K326A, K326D, K326N, K326G, K326F, K326E, K326F, K326Y, K326H, and K326M.

[00165] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E333 selected from the group Petition 870260062878, dated 06 / 26 / 2026, page 58 / 410 / 194 consisting of: E333S, E333A, E333T and E333G.

[00166] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at the E396 position selected from the group consisting of: E396L, E396I, E396V, E396Q, E396N, and E396A.

[00167] 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 the K326W and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W and E333T substitutions. In one embodiment of the invention... Petition 870260062878, dated 06 / 26 / 2026, page 59 / 410 / 194 invention, the polypeptide or antibody comprises the K326W and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326A and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326A and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326A and E333T substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the E333A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the E333S and P396L substitutions. In another embodiment of the invention, the polypeptide or antibody comprises the K326A, E333A, and P396L substitutions.In one embodiment of the invention, the polypeptide or antibody comprises the K326S, E333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333S, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333T, and P396L substitutions.

[00168] In one embodiment of the invention, the polypeptide or antibody comprises one or more C1q-binding substitutions at a position 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 a position 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 Petition 870260062878, dated 06 / 26 / 2026, page 60 / 410 / 194 S324. In one embodiment of the invention, the polypeptide or antibody comprises substitutions at positions H268 and S324. In another embodiment of the invention, the polypeptide or antibody comprises substitutions at positions S267, H268, and S324.

[00169] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at one or more position(s) 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 position(s) 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 substitutions at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions H268 and S324.In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and substitutions at positions S267, H268, and S324.

[00170] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at one or more position(s) 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 position(s) 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 substitutions at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at positions Petition 870260062878, dated 06 / 26 / 2026, page 61 / 410 / 194 In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and substitutions at positions H268 and S324.

[00171] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution 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 an S440Y or S440W substitution and a substitution at one or more positions, such as two or three position(s) selected from the group consisting of: S267, H268, and S324. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and substitutions at positions S267 and H268. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and substitutions at positions S267 and S324. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and substitutions at the H268 and S324 positions.In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and substitutions at positions S267, H268, and S324.

[00172] In one embodiment of the invention, one or more substitutions 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 Petition 870260062878, dated 06 / 26 / 2026, page 62 / 410 / 194 invention, the polypeptide or antibody comprises the S267E and H268F substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the S267E and S324T substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the H268F and S324T substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the S267E, H268F and S324T substitutions.

[00173] 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 substitution, b) at least one substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00174] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and at least one substitution selected from one of the following groups consisting of: i. K326A, Petition 870260062878, dated 06 / 26 / 2026, page 63 / 410 / 194 ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00175] Thus, embodiments are provided in which a replacement is in position E430. In one embodiment, the replacement in position E430 is selected from the group consisting of: E430G, E430S, E430F, E430T.

[00176] In one embodiment of the invention, the polypeptide or antibody comprises an E430G substitution and a substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, Petition 870260062878, dated 06 / 26 / 2026, page 64 / 410 / 194 xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00177] In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326A, E333A, and P396L substitution.

[00178] In one embodiment of the invention, the polypeptide or antibody comprises an E430S, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430S, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430S, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430S, K326A, E333A, and P396L substitution.

[00179] In one embodiment of the invention, the polypeptide or antibody comprises an E430F, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430F, K326A, and E333A substitution. 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 an E430F, K326A, E333A, and P396L substitution.

[00180] In one embodiment of the invention, the polypeptide or antibody comprises an E430T, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430T, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or Petition 870260062878, dated 06 / 26 / 2026, page 65 / 410 / 194. The antibody comprises an E430T, K333A and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430T, K326A, E333A and P396L substitution.

[00181] In one embodiment of the invention, a polypeptide or antibody comprises a substitution at position E345 and at least one substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, vi. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00182] Thus, modes are provided in which a replacement is in position E345. In one mode, the replacement in position E345 is selected from the group consisting of: E345K, E345Q, E345R, E345Y.

[00183] In one embodiment of the invention, the polypeptide or antibody comprises an E345K substitution and at least one substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, Petition 870260062878, dated 06 / 26 / 2026, page 66 / 410 / 194 iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00184] In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326A, E333A, and P396L substitution.

[00185] In one embodiment of the invention, the polypeptide or antibody comprises an E345R substitution and at least one substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T Petition 870260062878, dated 06 / 26 / 2026, page 67 / 410 / 194 viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00186] In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326A, E333A, and P396L substitution.

[00187] In one embodiment of the invention, the polypeptide or antibody comprises an E345Q, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Q, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Q, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Q, K326A, E333A, and P396L substitution.

[00188] In one embodiment of the invention, the polypeptide or antibody comprises an E345Y, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Y, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Y, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345Y, K326A, E333A, and P396L substitution.

[00189] In one embodiment of the invention, the polypeptide or antibody Petition 870260062878, dated 06 / 26 / 2026, page 68 / 410 / 194, comprises an S440Y or S440W replacement and at least one replacement selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00190] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y substitution and at least one substitution selected from one of the following groups consisting of: i. K326A, ii. E333A, iii. E333T iv. P396L, v. E333S, see. K326W, E333S vii. K326W, E333T viii. K326A, E333A, ix. K326A, K333A, P396L x. S267E, H268F, Petition 870260062878, dated 06 / 26 / 2026, page 69 / 410 / 194 xi. S267E, S324T, xii. H268F, S324T, xiii. S267E, H268F, S324T, and xiv. S324, I332.

[00191] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326W, and E333S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326A, and E333A substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K333A, and P396L substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326A, E333A, and P396L substitution.

[00192] 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.

[00193] 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.

[00194] In one embodiment of the invention, the polypeptide or antibody further comprises at least two substitutions selected from the group consisting of: i. G236A, I332E, and ii. S239D, I332E.

[00195] 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 substitution or Petition 870260062878, dated 06 / 26 / 2026, page 70 / 410 / 194 S440W eb) replacements selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00196] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E430 and the substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00197] In one embodiment of the invention, the polypeptide or antibody comprises an E430G substitution and substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00198] In one embodiment of the invention, the polypeptide or antibody comprises a substitution at position E345 and the substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00199] In one embodiment of the invention, the polypeptide or antibody comprises an E345K substitution and the substitutions selected from Petition 870260062878, dated 06 / 26 / 2026, page 71 / 410 / 194 from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00200] In one embodiment of the invention, the polypeptide or antibody comprises an E345R substitution and substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00201] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y or S440W substitution and the substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00202] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y substitution and substitutions selected from one of the following groups consisting of: i. H268F, S324T, G236A, I332E, ii. H268F, S324T, S239D, I332E, iii. S267E, H268F, S324T, G236A, I332E, and iv. S267E, H268F, S324T, S239D, I332E.

[00203] In one embodiment of the invention, the polypeptide or antibody comprises one or more additional substitutions. That is, in one embodiment of the invention the polypeptide or antibody according to any aspect or Petition 870260062878, dated 06 / 26 / 2026, page 72 / 410 / 194, the modality described herein includes one or more additional substitutions in the Fc region.

[00204] In one embodiment of the invention, the polypeptide or antibody comprises an additional substitution corresponding to the K439 position or where the Fc region does not comprise a substitution at the S440 position, the additional substitution may be at the S440 position.

[00205] In one embodiment of the invention, the polypeptide or antibody comprises an additional substitution in the polypeptide or antibody corresponding to the K439 or S440 position, provided that the substitution at S440 is not S440Y or S440W.

[00206] Polypeptides or antibodies comprising an Fc-Fc enhancing substitution and a C1q-binding substitution according to the present invention and an additional substitution at the S440 position, such as S440K, do not form oligomers with polypeptides or antibodies comprising a substitution at the S440 position, such as S440K. Polypeptides or antibodies comprising an Fc-Fc enhancing substitution and a C1q-binding substitution according to the present invention and an additional substitution at the K439 position, such as K439, do not form oligomers with polypeptides or antibodies comprising the mutation at the K439 position, such as K439E. In one embodiment of the invention, the additional substitution is selected from either S440K or K439E.

[00207] In one embodiment of the present invention, the Fc region comprises an additional substitution that is a hexamerization-inhibiting substitution corresponding to K439E or S440K in EU numbering of human IgG1. That is, in one embodiment of the present invention, the Fc region comprises an enhancing Fc-Fc substitution such as E430G and the hexamerization-inhibiting substitution K439E. In another embodiment of the present invention, the Fc region comprises an enhancing Fc-Fc substitution such as E345K and a hexamerization-inhibiting substitution K439E. In another Petition 870260062878, dated 06 / 26 / 2026, page 73 / 410 / 194. In one embodiment of the present invention, the Fc region comprises an enhancing Fc-Fc substitution such as E430G and a hexamerization-inhibiting substitution S440K. In another embodiment of the present invention, the Fc region comprises an enhancing Fc-Fc substitution such as E345K and a hexamerization-inhibiting substitution S440K. Embodiments are provided here that allow exclusive hexamerization between antibody combinations comprising a K439E substitution and antibodies comprising an S440 substitution. That is, the K439E and S440K inhibiting substitutions can be viewed as complementary substitutions. Antibody combinations with two different complementary hexamerization-inhibiting substitutions may be of particular interest in compositions having at least two antibodies with different specificities.

[00208] 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, provided that the substitution at S440 is not S440Y or S440W and b) a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396 and c) a K439E substitution.

[00209] 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 position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396 and c) an S440K substitution.

[00210] In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326W, E333S, and K439E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326A, E333A, and K439E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, Petition 870260062878, dated 06 / 26 / 2026, page 74 / 410 / 194 K333A, P396L and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E430G, K326A, E333A, P396L and K439E.

[00211] In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345K, K326W, E333S and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345K, K326A, E333A and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345K, K333A, P396L and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345K, K326A, E333A, P396L and K439E.

[00212] In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345R, K326W, E333S, and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345R, K326A, E333A, and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345R, K333A, P396L, and K439E. In one embodiment of the invention, the polypeptide or antibody comprises a substitution of E345R, K326A, E333A, P396L, and K439E.

[00213] In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326W, E333S, and K439E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326A, E333A, and K439E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K333A, P396L, and K439E substitution. In one embodiment of the invention, the polypeptide or antibody comprises an S440Y, K326A, E333A, P396L, and K439E substitution.

[00214] In one embodiment of the invention, the polypeptide or antibody comprises a) a substitution at a position selected from the Petition 870260062878, dated 06 / 26 / 2026, page 75 / 410 / 194 group consisting of: E430 and E345, and b) a replacement in one or more position(s) selected from the group of: G236, S239, S267, H268, S324 K326, I332, E333 and P396 and c) a replacement S440K.

[00215] In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326W, E333S, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326A, E333A, and S440S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K333A, P396L, and S440S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E430G, K326A, E333A, P396L, and S440K substitution.

[00216] In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326W, E333S, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326A, E333A, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K333A, P396L, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345K, K326A, E333A, P396L, and S440K substitution.

[00217] In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326W, E333S, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326A, E333A, and S440S substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K333A, P396L, and S440K substitution. In one embodiment of the invention, the polypeptide or antibody comprises an E345R, K326A, E333A, P396L, and S440K substitution.

[00218] The polypeptide or antibody according to the invention has at least one Fc-Fc enhancing substitution and one or more Petition 870260062878, dated 06 / 26 / 2026, p. 76 / 410 / 194 C1q linkage substitutions, but as described above it may also have additional substitutions to introduce additional functions into the polypeptide or antibody. In one embodiment, the polypeptide or antibody comprises a maximum of substitutions, such as nine substitutions, such as eight substitutions, such as seven substitutions, such as six substitutions, such as five substitutions, such as four substitutions, such as three substitutions or such as two substitutions.

[00219] Herein are provided embodiments that enable the polypeptides or antibodies of the invention to have additional substitutions, which introduce additional features into the polypeptide or antibody. Additionally, the additional substitutions also allow for variation in the Fc region at positions that are not involved in Fc-Fc interaction, as well as at positions not involved in Fc effector functions. Additionally, additional substitutions may also be due to allelic variations.

[00220] In one embodiment of the invention, the polypeptide or antibody has an Fc effector function increased by at least 20% compared with a parent polypeptide or parent antibody, which is identical to the antibody except that it does not comprise an enhancing FcFc substitution and a C1q-binding substitution in the Fc region.

[00221] In one embodiment of the invention, the polypeptide or antibody has an Fc effector function enhanced by at least 40%, at least 50%, or at least 60% compared with a parent polypeptide or parent antibody, which is identical to the antibody except that it does not comprise an enhancing Fc-Fc substitution and a C1q-binding substitution in the Fc region.

[00222] In one embodiment of the invention, the polypeptide or antibody comprises an enhanced Fc effector function.

[00223] In one embodiment of the invention, the effector function of Fc is Petition 870260062878, dated 06 / 26 / 2026, page 77 / 410 / 194 selected from the following group: complement-dependent cytotoxicity (CDC), cell-mediated complement-dependent cytotoxicity, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis, C1q binding, and FcyR binding. In one embodiment, the effector function of Fc is FcYRIIIa signaling. That is, the second mutation according to the invention is capable of diminishing at least one effector function of Fc.

[00224] In one embodiment of the invention, the polypeptide or antibody comprises agonistic activity. That is, the polypeptide or antibody comprises agonistic activity when compared with a parent polypeptide or parent antibody.

[00225] In one embodiment of the invention, the polypeptide or antibody comprises enhanced agonist activity. That is, the polypeptide or antibody comprises enhanced agonist activity when compared with a parental polypeptide or parental antibody. In another embodiment of the invention, the polypeptide or antibody comprises enhanced agonist activity when compared with a polypeptide or antibody comprising the same enhancing Fc-Fc mutation, but without the C1q-binding mutation.

[00226] Agonistic activity of TNFR-SF receptors requires exogenous cross-linking to achieve agonistic activity. This can be measured in surrogate assays using, for example, HEK293 cells containing NF-κB-directed secreted reporter genes (e.g., pMetLuc reporter gene expressing luciferase, Clontech) that are stably transfected with the TNFR-SF receptor of interest. Cross-linking of the receptor leads to promoter activation and secretion of, for example, luciferase protein into the medium. In desired time-point assays, luciferase activity can be measured by transferring the sample medium and adding substrate. Luciferase activity can be measured in a luminometer, which is a measure for the Petition 870260062878, dated 06 / 26 / 2026, p. 78 / 410 / 194 agonistic activity. For examples of OX40, see Zhang et al. J Biol Chem. December 30, 2016, 291(53):27134-27146

[00227] In one embodiment of the invention, the polypeptide or antibody comprises enhanced agonist activity. That is, the polypeptide or antibody comprises enhanced agonist activity when compared with a parent polypeptide or parent antibody.

[00228] 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.

[00229] The polypeptide of the invention is not limited to antibodies having a natural domain, for example a human Fc, but may also be an antibody having other mutations different from those of the present invention, such as, for example, mutations affecting glycosylation or allowing the antibody to be a bispecific antibody. By the term “natural antibody” is meant any antibody that does not comprise any genetically introduced mutations. An antibody comprising naturally occurring modifications, for example different allotypes, should thus be understood as being a “natural antibody” within the meaning of the present invention, and may thus be understood as a parental antibody. Such antibodies may serve as a template for at least two substitutions according to the present invention and thus provide the antibodies of the invention.An example of a parental antibody comprising substitutions other than those of the present invention is the bispecific antibody, as described in WO2011 / 131746 (Genmab), using reducing conditions to promote half-molecule exchange of two antibodies comprising IgG4-type CH3 regions, thus forming bispecific antibodies without concomitant aggregate formation. Others. Petition 870260062878, dated 06 / 26 / 2026, page 79 / 410 / 194 examples of parental antibodies include, but are not limited to, bispecific antibodies, such as heterodimeric bispecific antibodies: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat Neurosciences Corporation); FcΔAdp (Regeneron); knots-in-hole (Genentech); electrostatic direction (Amgen, Chugai, Oncomed); SEEDbodies (Merck); 'Azymetric' scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary formats of parental antibodies include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a human antibody, a humanized antibody, a chimeric antibody, or any combination thereof.

[00230] In one embodiment, the polypeptide or antibody comprises an Fc region comprising an R435H substitution.

[00231] In one embodiment, the present invention provides a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) a substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) a substitution at one or more position(s) selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, and c) an R435H substitution, wherein the positions correspond to human IgG1, according to EU numbering.

[00232] The polypeptide or antibody may be any human antibody of any isotype, for example IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA, optionally a full-length human antibody, such as a full-length human IgG1 antibody.

[00233] In one embodiment of the invention, the polypeptide or antibody is a human IgG1 antibody, for example the IgG1m(za) or IgG1m(f) allotype.

[00234] In one embodiment of the invention, the polypeptide or antibody Petition 870260062878, dated 06 / 26 / 2026, p. 80 / 410 / 194 has an Fc region that is a human isotype of IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA or a mixed isotype. That is, the Fc region of a polypeptide or antibody according to the invention has at least one first and one second mutation introduced into the Fc region corresponding to a human isotype of IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, IgA or a mixed isotype. In one embodiment of the invention, the Fc region is a mixed isotype selected from the following group: IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4 and IgG3 / IgG4. In a mixed isotype, the Fc region consists of an amino acid sequence from more than one isotype.

[00235] In one embodiment of the invention, the Fc region is a human isotype of IgG1, IgG2, IgG3, IgG4 or a human isotype.

[00236] In one embodiment of the invention, the Fc region is a human IgG.

[00237] In a preferred embodiment of the invention, the polypeptide or antibody has an Fc region that is a human IgG isotype.

[00238] In one embodiment of the invention, the polypeptide or antibody has an Fc region that is an allotype of IgG1m(f), IgG1m(a), IgG1m(z), IgG1m(x) or a mixed allotype.

[00239] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as specified in SEQ ID NO: 73, 74, 75, 76, 89, 168, 169 or 170, wherein the Fc region comprises a substitution at the position selected from the group corresponding to E430, E345 and S440, provided that the substitution at 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, I332, E333 and P396, wherein the positions correspond to human IgG1, according to EU numbering.

[00240] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as specified in SEQ ID NO:77, 78 or 90 Petition 870260062878, dated 06 / 26 / 2026, page 81 / 410 / 194, in which the Fc region comprises a substitution at one or more position(s) 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.

[00241] In one embodiment of the invention, the polypeptide or antibody comprises an Fc region as specified in SEQ ID NO: 80, 82, 83, 84, 87 or 88.

[00242] In one embodiment of the invention, the polypeptide is a human antibody, humanized antibody, or chimeric antibody. Multispecific antibodies

[00243] The polypeptide or antibody of the invention is not limited to antibodies having a natural domain, for example, a human Fc domain, but may also be an antibody having other mutations different from those of the present invention, such as, for example, mutations affecting glycosylation or allowing the antibody to be a multispecific or bispecific antibody. By the term “natural antibody” is meant any antibody that does not comprise any genetically introduced mutations. An antibody comprising naturally occurring modifications, for example different allotypes, should thus be understood as a “natural antibody” within the meaning of the present invention, and may thus be understood as a parental antibody. Such antibodies may serve as a template for at least two substitutions according to the present invention, and thus provide the antibodies of the invention.An example of a parental antibody comprising substitutions other than those of the present invention is the bispecific antibody, as described in WO2011 / 131746 (Genmab), using reduction conditions to promote the exchange of half molecules of two antibodies comprising IgG4-type CH3 regions, thus forming bispecific antibodies without concomitant aggregate formation. Other examples of parental antibodies include, but are not limited to, Petition 870260062878, dated 06 / 26 / 2026, p. 82 / 410 / 194 bispecific antibodies, such as heterodimeric bispecific antibodies: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat Neurosciences Corporation); FcΔAdp (Regeneron); nodes-in-holes (Genentech); electrostatic direction (Amgen, Chugai, Oncomed); SEEDbodies (Merck); 'Azymetric' scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech). Other exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a human antibody, a humanized antibody, a chimeric antibody, or any combination thereof.

[00244] It should be understood that any embodiment of the present invention described herein may be used in a multispecific antibody in an aspect described below.

[00245] Thus, in one embodiment, the variant of the present invention is an antibody selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[00246] In one particular embodiment, the bispecific antibody has the shape described in WO 2011 / 131746.

[00247] The bispecific antibody of the present invention is not limited to a particular shape and may be any of those described herein.

[00248] In another aspect, the invention relates to a polypeptide or antibody that is a bispecific polypeptide or antibody comprising a first heavy chain of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody comprising a second heavy chain of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same or different antigens, and wherein the first and / or second heavy chain comprise a) a replacement in a position selected from the group corresponding to E430, E345 and S440, with the condition that the Petition 870260062878, dated 06 / 26 / 2026, page 83 / 410 / 194 replacement in S440 is S440Y or S440W, b) one or more substitution(s) at a position selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333 and P396, and (c) wherein the first heavy chain comprises an additional substitution at the position selected from the group consisting of: K409, T366, L368, K370, D399, F405, and Y407; and the second heavy chain comprises an additional substitution at the position selected from the group consisting of: F405, T366, L368, K370, D399, Y407, and K409, and wherein the additional mutation in the first polypeptide is different from the additional mutation in the second polypeptide.

[00249] In one aspect, the present invention provides a polypeptide or antibody comprising an Fc region of a human IgG comprising a first heavy chain and a first antigen-binding region, a second heavy chain and a second antigen-binding region, wherein said first and second heavy chains comprise a) a substitution at a position selected from the group consisting of: E430, E345 and S440, provided that the substitution at S440 is S440Y or S440W and b) a substitution at one or more position(s) selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333 and P396 and c) an additional substitution at position F405 or K409; where the additional substitution is different from that of the first heavy chain and the second heavy chain, such that if the first heavy chain has a substitution at position F405, then the second heavy chain has a substitution at K409, and vice versa.

[00250] Thus, embodiments are provided in which the first heavy chain and the second heavy chain are not identical due to the (c) additional mutation not being located at the same position in the first and second chain Petition 870260062878, dated 06 / 26 / 2026, page 84 / 410 / 194 heavy.

[00251] It should be understood that any embodiment of the present invention described herein may be used in a multispecific antibody aspect described below.

[00252] Thus, in one embodiment, the variant of the present invention is an antibody selected from among monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[00253] In a particular embodiment, the bispecific antibody has the shape described in WO 2011 / 131746.

[00254] In a particular embodiment of the present invention, the first heavy chain comprises an additional substitution corresponding to K409, such as K409R; the second heavy chain comprises an additional substitution corresponding to F405, such as F405L.

[00255] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to position_E430, b) a substitution in one or more position(s) selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333 and P396, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00256] In one embodiment of the present invention, the first and / or second heavy chain comprise a) a replacement corresponding to position E345, b) a substitution in one or more position(s) selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333 and P396, and Petition 870260062878, dated 06 / 26 / 2026, page 85 / 410 / 194 c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00257] In one embodiment of the present invention, the first and / or second Fc heavy chain comprise a) a replacement corresponding to S440Y or S440W, b) a substitution in one or more position(s) selected from the group consisting of: G236, S267, H268, S324, K326, I332, E333 and P396, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00258] In one embodiment of the present invention, the first and / or second Fc heavy chain comprise a) a replacement corresponding to E430G, b) two substitutions corresponding to K326W, E333S, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00259] In one embodiment of the present invention, the first and / or second Fc heavy chains comprise a) a replacement corresponding to E430G, b) two substitutions corresponding to K326W, E333T, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00260] In one embodiment of the present invention, the first and / or second Fc region comprise a) a replacement corresponding to E430G, Petition 870260062878, dated 06 / 26 / 2026, p. 86 / 410 / 194 b) two substitutions corresponding to K326A, E333A, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00261] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E430G, b) two substitutions corresponding to K333A, P396L, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00262] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E430G, b) three substitutions corresponding to K326A, E333A, P396L, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00263] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E430G, b) three substitutions corresponding to K326A, E333T, P396L, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00264] In one embodiment of the present invention, the first and / or second heavy chains comprise Petition 870260062878, dated 06 / 26 / 2026, p. 87 / 410 / 194 a) a replacement corresponding to E345K, b) two substitutions corresponding to K326W, E333S, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00265] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345K, b) two substitutions corresponding to K326W, E333T, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00266] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345K, b) two substitutions corresponding to K326A, E333A, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00267] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345K, b) two substitutions corresponding to K333A, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00268] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345K, Petition 870260062878, dated 06 / 26 / 2026, page 88 / 410 / 194 b) three substitutions corresponding to K326A, E333A, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00269] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345K, b) three substitutions corresponding to K326A, E333T, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00270] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345R, b) two substitutions corresponding to K326W, E333S, and c) where the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00271] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345R, b) two substitutions corresponding to K326W, E333T, and c) where the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00272] In one embodiment of the present invention, the first and / or second heavy chains comprise Petition 870260062878, dated 06 / 26 / 2026, p. 89 / 410 / 194 a) a replacement corresponding to E345R, b) two substitutions corresponding to K326A, E333A, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00273] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345R, b) two substitutions corresponding to K333A, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00274] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to E345R, b) three substitutions corresponding to K326A, E333A, P396L, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00275] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to S440Y, b) two substitutions corresponding to K326W, E333S, and c) where the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00276] In one embodiment of the present invention, the first and / or second heavy chains comprise Petition 870260062878, dated 06 / 26 / 2026, p. 90 / 410 / 194 a) a replacement corresponding to S440Y, b) two substitutions corresponding to K326W, E333T, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00277] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to S440Y, b) two substitutions corresponding to K326A, E333A, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R, and the second heavy chain comprises an additional substitution corresponding to F405L.

[00278] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to S440Y, b) two substitutions corresponding to K333A, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L.

[00279] In one embodiment of the present invention, the first and / or second heavy chains comprise a) a replacement corresponding to S440Y, b) three substitutions corresponding to K326A, E333A, P396L, and c) wherein the first heavy chain comprises an additional substitution corresponding to K409R; and the second heavy chain comprises an additional substitution corresponding to F405L. Targets and method of use

[00280] The polypeptide or antibody according to the present Petition 870260062878, dated 06 / 26 / 2026, p. 91 / 410 / 194. The invention can bind to a target that activates a signal transduction pathway. In one embodiment, the target is a target that activates, inhibits, modulates, and / or regulates the signal transduction pathway. Examples of targets that may be particularly suitable as targets according to the present invention are cell surface receptors and ligands.

[00281] Cell surface receptors include, for example, 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. Several references that refer to receptors belonging to these receptor families and their characteristics are available and include, for example, Cooke B A., King RJ B., van der Molen H J. ed. New Comprehensive Biochemistry Vol. 18B “Hormones and Their Actions Part Π” 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: 10671070; 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: 959962; 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).

[00282] 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 (TNFR-SF) superfamily or G protein-coupled receptor (GPCR) superfamily. Petition 870260062878, dated 06 / 26 / 2026, p. 92 / 410 / 194

[00283] In one embodiment of the invention, the polypeptide or antibody binds to a cell surface receptor including, for example, hormone receptors and cytokine receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptor, lymphokine receptor, growth factor receptor, differentiation control factor receptor and the like. Examples of cytokine receptors include erythropoietin receptor (EPO), thrombopoietin receptor (TPO), granulocyte colony-stimulating factor receptor (G-CSF), macrophage colony-stimulating factor receptor (M-CSF), granular macrophage colony-stimulating factor receptor (GM-CSF), tumor necrosis factor receptor (TNF), interleukin-1 receptor (IL-1), interleukin-2 receptor (IL-2), interleukin-3 receptor (IL-3), interleukin-4 receptor (IL-4), interleukin-5 receptor (IL-5), interleukin-6 receptor (IL-6), and interleukin-7 receptor (IL-7).interleukin-9 receptor (IL-9), interleukin-10 receptor (IL-10), interleukin-11 receptor (IL-11), interleukin-12 receptor (IL-12), interleukin-13 receptor (IL-13), interleukin-15 receptor (IL-15), interferon-alpha receptor (IFN-alpha), interferon-beta receptor (IFN-beta), interferon-gamma receptor (IFN-gamma), growth hormone receptor (GH), insulin receptor, blood stem cell proliferating factor receptor (SCF), vascular epidermal growth factor receptor (VEGF), epidermal growth factor receptor (EGF), nerve growth factor receptor (NGF), fibroblast growth factor receptor (FGF), platelet-derived growth factor receptor (PDGF), transforming growth factor-beta receptor (TGF-beta), leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor receptor (CNTF), oncostatin M receptor (OSM),and Notch family receiver.

[00284] The tumor necrosis factor receptor (TNFRSF) superfamily is a group of receptors characterized by the ability to bind Petition 870260062878, dated 06 / 26 / 2026, page 93 / 410 / 194 ligands of the tumor necrosis factor superfamily (TNFRSF) via an extracellular cysteine-rich domain. TNF receptors form trimeric complexes in the plasma membrane. TNFRSF includes the following list of 29 proteins; TNFR1 (Uniprot P19438), FAS (Uniprot P25445), DR3 (Uniprot Q93038), DR4 (Uniprot O00220), DR5 (Uniprot O14763), DR6 (Uniprot O75509), NGFR (Uniprot P08138), EDAR (Uniprot Q9UNE0), DcR1 (Uniprot O14798), DcR2(Uniprot Q9UBN6), DcR3 (Uniprot O95407), OPG (Uniprot O00300), TROY (Uniprot Q9NS68), 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).

[00285] Some TNFRSFs are involved in apoptosis and contain an intracellular death domain, 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, especially in leukocytes.

[00286] 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.

[00287] 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. Petition 870260062878, dated 06 / 26 / 2026, page 94 / 410 / 194, which does not include an intracellular killing domain. In one embodiment of the invention, TNFR-SF is selected from the group of: OX40, CD40, CD30, CD27, 4-1BB, RANK, TACI, BLySR, BCMA, RELT, and GITR. In another embodiment of the invention, TNFR-SF is selected from the group of: FAS, DR4, DR4, TNFR1, DR6, DR3, EDAR, and NGFR.

[00288] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to OX40, wherein the Fc region of IgG1 comprises: a. an E430G replacement, and b. a replacement for K326W and E333S.

[00289] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to CD40, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00290] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to CD137, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00291] In one embodiment of the invention, the antibody comprises an antigen-binding region linking to GITR, wherein the Fc region of IgG comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00292] In one embodiment of the invention, the antibody comprises an antigen-binding region linking to GITR, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and Petition 870260062878, dated 06 / 26 / 2026, page 95 / 410 / 194 b. a replacement for K326W and E333S.

[00293] In one embodiment of the invention, the antibody comprises an antigen-binding region linking to GITR, wherein the Fc region of IgG2 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00294] The polypeptide or antibody according to the invention can bind to any target, examples of such targets or antigens according to the 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.

[00295] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to FAS, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00296] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to DR5, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement of K326A and E333A.

[00297] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to DR5, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement of K326A and E333T.

[00298] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to DR5, wherein the Fc region of IgG1 Petition 870260062878, dated 06 / 26 / 2026, page 96 / 410 / 194 includes, a. an E430G replacement, and b. a K326A replacement.

[00299] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to DR5, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement E333A.

[00300] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to DR5, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00301] In one embodiment of the invention, the antibody comprises an antigen-binding region binding to CD20, wherein the Fc region of IgG1 comprises, a. an E430G replacement, and b. a replacement for K326W and E333S.

[00302] In one embodiment of the invention, the antigen-binding region binds to a member of the tumor necrosis factor (TNF-SF) superfamily.

[00303] In one embodiment of the invention, the antigen-binding region binds to a member of TNF-SF selected from the group consisting of: lymphotoxin beta (TNF-C), OX40L, CD154, FasL, CD70, CD153, RANKL, APRIL and BAFF.

[00304] In one embodiment of the invention, the polypeptide or antibody binding to a cell surface receptor includes, for example, hormone receptors and cytokine receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptor, lymphokine receptor, Petition 870260062878, dated 06 / 26 / 2026, page 97 / 410 / 194 growth factor receptor, differentiation control factor receptor and similar. Examples of cytokine receptors include erythropoietin receptor (EPO), thrombopoietin receptor (TPO), granulocyte colony-stimulating factor receptor (G-CSF), macrophage colony-stimulating factor receptor (M-CSF), granular macrophage colony-stimulating factor receptor (GM-CSF), tumor necrosis factor receptor (TNF), interleukin-1 receptor (IL-1), interleukin-2 receptor (IL-2), interleukin-3 receptor (IL-3), interleukin-4 receptor (IL-4), interleukin-5 receptor (IL-5), interleukin-6 receptor (IL-6), interleukin-7 receptor (IL-7), interleukin-9 receptor (IL-9), interleukin-10 receptor (IL-10), interleukin-11 receptor (IL-11), interleukin-12 receptor (IL-12), and... interleukin-13 (IL-13), interleukin-15 receptor (IL-15),Interferon-alpha receptor (IFN-alpha), interferon-beta receptor (IFN-beta), interferon-gamma receptor (IFN-gamma), growth hormone (GH) receptor, insulin receptor, blood stem cell proliferating 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-beta (TGF-beta) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M (OSM) receptor, Notch family receptor.

[00305] 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.

[00306] The polypeptide or antibody according to the invention can bind to any target, examples of such targets or antigens according to are described above. Petition 870260062878, dated 06 / 26 / 2026, page 98 / 410 / 194 Methods for increasing the agonistic activity of a polypeptide or antibody

[00307] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region; a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00308] In one aspect, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody by introducing an enhancing Fc-Fc substitution and a C1q-binding substitution.

[00309] In one aspect, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, the method comprising a) introducing at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, where the position corresponds to human IgG1, according to EU numbering.

[00310] Introducing a) at least one substitution according to the invention that is in one of the following positions E430, E345 or S440 introduces the effect of enhanced Fc-Fc interactions of the polypeptide or antibody. Introducing b) one or more substitutions according to the invention, which is in one of the following positions G236, S239, S267, H268, S324, K326, I332, E333 and P396, introduces the effect of agonistic activity. Petition 870260062878, dated 06 / 26 / 2026, p. 99 / 410 / 194 increased in polypeptide or antibody.

[00311] In another aspect, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and wherein the method comprises b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the position corresponds to human IgG1, according to EU numbering.

[00312] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and wherein the method comprises b) introducing at least two substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the position corresponds to human IgG1, according to EU numbering.

[00313] Increasing the agonistic activity of a polypeptide or antibody according to the present invention should be understood as increasing the agonistic activity of the polypeptide or antibody compared with a parental polypeptide or antibody; alternatively, increasing the agonistic activity of the polypeptide or antibody may also refer to when the polypeptide or antibody is compared with a polypeptide or antibody comprising an enhancing Fc-Fc mutation but not a C1q-binding mutation. Thus, it should be understood that the polypeptide or Petition 870260062878, dated 06 / 26 / 2026, page 100 / 410 / 194: The antibody can be compared with a parental polypeptide or parental antibody having an identical antigen-binding region and an Fc region without an enhancing Fc-Fc substitution and without a C1q-binding substitution; alternatively, the polypeptide or antibody can be compared with a polypeptide or antibody having an identical antigen-binding region and an Fc region with an enhancing Fc-Fc substitution, but without the C1q-binding substitution.

[00314] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position G236 is not G236F, G236R, G236Y.

[00315] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position S267 is not S267H, S267I, S267K, S267G.

[00316] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position H268 is not H268K, H268D, H268E.

[00317] 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. Herein, embodiments are provided in which the substitution intensifies the Fc-Fc interactions.

[00318] In one embodiment of the invention, at least one substitution is selected from the group consisting of: E430G, E430S, E430F, E430T.

[00319] In one embodiment of the invention, at least one replacement is selected from the group consisting of: E345K, E345Q, Petition 870260062878, dated 06 / 26 / 2026, page 101 / 410 / 194 E345R, E345Y.

[00320] In one embodiment of the invention, the polypeptide or antibody has at least one E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345R substitution. In one embodiment of the invention, the polypeptide or antibody has at least one S440Y substitution.

[00321] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00322] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00323] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, the method comprising introducing one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00324] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, the method Petition 870260062878, dated 06 / 26 / 2026, page 102 / 410 / 194, involves introducing one or more replacement(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00325] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00326] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00327] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00328] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, wherein the method Petition 870260062878, dated 06 / 26 / 2026, page 103 / 410 / 194, involves introducing one or more substitution(s) in a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00329] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00330] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00331] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00332] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing one or more substitution(s) selected from Petition 870260062878, dated 06 / 26 / 2026, page 104 / 410 / 194 of the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00333] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00334] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00335] In one embodiment, the present invention relates to a method for increasing the agonistic 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 substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00336] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396. Petition 870260062878, dated 06 / 26 / 2026, page 105 / 410 / 194

[00337] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00338] In one embodiment, the present invention relates to a method for increasing the agonistic activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00339] Here are provided embodiments that allow enhanced agonistic properties of polypeptides or antibodies when antigen binds to the cell surface. In one embodiment, the polypeptides or antibodies comprise enhanced agonistic properties. In one embodiment, the polypeptides or antibodies comprise an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the aforementioned substitutions may be present in the first and / or the second heavy chain.

[00340] In one embodiment of the invention, one or more substitutions at a position 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 at a position selected from the group consisting of: K326, E333 and P396. In one embodiment of the invention, the Petition 870260062878, dated 06 / 26 / 2026, page 106 / 410 / 194. 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.

[00341] 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 K326W and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises K326W and P396L substitutions.In one embodiment of the invention, the polypeptide or antibody comprises K326A and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises K326A and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises K326A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E333A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises E333S and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises substitutions of... Petition 870260062878, dated 06 / 26 / 2026, page 107 / 410 / 194 K326A, E333A and P396L. In one embodiment of the invention, the polypeptide or antibody comprises the K326S, E333A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333S and P396L substitutions.

[00342] In one embodiment of the invention, one or more substitutions at a position 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 at a position 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.

[00343] In one embodiment of the invention, one or more substitutions at a position 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 S267E and H268F substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the S267E and S324T substitutions. Petition 870260062878, dated 06 / 26 / 2026, page 108 / 410 / 194 In one embodiment of the invention, the polypeptide or antibody comprises the substitutions H268F and S324T. In another embodiment of the invention, the polypeptide or antibody comprises the substitutions: S267E, H268F, and S324T.

[00344] In one embodiment, the present invention relates to a method in which the Fc region comprises one or more additional substitutions.

[00345] In one embodiment, the present invention relates to a method in which the Fc region comprises an additional substitution at the following positions in human IgG1 according to EU numbering: S440 or K439. In one embodiment of the invention, the Fc region comprises an additional substitution corresponding to one of the following positions S440 or K439, provided that an additional substitution is not at the S440 position if the Fc-Fc enhancing substitution is at S440. Polypeptides or antibodies comprising the Fc-Fc enhancing substitution and the C1q-binding substitution according to the present invention and an additional substitution at the S440 position such as S440K, do not form oligomers with polypeptides or antibodies comprising the mutation at the S440 position such as S440K.Polypeptides or antibodies, comprising the enhancing Fc-Fc substitution and the C1q-binding substitution according to the present invention and an additional substitution at the K439 position such as K439E, do not form oligomers with polypeptides or antibodies comprising the mutation at the K439 position such as K439E. Here, a method is provided that allows the formation of oligomers between polypeptides or antibodies in which a first polypeptide or antibody comprises a K439E substitution and a second polypeptide or antibody comprises an S440K substitution. In this way, oligomers such as, for example, hexamers can be forced to be formed in some patterns of the first and second polypeptides. This may be of interest in methods where polypeptides bind to different targets or epitopes and oligomers must. Petition 870260062878, dated 06 / 26 / 2026, page 109 / 410 100 / 194 can be formed in combinations of these different targets or epitopes.

[00346] In one embodiment, the present invention relates to a method in which the additional replacement is selected from either S440K or K439E.

[00347] In one embodiment, the present invention relates to a method for increasing agonist activity wherein the agonist activity is increased by at least 20% compared with a parent polypeptide or parent antibody that is identical to the polypeptide or antibody or alternatively to the polypeptide or antibody with an identical Fc-Fc enhancing substitution, but without the C1q binding substitution. In another embodiment of the invention, the polypeptide or antibody has an increased agonist activity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared with a parent polypeptide or parent antibody or alternatively with the polypeptide or antibody with an identical Fc-Fc enhancing substitution, but without the C1q binding substitution. Methods to increase CDC activity

[00348] In one aspect, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody by introducing an enhancing Fc-Fc substitution and a C1q-binding substitution.

[00349] In one aspect, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, the method comprising a) introducing at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, where the position corresponds to human IgG1, according to EU numbering. Petition 870260062878, dated 06 / 26 / 2026, p. 110 / 410 101 / 194

[00350] Introducing a) at least one substitution according to the invention that is in one of the following positions E430, E345 or S440 introduces the effect of enhanced Fc-Fc interactions of the polypeptide or antibody. Introducing b) one or more substitutions according to the invention that is in one of the following positions G236, S239, S267, H268, S324, K326, I332, E333 and P396 introduces the effect of increased CDC activity in the polypeptide or antibody.

[00351] In another aspect, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and wherein the method comprises b) introducing one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the position corresponds to human IgG1, according to EU numbering.

[00352] In one embodiment, the present invention relates to a method for increasing the CDC activity of a polypeptide or antibody comprising an Fc region of a human immunoglobulin and an antigen-binding region, wherein the Fc region comprises a) at least one substitution at a position selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and wherein the method comprises b) introducing at least two substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein the position corresponds to human IgG1, according to EU numbering.

[00353] Increasing the agonistic activity of a polypeptide or antibody according to the present invention should be understood as Petition 870260062878, dated 06 / 26 / 2026, page 111 / 410 102 / 194 Increasing the CDC activity of the polypeptide or antibody compared to a parental polypeptide or antibody, alternatively increasing the agonist activity of the polypeptide or antibody, may also refer to when the polypeptide or antibody is compared to a polypeptide or antibody comprising an enhancing Fc-Fc mutation, but not a C1q-binding mutation. Thus, it should be understood that the polypeptide or antibody may be compared to a parental polypeptide or antibody having the identical antigen-binding region and an Fc region without an enhancing Fc-Fc substitution and without a C1q-binding substitution; alternatively, the polypeptide or antibody may be compared to a polypeptide or antibody having an identical antigen-binding region and an Fc region with an enhancing Fc-Fc substitution, but without the C1q-binding substitution.

[00354] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position G236 is not G236F, G236R, G236Y.

[00355] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position S267 is not S267H, S267I, S267K, S267G.

[00356] In one embodiment of the invention, one or more substitution(s) at a position selected from the group of G236, S239, S267, H268, S324, K326, I332, E333 and P396, provided that the substitution at position H268 is not H268K, H268D, H268E.

[00357] 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. Embodiments are provided herein in which the substitution intensifies... Petition 870260062878, dated 06 / 26 / 2026, page 112 / 410 103 / 194 Fc-Fc interactions.

[00358] In one embodiment of the invention, at least one substitution is selected from the group consisting of: E430G, E430S, E430F, E430T.

[00359] In one embodiment of the invention, at least one substitution is selected from the group consisting of: E345K, E345Q, E345R, E345Y.

[00360] In one embodiment of the invention, the polypeptide or antibody has at least one E430G substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345K substitution. In one embodiment of the invention, the polypeptide or antibody has at least one E345R substitution. In one embodiment of the invention, the polypeptide or antibody has at least one S440Y substitution.

[00361] 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00362] In one embodiment, the present invention relates to a method for increasing 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 substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00363] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, wherein the method Petition 870260062878, dated 06 / 26 / 2026, p. 113 / 410 104 / 194 involves introducing one or more substitution(s) in a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00364] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00365] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E430G substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00366] In one embodiment, the present invention relates to a method for increasing 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00367] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody wherein the Fc region comprises at least one substitution selected from Petition 870260062878, dated 06 / 26 / 2026, page 114 / 410 105 / 194 of the group consisting of: E345K, E345Q, E345R and E345Y, the method of which comprises introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00368] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, the method comprising introducing one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00369] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00370] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345K substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00371] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing one or more substitution(s) at a position Petition 870260062878, dated 06 / 26 / 2026, page 115 / 410 106 / 194 selected from the group of: G236, S239, S267, H268, S324 K326, I332, E333 and P396.

[00372] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00373] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an E345R substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00374] In one embodiment, the present invention relates to a method for increasing 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 substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00375] In one embodiment, the present invention relates to a method for increasing 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 substitution(s) selected from the group of: Petition 870260062878, dated 06 / 26 / 2026, page 116 / 410 107 / 194 K326A, K326W, E333A, E333S, E333T, and P396L.

[00376] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising one or more substitution(s) at a position selected from the group of: G236, S239, S267, H268, S324, K326, I332, E333 and P396.

[00377] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising introducing one or more substitution(s) selected from the group of: K326A, K326W, E333A, E333S, E333T, and P396L.

[00378] In one embodiment, the present invention relates to a method for increasing CDC activity of a polypeptide or antibody in which the Fc region comprises an S440Y substitution, the method comprising introducing the substitution(s) of one of the groups consisting of: i) K326W and E333S, ii) K326W and E333T, iii) K326A and E333A, iv) K326A, E333A and P396L, v) K326W, vi) E333S, and vii) E333T.

[00379] Embodiments are provided that enable enhanced CDC activity of polypeptides or antibodies by binding to antigen on the cell surface. In one embodiment, the polypeptides or antibodies comprise enhanced CDC activity. In one embodiment, the polypeptides or antibodies comprise an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the aforementioned substitutions may be present in the first and / or Petition 870260062878, dated 06 / 26 / 2026, page 117 / 410 108 / 194 second heavy chain.

[00380] In one embodiment of the invention, one or more substitutions at a position 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 at a position 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.

[00381] In one embodiment of the invention, the polypeptide or antibody comprises one or more, as well 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 K326W and E333S substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises K326W and P396L substitutions.In one embodiment of the invention, the polypeptide or antibody comprises K326A and E333A substitutions. In one embodiment of the invention, the polypeptide or antibody comprises K326A substitutions. Petition 870260062878, dated 06 / 26 / 2026, page 118 / 410 109 / 194 and E333S. In one embodiment of the invention, the polypeptide or antibody comprises the K326A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the E333A and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the E333S and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326A, E333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326S, E333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333A, and P396L substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the K326W, E333S, and P396L substitutions.

[00382] In one embodiment of the invention, one or more substitutions at a position 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 at a position 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.

[00383] In one embodiment of the invention, one or more substitutions at a position 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 a substitution Petition 870260062878, dated 06 / 26 / 2026, page 119 / 410 110 / 194 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 S267E and H268F substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the S267E and S324T substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the H268F and S324T substitutions. In one embodiment of the invention, the polypeptide or antibody comprises the substitutions: S267E, H268F, and S324T. Compositions

[00384] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region; a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00385] The invention also relates to compositions comprising polypeptides or antibodies described herein and variations thereof. Specific aspects and embodiments will be described below. Furthermore, such polypeptide or antibody can be obtained according to any method described herein.

[00386] In one aspect, the present invention relates to a composition comprising at least one polypeptide or antibody described herein.

[00387] In one embodiment of the present invention, the composition comprises one or more polypeptides or antibodies according to any Petition 870260062878, dated 06 / 26 / 2026, pp. 120 / 410 111 / 194 aspect or modality described here.

[00388] In one embodiment of the present invention, the composition comprises a first polypeptide or antibody and a second polypeptide or antibody as described in any aspect or embodiment herein.

[00389] In one aspect of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies comprise an Fc region comprising, (i) at least one substitution, which is an enhancing Fc-Fc mutation; and (ii) one or more substitutions, which are C1q-binding substitutions; and (iii) an additional mutation, which prevents oligomerization between Fc regions having the identical additional mutation, wherein the first and second polypeptides or antibodies do not comprise the same additional mutation.

[00390] In one embodiment of the present 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 the group consisting of: E430, E345 or S440Y or S440W substitution, and ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396 and iii) an additional mutation wherein the first and second polypeptides or antibodies do not comprise the same additional mutation. 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.

[00391] In one embodiment of the invention, the composition comprises Petition 870260062878, dated 06 / 26 / 2026, page 121 / 410 112 / 194 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) at least one or more substitutions selected from the group consisting of: E430, E345 or S440Y or S440W substitution; and (ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396; and (iii) an additional substitution at position K439 or S440, with the condition that if the additional substitution is at S440, then the substitution according to (i) is not at S440, with the condition that the first and second Fc regions do not comprise an additional substitution according to (iii) at the same amino acid position, (iv) wherein the substitutions correspond to the amino acid positions in human IgG1, according to EU numbering.

[00392] 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, (iii) an additional mutation, wherein the mutations correspond to the following amino acid positions in human IgG1, according to EU numbering: (i) at least one or more replacements selected from the group consisting of: E430, E345 or an S440Y or S440W replacement; and; (ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, Petition 870260062878, dated 06 / 26 / 2026, page 122 / 410 113 / 194 E333, and P396; and (iii) an additional substitution at position K439 in the first Fc region and an additional substitution at position S440 in the second Fc region, or vice versa, with the condition that if the additional substitution is at position S440, then the substitution is not at S440; (iv) wherein the substitutions correspond to the amino acid positions in human IgG1, according to EU numbering.

[00393] 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) at least one or more substitutions selected from the group consisting of: E430, E345 or an S440Y or S440W substitution; and (ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396; and (iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, and (iv) wherein the substitutions correspond to the amino acid positions in human IgG1, according to EU numbering.

[00394] 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) at least one or more replacements selected from the group consisting of: replacement E430, E345 or S440Y or S440W; and Petition 870260062878, dated 06 / 26 / 2026, page 123 / 410 114 / 194 (ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396; and (iii) an additional S440K substitution in the first Fc region and an additional K439E substitution in the second Fc region; (iv) wherein the substitutions correspond to the amino acid positions in human IgG1, according to EU numbering.

[00395] Thus, embodiments are provided in which one or both of the first and second polypeptides or antibody have increased agonist activity and / or CDC activity, or only the first or second polypeptide has increased agonist activity and / or CDC activity.

[00396] 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 substitution at the amino acid position corresponding to E430, and (ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396; and (iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00397] 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 substitution at the amino acid position corresponding Petition 870260062878, dated 06 / 26 / 2026, page 124 / 410 115 / 194 to E345, and ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396; and iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00398] 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) an E430G, and 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 iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00399] 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) an E430G, and ii) one or more substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and (iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00400] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first region Petition 870260062878, dated 06 / 26 / 2026, page 125 / 410 116 / 194 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) an E430G, and ii) at least two substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00401] 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) an E430G replacement, and ii) a K326W and E333S replacement; and iii) an additional K439E replacement in the first Fc region and an additional S440K replacement in the second Fc region, or vice versa.

[00402] 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) an E345K, and 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 iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00403] In one embodiment of the invention, the composition comprises Petition 870260062878, dated 06 / 26 / 2026, page 126 / 410 117 / 194 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) an E345K, and ii) one or more substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00404] 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) an E345K, and ii) at least two substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00405] 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) an E345K replacement, and ii) a K326W and E333S replacement; and iii) an additional K439E replacement in the first Fc region and an additional S440K replacement in the second Fc region, or vice versa.

[00406] In one embodiment of the invention, the composition comprises Petition 870260062878, dated 06 / 26 / 2026, page 127 / 410 118 / 194 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) an E345R, and 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 iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00407] 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) an E345R, and ii) one or more substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and iii) an additional K439E substitution in the first Fc region and an additional S440K substitution in the second Fc region, or vice versa.

[00408] 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) an E345R, and ii) at least two substitutions selected from the group consisting of: K326W, K326A, E333S, E333A and P396L; and iii) an additional K439E substitution in the first Fc region and Petition 870260062878, dated 06 / 26 / 2026, page 128 / 410 119 / 194 an additional S440K replacement in the second Fc region, or vice versa.

[00409] 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) an E345R replacement, and ii) a K326W and E333S replacement; and iii) an additional K439E replacement in the first Fc region and an additional S440K replacement in the second Fc region, or vice versa.

[00410] In another embodiment of the invention, the composition comprises a first and a second polypeptide or antibody, wherein the first and second polypeptides or antibodies comprise an Fc region comprising, i) at least one substitution, which is a mutation intensifying Fc-Fc; ii) an additional mutation, which prevents oligomerization between Fc regions having the identical additional mutation, wherein the first and second polypeptides or antibodies do not comprise the same additional mutation, iii) and either 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 a second mutation that diminishes the effector functions of Fc.

[00411] 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 Petition 870260062878, dated 06 / 26 / 2026, page 129 / 410 120 / 194 i) at least one or more substitutions selected from the group consisting of: E430, E345, or S440Y or S440W substitution, ii) an additional K439E or S440K mutation, wherein the first and second Fc regions do not comprise the same additional substitution, and wherein if at least one substitution is S440Y or S440W, then the additional mutation is not S440K; iii) and either the first or second Fc region comprises one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396.

[00412] 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) at least one or more substitutions selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396, and iii) an additional K439E mutation; and the second Fc region comprises i) at least one or more substitutions selected from the group consisting of: E430, E345 or S440Y or S440W substitution, and an additional S440K mutation.Hereby, embodiments are provided in which only the first polypeptide or antibody has increased agonist activity and / or increased CDC activity.

[00413] 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) at least Petition 870260062878, dated 06 / 26 / 2026, page 130 / 410 121 / 194 one or more substitutions selected from the group consisting of: E430, E345, and ii) one or more substitutions at a position selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333, and P396, and iii) an additional S440K mutation; and the second Fc region comprises i) at least one or more substitutions selected from the group consisting of: E430, E345 or an S440Y or S440W substitution, and an additional K439E mutation. Hereby, embodiments are provided in which only the first polypeptide or antibody has increased agonist activity and / or increased CDC activity.

[00414] 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) an additional K439E substitution; and the second Fc region comprises i) an E430G substitution, and an additional S440K substitution.

[00415] 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) an additional S440K substitution; and the second Fc region comprises i) an E430G substitution, and an additional K439E substitution.

[00416] In one embodiment of the invention, the composition comprises a first polypeptide or antibody comprising a first region Petition 870260062878, dated 06 / 26 / 2026, page 131 / 410 122 / 194 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) an additional S440K substitution; and the second Fc region comprises i) an E430G substitution, and an additional K439E substitution. 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) a K326W and E333S substitution, and iii) an additional K439E substitution; and the second Fc region comprises i) an E430G substitution and an additional S440K substitution.

[00417] In one embodiment of the present invention, the composition comprises a polypeptide or antibody capable of binding to a member of the tumor necrosis factor receptor (TNFR-SF) superfamily or G protein-coupled receptor (GPCR) superfamily.

[00418] In one embodiment of the present 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.

[00419] In one embodiment of the present invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member with an intracellular killing domain selected from the following group consisting of: TNFR1, FAS, DR3, DR4, DR5, DR6, NGFR and EDAR. Petition 870260062878, dated 06 / 26 / 2026, p. 132 / 410 123 / 194

[00420] In one embodiment of the present invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member without an intracellular killing domain selected from the following group consisting of: DcR1, DcR2, DcR3, OPG, TROY, XEDAR, LTbR, HVEM, TWEAKR, CD120b, OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR, RELT.

[00421] In one embodiment of the present invention, the composition comprises a polypeptide or antibody capable of binding to a TNFR-SF member belonging to the immune activator group consisting of: OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[00422] In one embodiment of the present invention, the composition comprises a polypeptide or antibody wherein a first polypeptide and a second polypeptide bind to different epitopes on one or more members of TNFR-SF without an intracellular killing domain, selected from the following group consisting of: OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[00423] In one embodiment of the present invention, the composition comprises a polypeptide or antibody wherein a first polypeptide binding to a TNFR-SF member without an intracellular killing domain selected from the following group consisting of: OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT does not block the binding of said second antibody binding to a TNFR-SF member without an intracellular killing domain selected from the following group consisting of: OX40, CD40, CD27, CD30, 4-1BB, RANK, TACI, BLySR, BCMA, GITR and RELT.

[00424] In one embodiment of the present invention, the composition comprising a first polypeptide or antibody and a second polypeptide or antibody are present in the composition in a molar ratio of 1:49 to 49:1, such as a molar ratio of 1:1, a molar ratio of 1:2, Petition 870260062878, dated 06 / 26 / 2026, page 133 / 410 124 / 194 a molar ratio of 1:3, a molar ratio of 1:4, a molar ratio of 1:5, a molar ratio of 1:6, a molar ratio of 1:7, a molar ratio of 1:8, a molar ratio of 1:9, a molar ratio of 1:10, a molar ratio of 1:15, a molar ratio of 1:20, a molar ratio of 1:25, a molar ratio of 1:30, a molar ratio of 1:35, a molar ratio of 1:40, a molar ratio of 1:45, a molar ratio of 1:50, a molar ratio of 50:1, a molar ratio of 45:1, a molar ratio of 40:1, a molar ratio of 35:1, a molar ratio of 30:1, a molar ratio of 25:1, a ratio molar ratio of 20:1, molar ratio of 15:1, molar ratio of 10:1, molar ratio of 9:1, molar ratio of 8:1, molar ratio of 7:1, molar ratio of 6:1, molar ratio of 5:1, molar ratio of 4:1, molar ratio of 3:1, molar ratio of 2:1.

[00425] In one embodiment of the present invention, the composition comprising a first polypeptide and a second polypeptide and / or any additional polypeptide are present in the composition in an equimolar ratio.

[00426] In one embodiment of the present invention, the composition according to any aspect or embodiment is a pharmaceutical composition. THERAPEUTIC APPLICATIONS

[00427] Polypeptides, antibodies, bispecific antibodies or compositions according to any aspect or embodiment of the present invention can be used as a medicament, that is, for therapeutic applications.

[00428] In one aspect, the present invention provides a polypeptide, antibody or a composition according to any aspect or embodiment described herein for use as a medicament.

[00429] In another aspect, the present invention provides a polypeptide, antibody or a composition according to any aspect or embodiment described herein for use in the treatment of cancer, autoimmune disease, inflammatory disease or infectious disease. Petition 870260062878, dated 06 / 26 / 2026, p. 134 / 410 125 / 194

[00430] In another aspect, the present invention relates to a method of treating an individual having a disease comprising administering to the individual an effective amount of a polypeptide, antibody or composition according to any aspect or embodiment described herein.

[00431] In one embodiment of the invention, the disease is selected from the group of: cancer, autoimmune disease, inflammatory disease and infectious disease.

[00432] In one embodiment of the invention, the method according to any aspect or embodiment described herein refers to the further administration of an additional therapeutic agent.

[00433] In one embodiment of the invention, the additional therapeutic agent is one or more anticancer agent(s) 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-modulating agents (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), nutraceuticals, cytokines (including but not limited to IFN-γ), antibodies or antibody mimetics (including but not limited to antibodies). anti-EGFR, anti-IGF-1R, anti-VEGF, antiCD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137,anti-GITR and antibody mimetics), antibody-drug conjugates. PARTS KIT

[00434] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and a Petition 870260062878, dated 06 / 26 / 2026, page 135 / 410 126 / 194 antigen-binding region, a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00435] The invention also relates to kits of parts for simultaneous, separate or sequential use in therapy comprising polypeptides or antibodies described herein. Furthermore, such variants can be obtained according to any method described herein.

[00436] In one aspect, the present invention relates to a kit of parts comprising a polypeptide, antibody or composition according to any aspect or embodiment described herein, wherein said polypeptide, antibody or composition is in one or more containers, such as small vials.

[00437] In one embodiment of the present invention, the kit of parts comprises a polypeptide, antibody, or a composition according to any aspect or embodiment described herein, for simultaneous, separate, or sequential use in therapy. In another aspect, the present invention relates to the use of a polypeptide, an antibody, a composition, or a kit of parts according to any of the embodiments described herein for use in a diagnostic method.

[00438] In another aspect, the present invention relates to a diagnostic method comprising administering a polypeptide, antibody, a composition or a kit of parts according to any of the embodiments described herein to at least one part of the body of a human or other mammal.

[00439] In another aspect, the present invention relates to the use of a polypeptide, an antibody, a composition or kit of parts according to any of the embodiments described herein in hair imaging. Petition 870260062878, dated 06 / 26 / 2026, p. 136 / 410 127 / 194 minus a part of the body of a human or other mammal.

[00440] In another aspect, the present invention relates to a method for imaging at least one part of the body of a human or other mammal, comprising administering a variant, a composition or a kit of parts according to any of the embodiments described herein. ADDITIONAL USES

[00441] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region; a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00442] In a further aspect, the invention relates to a polypeptide, antibody of the invention as described above for use as a medicament, in particular for use as a medicament for the treatment of diseases or disorders. Examples of such diseases and disorders include, without limitation, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, bacterial, viral or fungal infections.

[00443] In another aspect, the present invention relates to the polypeptide, antibody, bispecific antibodies, compositions and kit of parts described herein, for the treatment of a disease, such as cancer.

[00444] In another aspect, the present invention relates to a method for treating a human disease, comprising administering a variant, a composition or a kit of parts described herein.

[00445] In another aspect, the present invention relates to a method for treating cancer in a human comprising administering Petition 870260062878, dated 06 / 26 / 2026, p. 137 / 410 128 / 194 a variant, a composition or a kit of parts.

[00446] “Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention with the aim of easing, improving, stopping or eradicating (curing) the symptoms or disease states.

[00447] An “effective amount” or “therapeutically effective amount” refers to an effective quantity, in dosages and for periods of time necessary to achieve a desired therapeutic result. A therapeutically effective amount of an antibody may vary according to factors such as the disease status, 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 harmful effects of the antibody or portion of the antibody are outweighed by the therapeutically beneficial effects. DOSAGES

[00448] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region; a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00449] Effective dosages and dosing regimens for the antibody depend on the disease or pathology being treated and can be determined by those skilled in the art. An exemplary and non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, for example about 0.1 to 20 mg / kg, such as about 0.1 to 10 mg / kg, for example, about 0.5, about Petition 870260062878, dated 06 / 26 / 2026, p. 138 / 410 129 / 194 such as 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[00450] Polypeptides or antibodies of the present invention can also be administered in combination therapy, that is, combined with other therapeutic agents relevant to the disease or condition to be treated. Consequently, in one embodiment, the antibody-containing drug is for combination with one or more additional therapeutic agents, such as cytotoxic, chemotherapeutic or anti-angiogenic agents. Such combined administration may be simultaneous, separate or sequential.

[00451] In a further embodiment, the present invention provides a method for treating or preventing diseases, such as cancer, which method comprises administering to an individual in need thereof a therapeutically effective amount of a variant or pharmaceutical composition of the present invention, in combination with radiotherapy and / or surgery. Preparation method

[00452] It should be understood that the embodiments described below with reference to a polypeptide or antibody refer to a polypeptide or antibody comprising an Fc region of an immunoglobulin and an antigen-binding region; a polypeptide or antibody may also be a multispecific polypeptide or antibody having a first Fc region of an immunoglobulin and a first antigen-binding region, and a second polypeptide or antibody having a second Fc region of an immunoglobulin and a second antigen-binding region.

[00453] The invention also provides isolated nucleic acids and vectors encoding a variant according to any of the aspects described above, as well as vectors and expression systems encoding the variants. Nucleic acid constructs, vectors, and expression systems suitable for antibodies and variants thereof are known in the art and are described in the Examples. In embodiments where the variant Petition 870260062878, dated 06 / 26 / 2026, page 139 / 410 130 / 194 comprises not only a heavy chain (or an Fc-containing fragment thereof) but also a light chain; the nucleotide sequences encoding the heavy and light chain portions may be present in the same or different nucleic acids or vectors.

[00454] The invention also provides a method for producing, in a host cell, a polypeptide or antibody according to any of the aspects described above, wherein said polypeptide or antibody comprises at least the Fc region of a heavy chain, said method comprising the following steps: a) provide a nucleotide construct encoding the said Fc region of the said variant, b) to express said nucleotide construct in a host cell and c) recover said antibody variant from a cell culture of said host cell.

[00455] In some embodiments, the antibody is a heavy chain antibody. In most embodiments, however, the antibody will also contain a light chain, and thus the said host cell will still express a construct encoding the light chain, in the same vector or in a different vector.

[00456] Host cells suitable for recombinant antibody expression are well known in the art and include CHO, HEK-293, Expi293, PER-C6, NS / 0, and Sp2 / 0 cells. In one embodiment, said host cell is a cell capable of glycosylation of Asn-linked proteins, e.g., a eukaryotic cell, such as a mammalian cell, for example, a human cell. In a further embodiment, said host cell is a non-human cell that is genetically modified to produce glycoproteins having human-type or human-like glycosylation. Examples of such cells are genetically modified Pichia pastoris cells. Petition 870260062878, of 26 / 06 / 2026, p. 140 / 410 131 / 194 (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 2006) 1591-1597).

[00457] In one embodiment, said host cell is a host cell that is not capable of efficiently removing K447 residues from the C-terminal lysine of antibody heavy chains. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97: 2426 (incorporated herein by reference) lists some such antibody production systems, e.g., Sp2 / 0, NS / 0, or transgenic mammary gland (goat), in which only partial removal of C-terminal lysines is achieved. In another embodiment, the host cell is a host cell with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells in which variants of the invention can be expressed to produce an antibody with altered glycosylation. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03 / 035835; and WO99 / 54342.Additional methods for generating engineered glycoforms are known in the art, and include, but are not limited to, those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc., Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[00458] The invention also relates to an antibody obtained or obtainable by the method of the invention described above.

[00459] In a further aspect, the invention relates to a host cell capable of producing a polypeptide or antibody of the invention. In Petition 870260062878, dated 06 / 26 / 2026, pp. 141 / 410 132 / 194 In one embodiment, the host cell was transformed or transfected with a nucleotide construct of the invention.

[00460] The present invention is further illustrated by the following examples which should not be interpreted as being limiting. TABLE 1 SEQ ID NO: Nome Sequência Clone SEQ ID NO: 1 VH hDR5-01G56T CDR1 GFNIKDTF hDR501-G56T SEQ ID NO: 2 VH hDR5-01G56T CDR2 IDPANTNT SEQ ID NO: 3 VH hDR5-01G56T CDR3 VRGLYTYYFDY SEQ ID NO: 4 VH hDR5-01G56T EVQLQQSGAEVVKPGASVKLSCKASGFNIKDTFIH WVKQAPGQGLEWIGRIDPANTNTKYDPKFQGKATI TTDTS SNTAYMELS SLRSEDTA VYYC VRGLYTYYF DYWGQGTLVTVSS SEQ ID NO: 5 HC hDR5-01G56T EVQLQQSGAEVVKPGASVKLSCKASGFNIKDTFIH WVKQAPGQGLEWIGRIDPANTNTKYDPKFQGKATI TTDTS SNTAYMELS SLRSEDTA VYYCVRGLYTYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK FAS SEQ ID NO: 7 VL hDR5-01 CDR3 QQGNSWPYT SEQ ID NO: 8 VL hDR5-01 EIVMTQSPATLSVSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKFASQSITGIPARFSGSGSGTEFTLT ISSLQSEDFAVYYCQQGNSWPYTFGQGTKLEIK SEQ ID NO: 9 LC hDR5-01 EIVMTQSPATLSVSPGERATLSCRASQSISNNLHWY QQKPGQAPRLLIKFASQSITGIPARFSGSGSGTEFTLT ISSLQSEDFAVYYCQQGNSWPYTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10 VH hDR5-05 CDR1 GFNIKDTH hDR5-05 SEQ ID NO: 11 VH hDR5-05 CDR2 IDPANGNT SEQ ID NO: 12 VH hDR5-05 CDR3 ARWGTNVYFAY SEQ ID NO: 13 VH hDR5-05 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTHM HWVRQAPGQRLEWIGRIDPANGNTEYDQKFQGRV TITVDTSASTAYMELSSLRSEDTAVYYCARWGTNV YFAY WGQGTLVTVS S SEQ ID NO: HC hDR5-05 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTHM Petition: 870260062878, on June 26, 2026, page. 142 / 410 133 / 194 14 HWVRQAPGQRLEWIGRIDPANGNTEYDQKFQGRV TITVDTSASTAYMELSSLRSEDTAVYYCARWGTNV YFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 15 VL hDR5-05 CDR1 SSVSY VL hDR5-05 CDR2 RTS SEQ ID NO: 16 VL hDR5-05 CDR3 QQYHSYPPT SEQ ID NO: 17 VL hDR5-05 DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQ QKPGKAPKPWIYRTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIK SEQ ID NO: 18 LC hDR5-05 DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQ QKPGKAPKPWIYRTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 19 VH CONAC49W-CDR1 GGSISSGDYF IgG1- CONAC49W SEQ ID NO: 20 VH CONAC49W-CDR2 IHNSGTTSEQ ID NO: 21 VH CONAC49W-CDR3 ARDRGGDYYYGMDV SEQ ID NO: 22 VH CONAC49W-C49W QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFW SWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISV DTSKKQFSLRLSSVTAADTAVYYCARDRGGDYYY GMDVWGQGTTVTVS S SEQ ID NO: 23 HC CONAC49W QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFW SWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISV DTSKKQFSLRLSSVTAADTAVYYCARDRGGDYYY GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 24 VL CONAC49W-CDR1 QGISRSY VL CONAC49W-CDR2 GAS SEQ ID NO: 25 VL CONAC49W-CDR3 QQFGSSPWT SEQ ID NO: 26 VL CONAC49W EIVLTQSPGTLSLSPGERATLSCRASQGISRSYLAWY QQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIK SEQ ID LC CONA- EIVLTQSPGTLSLSPGERATLSCRASQGISRSYLAWY Petition 870260062878, dated 06 / 26 / 2026, pp. 143 / 410 134 / 194 NO:27 C49W QQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIK EIVLTQSPGTLSLSPGERATLSCRASQGISRSYLAWY QQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28 VH 7D8 CDR1 GFTFHDYA 7D8 SEQ ID NO: 29 VH 7D8 CDR2 ISWNSGTI SEQ ID NO: 30 VH 7D8 CDR3 AKDIQYGNYYYGMDV SEQ ID NO: 31 VH 7D8 EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMH WVRQAPGKGLEWVSTISWNSGTIGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGN YYYGMDVWGQGTTVTVSS SEQ ID NO: 32 HC 7D8 EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMH WVRQAPGKGLEWVSTISWNSGTIGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGN YYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPS S SLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 VL 7D8 CDR1 QSVSSY VL 7D8 CDR2 DAS SEQ ID NO: 34 VL 7D8 CDR3 QQRSNWPIT SEQ ID NO: 35 VL 7D8 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWY QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK SEQ ID NO: 36 LC 7D8 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWY QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 37 VH 11B8 CDR1 GFTFSYHA 11B8 SEQ ID NO: 38 VH 11B8CDR2 IGTGGVT SEQ ID NO: 39 VH 11B8CDR3 ARDYYGAGSFYDGLYGMDV SEQ ID NO: 40 VH 11B8 EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMH WVRQAPGKGLEWVSIIGTGGVTYYADSVKGRFTIS RDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAG SFYDGLYGMDVWGQGTTVTVS S SEQ ID NO: 41 HC 11B8 EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMH WVRQAPGKGLEWVSIIGTGGVTYYADSVKGRFTIS RDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAG SFYDGLYGMDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV Petition 870260062878, dated 06 / 26 / 2026, page 144 / 410 135 / 194 NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 42 VL 11B8CDR1 QSVSSY VL 11B8 CDR2 DAS SEQ ID NO: 43 VL 11B8 CDR3 QQRSDWPLT SEQ ID NO: 44 VL 11B8 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWY QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIK SEQ ID NO: 45 LC 11B8 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWY SEQ ID NO: 46 VH ALEM CDR1 GFTFTDFY alemtuzu mab SEQ ID NO: 47 VH ALEM CDR2 IRDKAKGYTT SEQ ID NO: 48 VH ALEM CDR3 AREGHTAAPFDY SEQ ID NO: 49 VH ALEM QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMN WVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRV TMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHTA APFDYWGQGSLVTVSS SEQ ID NO: 50HC ALEM QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMN WVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRV TMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHTA APFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 51 VL ALEM CDR1 QNIDKY VL ALEM CDR2 NTN SEQ ID NO: 52 VL ALEM CDR3 LQHISRPRT SEQ ID NO: 53 VL ALEM DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWY QQKPGKAPKLLIYNTNNLQTGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCLQHISRPRTFGQGTKVEIK SEQ ID NO:54 LC ALEM DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWY QQKPGKAPKLLIYNTNNLQTGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCLQHISRPRTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 55 VH 2F8 CDR1 GFTFSTYG 2F8 Petition 870260062878, dated 06 / 26 / 2026, pp. 145 / 410 136 / 194 SEQ ID NO: 56 VH 2F8 CDR2 IWDDGSYK SEQ ID NO: 57 VH 2F8 CDR3 ARDGITMVRGVMKDYFDY SEQ ID NO: 58 VH 2F8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMH WVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITM VRGVMKDYFDYWGQGTLVTVS S SEQ ID NO: 59 HC 2F8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMH WVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITM VRGVMKDYFDYWGQGTLVTVS SASTKGPS VFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 60 VL 2F8 CDR1 QDISSA VL 2F8 CDR2 DAS SEQ ID NO: 61 VL 2F8 CDR3 QQFNSYPLT SEQ ID NO: 62 VL 2F8 AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQ QKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO:63 LC 2F8 AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLT ISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 64 VH b12 CDR1 GYRFSNFV b12 SEQ ID NO: 65 VH b12 CDR2 INPYNGNK SEQ ID NO:66 VH b12 CDR3 ARVGPYSWDDSPQDNYYMDV SEQ ID NO: 67 VH b12 QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIH WVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVT FTADTSANTAYMELRSLRSADTAVYYCARVGPYS WDDSPQDNYYMDVWGKGTTVIVSS SEQ ID NO:68 HC b12 QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIH WVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVT FTADTSANTAYMELRSLRSADTAVYYCARVGPYS WDDSPQDNYYMDVWGKGTTVIVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO:69 VL b12 CDR1 HSIRSRR Petition 870260062878, dated 06 / 26 / 2026, pp. 146 / 410 137 / 194 VL b12 CDR2 GVS SEQ ID NO: 70 VL b12 CDR3 QVYGASSYT SEQ ID NO: 71 VL b12 EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWY QHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFT LTITRVEPEDFALYYCQVYGASSYTFGQGTKLERK SEQ ID NO:72 LC b12 EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWY QHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFT LTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:73 Fc IgG1m(f) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO:74 Fc IgG1m(z) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQ7GNSLQNFSGTSVMHEQAL Fc IgG1m(a) ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVTVP SS SLGTQTYICNVNHKPSNTKVDKPVPKSCDKTHT CPPCPAPELLGPGVDTKVFLIPKSCDKTH VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYCKVSNKALPA PIECTISCAKGQPREPQVTLPPSRDELTKNQVSLTC LVKGFYPSDIAVETTNGQVPENGSSDNYK FFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 76 Fc IgG1m(x) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSSSVVTV SLGTQTYICNVNHKPSNTKVDKPVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGVKEYKPACK PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEGLHYT QKSLSLSPGK SEQ ID NO: 73m FcGf) ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEWKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKKKKKKVSPACK PIEKTISCAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYCTTTPPVLDSDGS Petition 870260062878, of 26 / 06 / 2026, p. 147 / 410 138 / 194 FFLYSKLTVDKSRWQGGNVFSCSVMHGALHNHYT QKSLSLSPGK SEQ ID NO: 78 Fc IgG1m(f)E345K ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVSLQVSSVGLY SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVSVLTVLHQDWLVNGKEY PIEKTISKAKGQPRKPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 72A / Fc P396L ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFRTKPEVKDTTVLMIS VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYCKVSNAALPA PIACTISCAKGQPREPQVTLPPSREEMTKNQVSLT CLVKGFYPSDIAVETTPLQLDVPENNYK GSFFLYSKLTVDKSRWQGGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 80 Fc IgG1m(f)K326A / E333A / P396L / E430G ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALPA PIAKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNH YTQKSLSLSPGK SEQ ID NO:81 Fc IgG1m(f)K326A / E333A ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALPA PIAKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO:82 Fc IgG1m(f)K326A / E333A / E430G ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALPA PIAKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNH YTQKSLSLSPGK SEQ ID NO: 83 Fc IgG1m(f)K326A / P396L / E430GASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYT Petition 870260062878, de 26 / 06 / 2026, pág. 148 / 410 139 / 194 QKSLLSPGK SEQ ID NO: 84 Fc IgG1m(f)E333A / P396L / E 430G ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVTHVTHVRTVRTVP SSNTSLKVDCKTY CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYCKVSNKALPA PIAKTISKAKGQPREPQVYTLPPKPKDT CLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSD GSFFLYSKLTVDKSRWQGGNVFSCSVMHGALHNH YTQKSLSLSPGK SEQ ID NO: 85 Fc IgG1m(f)I253D / K322A ASTKGPCLSTVGFPLAPSALSK VTVSWNSGALTSGVHTFPAVLQSSGLYSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMDSRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVEEQYKTV NSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQGGNSLQNFSGTSVMHEQAL Fc IgG1m(f)K326W / E333S ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSVTVP SSSLGTQTYRTYICPYICNVNHKPSNTKVDKRVEKSVDTPCDPAKTHT VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNWALPA PISKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQGNVSLHCSVMHEALPA IgG1m(f)K326W / E333S / E430G ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKNHKNHKPSNTKVDKRTEKSCD CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYCKVSNWALPA PISKTISKAKGQPREPQVYTLPPPSTREMTK LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYT QKSLSLSPGK SEQ ID NO: 88 Fc IgG1m(f)S267E / H268F / S 320G / E434T / E434T / E434T / E434T / E434 ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVEFEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKVTNKALPA PIECTISCAKGQPREPQVTLPPSREMTKNQVSLTC LVKGFYPSDIAVETTNGQVPENGSSDNYK FFLYSKLTVDKSRWQGGNVFSCSVMHGALHNHYT QKSLSLSPGK SEQ ID NO:89 Fc IgG113F [Fc IgG1(f)mK274Q / N276K / Y300F / A339T / N384S / K392N / V397M / V422I] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQY NSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSF Petition 870260062878, dated 06 / 26 / 2026, pp. 149 / 410 140 / 194 FLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 90 Fc IgG113FE430G ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQY NSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSF FLYSKLTVDKSRWQQGNIFSCSVMHGALHNHYTQK SLSLSPGK SEQ ID NO: 91 VH BMS663513 CDR1 GGSFSGYY BMS663513 SEQ ID NO: 92 VH BMS663513 CDR2 INHGGYV SEQ ID NO: 93 VH BMS663513 CDR3 ARDYGPGNYDWYFDL SEQ ID NO: 94 VH BMS663513 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS WIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVD TSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDW YFDLWGRGTLVTVSS SEQ ID NO:95 VL BMS663513 CDR1 QSVSSY VL BMS663513 CDR2 DAS SEQ ID NO: 96 VL BMS663513 CDR3 QQRSNWPPALT SEQ ID NO: 97 VL BMS663513 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWY QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTIS SLEPEDFAVYYCQQRSNWPPALTFGGGTKVEI K SEQ ID NO: 98 VH CD134-SF2 CDR1 GYTFKDYT CD134SF2SEQ ID NO: 99 VH CD134-SF2 CDR2 IYPNNGGS SEQ ID NO: 100 VH CD134-SF2 CDR3 ARMGYHGPHLDFDV SEQ ID NO: 101 VH CD134-SF2 QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTM HWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRV TLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHG PHLDFDVWGQGTTVTVSS SEQ ID NO: 102 VL CD134-SF2 CDR1 QDVGAA VL CD134-SF2 CDR2 WAS SEQ ID NO: 103 VL CD134-SF2 CDR3 QQYINYPLT SEQ ID NO: 104 VL CD134-SF2 DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAW YQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIK SEQ ID NO: 105 VH CD137MOR7480 CDR1 GYSFSTYW CD137MOR748 0 SEQ ID NO: 106 VH CD137MOR7480 CDR2 IYPGDSYT SEQ ID NO: 107 VH CD137MOR7480 CDR3 ARGYGIFDY SEQ ID NO: VH CD137- EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISW Petição 870260062878, de 26 / 06 / 2026, pág. 150 / 410 141 / 194 108 MOR7480 VRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTIS ADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYW GQGTLVTVSS SEQ ID NO: 109 VL CD137MOR7480 CDR1 NIGDQY VL CD137MOR7480 CDR2 QDK SEQ ID NO: 110 VL CD137MOR7480 CDR3 ATYTGFGSLAV SEQ ID NO:111 VL CD137MOR7480 SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQ QKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLT ISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL SEQ ID NO: 112 VH CD40CP870893 CDR1 GYTFTGYY CD40CP87089 3 SEQ ID NO: 113 VH CD40CP870893 CDR2 INPDSGGT SEQ ID NO: 114 VH CD40CP870893 CDR3 ARDQPLGYCTNGVCSYFDY SEQ ID NO: 115 VH CD40CP870893 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM HWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR VTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPL GYCTNGVCSYFDYWGQGTLVTVSS SEQ ID NO: 116 VL CD40CP870893 CDR1 QGIYSW VL CD40CP870893 CDR2 TAS SEQ ID NO: 117 VL CD40CP870893 CDR3 QQANIFPLT SEQ ID NO: 118 VL CD40CP870893 DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWY QQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK SEQ ID NO: 119 VH CD40- SGN40 CDR1 GYSFTGYY CD40- SGN40 SEQ ID NO:120 VHCD40- SGN40 CDR2 VIPNAGGT SEQ ID NO: 121 VH CD40- SGN40 CDR3 AREGIYW SEQ ID NO: 122 VH CD40SGN40 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIH WVRQAPGKGVIGGFTGFTGFTGGFTGGYYYW SEQ ID NO: 122 VH LSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYW WGQGTLVTVSS SEQ ID NO: 123 VL CD40- SGN40 CDR1 QSLVHSNGNTF VL CD40SGN40 CDR2 TVS SEQ ID NO:124 VL CD40SGN40 CDR3 SQT SEQT IDL NO: 125 VWT NO: CD40SGN40 DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTF LHWYQQKPGKAPKLLIYTVSNRFSGVPSRFSGSGSG TDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKV EIK SEQ ID NO: VH CD95- GFNTNA CD95-TFNTNA Petition 870260062878, of 26 / 06 / 2026, p. 151 / 410 142 / 194 126 APO1 CDR1 APO1 SEQ ID NO: 127 VH CD95- APO1 CDR2 IRSKSNNYAT SEQ ID NO: 128 VH CD95- APO1 CDR3 VTDGYY SEQ ID NO: 129 VH CD95APO1 EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMN WVRQAPGKGLEWVARIRSKSNNYATYYAESVKDR FTISRDDSQSMLYLQMNNLKAEDTAMYYCVTDGY YWGQGTTLTVSS SEQ ID NO: 130 VL CD95- APO1 CDR1 ESVEYYGTSL VL CD95- APO1 CDR2 VAS SEQ ID NO: 131 VL CD95- APO1 CDR3 QQSTKVPWT SEQ ID NO: 132 VL CD95- APO1 DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSL MQWYQQKPGQPPKLLIYVASNVESGVPARFSGSGS GTDFSLNIHPVEEDDIAMYFCQQSTKVPWTFGGGTK LEIK SEQ ID NO: 133 VH CD95- HFE7A CDR1 GYTFTSYW CD95- HFE7A SEQ ID NO: 134 VH CD95HFE7A CDR2 IDPSDSYT SEQ ID NO: 135 VH CD95- HFE7A CDR3 ARNRDYSNNWYFDV SEQ ID NO: 136 VH CD95HFE7A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWM QWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKAT LTVDTS S STAYMQLS SLTSEDSAVYYCARNRDYSN NWYFDVWGTGTTVTVS S SEQ ID NO: 137 VL CD95- HFE7A CDR1 QSVDYDGDSY VL CD95HFE7A CDR2 AAS SEQ ID NO: 138 VL CD95HFE7A CDR3 QQSNEDPRT SEQ ID NO: 139 VL CD95HFE7A DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSG TDFTLNIHPVEEEDAATYYCQSNEDPRTFGGGTKL EIK SEQ ID NO: 140 VH DR4chCTB007 CDR1 GFNIKDTY DR4chCTB0 07 SEQ ID NO: 141 VH DR4chCTB007 CDR2 IDPANGNT SEQ ID NO: 142 VH DR4chCTB007 CDR3 AYYYVSNAWFTY SEQ ID NO: 143 VH DR4chCTB007 EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMH WVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATI TADTSSNTAYLQLSSLTSEDTAVYYCAYYYVSNAW FTYWGQGTLVTVSA SEQ ID NO: 144 VL DR4chCTB007 CDR1 ENIYSN VL DR4chCTB007 CDR2 AAT SEQ ID NO: 145 VL DR4chCTB007 CDR3 QHFWGTWT Petition: 870260062878, on June 26, 2026, page. 152 / 410 143 / 194 FAS-E09 CDR1 GASISANSYY FAS-E09 SEQ ID NO: 148 VH FAS-E09 CDR2 IAYRGNSNSGST SEQ ID NO: 149 VH FAS-E09 CDR3 ARRQLLDDGTGYQWAAFDV SEQ ID NO: 150 VH FAS-E09 QLQLQESGPGLVKPSETLSLTCTVSGASISANSYYG VWVRQSPGKGLEWVGSIAYRGNSNSGSTYYNPSLK SRATVSVDTSKNQVSLRLTSVTAADTALYYCARRQ LLDDGTGYQWAAFDVWGQGTMVTVSS SEQ ID NO: 151 VL FAS-E09 CDR1 SFNIGRYP VL FAS-E09 CDR2 YNN SEQ ID NO: 152 VL FAS-E09 CDR3 STWDDTLKGWV SEQ ID NO: 153 VL FAS-E09 QSVLTQPPSVSEAPRQTVTISCSGNSFNIGRYPVNW YQQLPGKAPKLLIYYNNLRFSGVSDRFSGSKSGTSA SLAIRDLLSEDEADYYCSTWDDTLKGWVFGGGTKV TVL SEQ ID NO: 154 VH GITR-36E5 CDR1 GFTFSSYA GITR36E5 SEQ ID NO:155 VH GITR-36E5 CDR2 ISSGGTT SEQ ID NO: 156 VH GITR-36E5 CDR3 ARVGGYYDSMDY SEQ ID NO: 157 VH GITR-36E5 EVNLVESGGGLVKPGGSLKVSCAASGFTFSSYAMS WVRQTPEKRLEWVASISSGGTTYYPDSVKGRFTISR DNARNILYLQMS SLRSEDTAMYYC ARVGGYYDSM DYWGQGISVTDSS SEQ ID NO: 158 VLGITR-36E5 CDR1 ESVDNYGVSF VL GITR-36E5 CDR2 AAS SEQ ID NO: 159 VL GITR-36E5 CDR3 QQTKEVTWT SEQ ID NO: 160 VL GITR-36E5 DIVLTQSPASLAVSLGQRATISCRASESVDNYGVSF MNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGS GTDFSLNIHPMEEDDTAMYFCQQTKEVTWTFGGGT KLEIK SEQ ID NO: 161 VH GITRINCAGNO1876 CDR1 GYTFTDYA GITRINCAG NO1876 SEQ ID NO: 162 VH GITRINCAGNO1876 CDR2 IRTYSGDV SEQ ID NO: 163 VH GITRINCAGNO1876 CDR3 AKSGTVRGFAY SEQ ID NO: 164 VH GITRINCAGNO1876 QVQLLQSGTELVRPGVSVKISCCKGSGYTFTDYAMY WVKQSHAKSLEWIGVIRTYSGDVTYNQKFKDKAT MTVDKSSSIAYMELARLSSEDSAIYYCAKSGTVRGF AYWGQGTLVTVSS SEQ ID NO: 165 VL GITRINCAGN01876 CDR1 QSLLNSGNQKNY VL GITRINCAGN01876 WAS Petition: 870260062878, on June 26, 2026, page. 153 / 410 144 / 194 CDR2 SEQ ID NO: 166 VL GITRINCAGN01876 CDR3 QNDYSYPYT SEQ ID NO: 167 VL GITRINCAGN01876 DIVMTQSPSSLTVTAGEKVIMSCKSSQSLLNSGNQK NYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGS GSGTDFTLTISSVQAEDLAVYHCQNDYSYPYTFGGG TKLEIK SEQ ID NO: 168 Fc IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK IgG2 humano SEQ ID NO:169 Fc IgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDT THTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNA KTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTT PPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMH EALHNRFTQKSLSLSPGK IgG3 HumanoSEQ ID NO: 170 Fc IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPS CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK human IgG4 SEQ ID NO: 171 FcRnECDHisB AP AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQY LSYNSLRGEAEPCGAWVWENQVSWYWEKETTDLR IKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSV PTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQR WQQQDKAANKELTFLLFSCPHRLREHLERGRGNLE WKEPPSMRLKARPSSPGFSVLTCSAFSFYPPELQLRF LRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDE HHYCCIVQHAGLAQPLRVELESPAKSSPGS SSHHHH HHPGGGLNDIFEAQKIEWHE FcRn Human SEQ ID NO: 172 B2M IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEV DLLKNGERIEKVEHSDLFSKDWSFYLLYYTEFTPT EKDEYACRVNHVTLSQPKIVKWDRDM B2M EXAMPLES Example 1: Antibody generation, production, and purification. Expression constructs for antibodies

[00461] For antibody expression, variable heavy chain (VH) and variable light chain (VL) sequences were prepared by synthesis of Petition 870260062878, dated 06 / 26 / 2026, page 154 / 410 145 / 194 genes (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) were cloned into pcDNA3.3 expression vectors (ThermoFisher Scientific, US) containing constant regions of IgG1 heavy chain (HC) and light chain (LC). Desired mutations were introduced either by gene synthesis or site-directed mutagenesis. Antibodies mentioned in this application have VH and VL sequences derived from previously described DR5 antibodies hDR5-01, hDR5-05 (WO2014 / 009358) and conatumumab (US7521048 B2 and WO2010 / 138725), DR4 antibody chCTB007 (US 2009 / 0136503), FAS antibodies 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), antibodies 4-1BB MOR7480 (WO 2012 / 032433) and BMS663513 (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 2F8 antibody (WO2002 / 100348).In some of the examples, the human IgG1 antibody b12, a specific antibody gp120, was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-23). Transient expression

[00462] Antibodies were expressed as mixtures of IgG1,K plasmid DNA encoding both heavy and light antibody chains that were transiently transfected into Expi293 cells (Life / Thermo Scientific, USA) using Expifectamine (Invitrogen, US) essentially as described by the manufacturer. Protein purification and analysis

[00463] Antibodies were purified by protein A affinity chromatography. Culture supernatants were filtered through a 0.20 pM dead-end filter and loaded onto 5 mL MabSelect SuRe columns (GE Healthcare), washed, and eluted with 0.02 M sodium citrate-NaOH, pH 3. Petition 870260062878, dated 06 / 26 / 2026, page 155 / 410 146 / 194 The eluates were loaded onto a HiPrep desalting column (GE Healthcare) immediately after purification, and the antibody buffer was changed to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B. Braun or Thermo Fisher). After buffer change, samples were sterilely filtered through 0.2 μm dead-end filters. Purified proteins were analyzed by various bioanalytical assays including capillary electrophoresis on sodium dodecyl sulfate-polyacrylamide (CE-SDS) gels and high-performance size exclusion chromatography (HP-SEC). Concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2–8°C. Generation of bispecific antibodies

[00464] Bispecific IgG1 antibodies were generated by Fab-arm exchange under controlled reduction conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific test conditions as described in WO2011 / 131746. The F405L and K409R mutations (EU numbering) were introduced into anti-DR5 IgG1 antibodies to create antibody pairs with complementary CH3 domains. The F405L mutation was introduced into IgG1b12-K326A / E333A / P396L / E430G and IgG1-CONA-C49W-K326W / E333S / E430G; The K409R mutation was introduced into IgG1-b12K326W / E333S / E430G and IgG1-hDR5-01-G56T-K326A / E333A / P396L / E430G. To generate bispecific antibodies, two complementary parental antibodies, each antibody at a final concentration of 0.5 mg / mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 pL PBS at 31°C for 5 hours.The reduction reaction was stopped by removing the reducing agent 2-MEA using centrifuge columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's protocol. The antibody buffer was changed to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun or Thermo). In this way the antibodies... Petition 870260062878, dated 06 / 26 / 2026, page 156 / 410 147 / 194 bispecific IgG1-hDR5-01-G56T-K326A / E333A / P396L / K409R / E430G x IgG1-b12-K326A / E333A / P396L / F405L / E430G said to be BsAb (hDR5-01G56T-K409Rxb12-F405L)-K326A / E333A / P396L / E430G and IgG1-CONAC49W-F405L-K326W / E333S / E430G x IgG1-b12-K409RK326W / E333S / E430G, said to be BsAb (IgG1-CONA-C49W-F405L x IgG1-b12-K409R)-K326W / E333S / E430G were generated. Example 2: Effect of combining E430G and K326A / E333A / P396L on the efficacy of agonistic anti-DR5 antibodies.

[00465] A feasibility test was performed to evaluate the effect of the Fc-Fc enhancing substitution combination of 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 IgG1-hDR5-01-G56T and IgG1-hDR-05 antibodies in DR5-positive BxPC3 cells (ATCC, CRL-1687). Cells were collected by trypsinization and passed through a cell filter. Cells were granulated by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium (RPMI 1640 with 25mM Hepes and L-Glutamine (Lonza catalog number BE12-115F) + 10% heat-inactivated bovine donor serum with iron (DBSI; Life Technologies catalog number 10371-029) + 50 U / mL Penicillin / Streptomycin (Pen / Strep; Lonza; catalog number DE17-603E) at a concentration of 0.5x10⁵ cells / mL.000 cells per well) were cultured in 96-well flat-bottomed polystyrene plates (Greiner Bio-One, catalog number 655182) and allowed to adhere overnight at 37°C. Then, 50 μL of a series of serial dilution antibody preparations (final concentration range of 0.0003 to 20.000 ng / mL in 4-fold dilutions) were added and incubated for 3 days at 37°C. As a negative and positive control, cells were incubated without antibody or with 5 μM of staurosporine (Sigma Aldrich, . Petition 870260062878, dated 06 / 26 / 2026, page 157 / 410 148 / 194 catalog number S6942), respectively. The viability of the cell cultures was determined using a CellTiter-Glo luminescent cell viability assay (Promega, catalog number G7571) which quantifies the ATP present, an indicator of metabolically active cells. From the kit, 20 μL of luciferin solution reagent were added per well and mixed by shaking the plate for 2 minutes at 500 rpm. The plates were then incubated for 1.5 hours at 37°C. 100 μL of supernatant were transferred to a blank OptiPlate-96 (Perkin Elmer, catalog number 6005299) and luminescence was measured using an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software.Figure 1 shows the percentage of viable cells, as calculated using the following formula: % viable cells = [luminescence in antibody sample - luminescence in staurosporine sample) / (no luminescence in antibody sample - luminescence in staurosporine sample)]*100.

[00466] Figure 1 shows that the combination of the enhancing Fc-Fc E430G substitution and the three K326A / E333A / P396L substitutions resulted in induction of killing efficacy for the anti-DR5 antibodies IgG1-hDR5-01-G56T (Figure 1A) and IgG1-hDR5-05 (Figure 1B) when tested as a single agent in an in vitro viability assay in adherent human pancreatic cancer BxPC-3 cells. In contrast, these antibodies did not show killing efficacy in these non-adherent BxPC-3 cells when only E430G or K326A / E333A / P396L were present. Also for the non-cross-blocking antibody combination IgG1hDR5-01-G56T + IgG1-hDR5-05, the introduction of the combined substitutions K326A / E333A / P396L / E430G resulted in more effective killing of non-adherent BxPC3 cells (Figure 1C).

[00467] These data show that the replacements Petition 870260062878, dated 06 / 26 / 2026, page 158 / 410 149 / 194 K326A / E333A / P396L / E430G induced strong agonistic activity for anti-DR5 antibodies in adherent BxPC-3 cells. Example 3: Efficacy of monovalent anti-DR5 antibody containing K326A / E333A / P396L / E430G.

[00468] A feasibility test was performed on human colon cancer cells COLO 205 and pancreatic cancer cells BxPC-3 to study the efficacy of monovalent anti-DR5 antibody containing K326A / E333A / P396L / E430G. The monovalent anti-DR5 antibody was generated by controlled Fab-arm exchange between IgG1-hDR5-01-G56T-K326A / E333A / P396L / K409R / E430G and IgG1b12-K326A / E333A / P396L / F405L / E430G as described in Example 1. The generated bispecific antibody, referred to as BsAb (hDR5-01-G56TK409Rxb12-F405L)-K326A / E333A / P396L / E430G, contains a DR5-specific arm and a non-specific arm against the HIV glycoprotein gp120, resulting in monovalent DR5 binding to DR5-positive human cancer cells. BxPC-3 cells were collected as described in Example 2. COLO 205 (ATCC, CCL-222) cells were collected by pooling the culture supernatant containing non-adherent cells and trypinized adherent COLO 205 cells.Cells were granulated by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium (RPMI 1640 with 25 mM Hepes and L-Glutamine + 10% heat-inactivated DBSI + 50 U / mL Pen / Strep at a concentration of 0.5 x 10⁵ cells / mL). 100 μL of the single cell suspensions (5,000 cells per well) were cultured in 96-well flat-bottomed polystyrene plates and allowed to adhere overnight at 37°C. Then, 50 μL of a series of serial dilution antibody preparations (final concentration range of 0.0024 to 10,000 ng / mL in 4-fold dilutions) were added and incubated for 3 days at 37°C. As a negative and positive control, cells were cells were incubated without antibody or with 5 μM of staurosporine, respectively. The viability of the cultured cells was determined at one point. Petition 870260062878, dated 06 / 26 / 2026, page 159 / 410 150 / 194 CellTiter-Glo luminescent cell viability test as described in Example 2.

[00469] Figure 2 shows that in the presence of the K326A / E333A / P396L / E430G mutations, the monovalent variant of IgG1-hDR5-01G56T could further induce cell death in human colon cancer cells (COLO 205) and pancreatic cancer cells (BxPC-3). Example 4: Effect of combining E430G and K326A / E333A, K326A / P396L or E333A / P396L on C1q binding and the efficacy of agonistic anti-DR5 antibodies.

[00470] A viability test was performed to study the effect of the Fc-Fc enhancement substitution combination of E430G with two of the three K326A / E333A / P396L substitutions on the agonistic activity of anti-DR5 IgG1-hDR5-01-G56T antibody in DR5-positive BxPC-3 and COLO 205 cells. As a reference, the combination of E430G with all three K326A / E333A / P396L substitutions as described in Example 2 was included in the experiment. The viability test was performed as described in Example 3. The viability of the cultured cells was determined in a CellTiter-Glo luminescent cell viability assay, as described in Example 2.

[00471] Figure 3 shows that the combination of the enhancing Fc-Fc E430G substitution and two K326A / E333A / P396L substitutions (E333A / P396L, K326A / E333A, or K326A / P396L) resulted in the induction of killing efficacy for the anti-DR5 antibody IgG1-hDR5-01-G56T when tested as a single agent in an in vitro viability assay in adherent human pancreatic BxPC-3 (Figure 3A) and colon COLO 205 (Figure 3B) cells. In contrast, no killing in these pre-adherent cancer cells was observed when only E430G was present. More efficient killing was observed when E430G was combined with all three K326A / E333A / P396L mutations. Petition 870260062878, dated 06 / 26 / 2026, page 160 / 410 151 / 194

[00472] A linkage ELISA was performed to evaluate the effect of different substitutions on C1q binding. Antibody samples purified from IgG-hDR5-01-G56T variants containing the E430G substitution in combination with the K326A / E333A, K326A / P396L substitutions,E333A / P396L or K326A / E333A / P396L were tested and compared with WT IgG-hDR5-01-G56T and IgG-hDR5-01-G56T-E430G. IgG-2F8I253D / K322A was used as a negative control for C1q binding. Coating of 96-well Microlon ELISA plates (Greiner catalog number 655092) was performed by overnight incubation at 4°C with 1 μg / mL of antibody samples in 100 μL PBS. The plates were washed and blocked for 1 hour at RT with 200 μL / well 0.5x PBS supplemented with 0.025% Tween 20 and 0.1% gelatin while shaking.With washes between incubations, the plates were incubated sequentially with 100 μL per well of a series of serial dilutions of purified C1q (Quidel catalog number A400; final concentration range of C1q 30 0.010 μg / mL in 3-fold dilutions) for 1 ha at 37°C, 100 μL rabbit anti-human C1q per well (DAKO, product # A0136, 1 / 4,000) for 1 ha RT, and with 100 μL / well porcine anti-rabbit IgG-HRP (DAKO, P0399, 1:10,000) as a detection antibody for 1 ha RT, and finally 100 μL / well of substrate with 1 mg / mL of 2,2'-azinobisic acid. (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche catalog number 11112 597001) for approximately 15 min at RT. The reaction was stopped by the addition of 100 μL of 2% oxalic acid. Absorbance was measured at 405 nm using a BioTek EL808 Microplate Reader (BioSPX). Logarithmically transformed data were analyzed by plotting variable-slope sigmoidal dose-response curves using GraphPad Prism software.

[00473] Figure 3C shows that the introduction of the enhancing Fc-Fc E430G substitution did not affect the apparent C1q binding affinity for 1 μg / mL of coated IgG1-hDR5-01-G56T antibody, while the Petition 870260062878, dated 06 / 26 / 2026, page 161 / 410 152 / 194 antibody variants containing the combination of the E430G substitution and the K326A / E333A, K326A / P396L, E333A / P396L or substitutions K326A / E333A / P396L showed enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G (Table 2). Table 2: EC50 values ​​of C1q binding for IgG1hDR5-01-G56T antibody variants (ELISA) IgG1-hDR5-01-G56T antibody variant (1 pg / mL) C1q binding EC50 (pg / mL) SD n Antibody variant versus WT1 Antibody variant versus E430G1 WT 18.8 9.9 6 Not applicable Not significant E430G 20.7 12.6 6 Not significant Not applicable K326A / E333A / P396L / E430G 1.3 0.2 3 p<0.05 p<0.05 K326A / E333A / E430G 0.8 0.1 3 p<0.05 p<0.05 K326A / P396L / E430G 0.6 0.1 3 p<0.05 p<0.05 E333A / P396L / E430G 1.9 0.8 3 p<0.05 p<0.05 One-way ANOVA p-value = 0.0022; Bonferroni post-hoc test Ab versus WT: p < 0.05 as indicated.

[00474] Together, these data showed that the combination of the enhancing Fc-Fc E430G substitution with K326A / E333A, K326A / P396L, E333A / P396L or K326A / E333A / P396L substitutions resulted in increased C1q binding and increased agonist activity of the anti-DR5 antibody IgG1-hDR5-01-G56T-E430G with only the enhancing Fc-Fc E430G mutation. Example 5: Effect of the combination of E430G and K326W / E333S on C1q binding and the efficacy of agonistic anti-DR5 antibodies.

[00475] A linkage ELISA was performed to evaluate the effect of K326A / E333A and K326W / E333S on C1q binding to an antibody containing the enhancing Fc-Fc E430G mutation. Purified antibody samples of IgG1-CONA-C49W variants containing the E430G substitution in combination with the K326A / E333A or K326W / E333S mutations were tested and compared with WT IgG1-CONA-C49W and IgG1-CONA-C49W-E430G. IgG1-CONA-C49W-K326W / E333S without the E430G substitution was also tested. IgG1-2F8-I253D / K322A was used as the negative control for C1q binding. The C1q linkage ELISA was performed on ELISA plates. Petition 870260062878, dated 06 / 26 / 2026, page 162 / 410 153 / 194 coated with 1 μg / mL of antibody as described in Example 4.

[00476] Strong enhancement of C1q binding by the introduction of the K326W / E333S substitution was confirmed when compared to the WT antibody (Figure 4A). In contrast, the introduction of the Fc-Fc E430G enhancing mutation did not affect the apparent C1q binding affinity for 1 μg / mL of coated IgG1-CONA-C49W antibody. Antibody variants containing the combination of the E430G substitution and the K326A / E333A or K326W / E333S substitution showed strongly enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56TE430G (Table 3). Table 3: EC50 values ​​of C1q binding for antibody variants IgG1-CONA-C49W (ELISA) _______________________ IgG1-CONA-C49W antibody variant (1 μg / mL) C1q binding EC50 (μg / mL) SD n Antibody variant versus WT1 Antibody variant versus E430G1 WT 15.2 12.2 3 Not applicable Not significant E430G 15.4 6.1 3 Not significant Not applicable K326W / E333S 0.3 0.1 3 p<0.05 p<0.05 K326A / E333A / E430G 0.8 0.3 3 p<0.05 p<0.05 K326W / E333S / E430G 0.5 0.1 3 p<0.05 p<0.05 'One-way ANOVA p-value = 0.0013; Bonferroni post-hoc test Ab versus WT: p < 0.05 as indicated.'

[00477] A viability test was performed to study the effect of the combination of Fc-Fc E430G mutation enhancing with C1q K326A / E333A or K326W / E333S linking substitutions on the agonistic activity of anti-DR5 IgG1-hDR5-01-G56T antibody in DR5-positive BxPC-3 and COLO 205 cells. The viability test was performed as described in Example 3. The viability of the cultured cells was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 2.

[00478] Figure 4B / C shows that the combination of the Fc-Fc E430G enhancing substitution and the two K326W / E333S mutations resulted in the induction of strong killing efficacy for the anti-DR5 IgG1-hDR5-01G56T antibody when tested as a single agent in an in vitro viability assay. Petition 870260062878, dated 06 / 26 / 2026, page 163 / 410 154 / 194 in adherent human pancreatic cancer cells BxPC-3 (Figure 4B) and colon cancer cells COLO 205 (Figure 4C). In contrast, the WT antibody and IgG1-hDR501-G56T-E430G showed no efficacy. The killing efficacy of IgG1-hDR501-G56T-K326W / E333S / E430G was better than that of IgG1-hDR501-G56T-K326A / E333A / E430G in both BxPC-3 and COLO 205 cancer cells.

[00479] Together, these data showed that the combination of the enhancing Fc-Fc E430G substitution with the K326A / E333A or K326W / E333S substitutions resulted in increased C1q binding and increased agonist activity of anti-DR5 IgG1-CONA-C49W-E430G antibody only with the E430G hexamerization enhancing mutation. Example 6: Effect of combining E430G with other Fc variants on C1q binding and the efficacy of agonistic anti-DR5 antibodies.

[00480] A C1q binding ELISA was performed to study the effect of C1q-binding substitutions S267E / H268F / S324T or the 113F variant of the chimeric IgG1 / IgG3 isotype on C1q binding to an antibody containing the enhancing Fc-Fc E430G substitution (Tammen et al., J Immunol. 2017). Purified antibody samples of IgG1hDR5-01-G56T variants with and without these substitutions were tested and compared with WT IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T-E430G. IgG1-2F8I253D / K322A was used as a negative control for C1q binding. The C1q binding ELISA was performed on ELISA plates coated with 1 μg / mL of antibody as described in Example 4.

[00481] Figure 5A shows that the introduction of the enhancing Fc-Fc E430G substitution did not affect the apparent C1q binding affinity for 1 μg / mL of coated IgG1-hDR5-01-G56T antibody. In contrast, the antibody variant containing the combination of the E430G substitution and the S267E / H268F / S324T substitutions showed strongly enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56T Petition 870260062878, dated 06 / 26 / 2026, page 164 / 410 155 / 194 E430G, while the introduction of the E430G substitution in the IgG113F-hDR5-01-G56T format variant resulted in slightly enhanced C1q binding compared to IgG1-hDR5-01-G56T and IgG1-hDR5-01-G56TE430G (Table 4). Table 4: EC50 values ​​of C1q binding for antibody variants IgG1-hDR5-01-G56T (ELISA) IgG1-hDR501-G56T antibody variant (1 μg / mL) EC50 binding to C1q (pg / mL) SD n Antibody variant versus WT1 Antibody variant versus E430G1 WT 15.2 12.2 3 Not applicable Not significant E430G 15.4 6.1 3 Not significant Not applicable S267E / H268F / S324T / E430G 0.5 0.1 3 p<0.05 p<0.05 IgG113F-E430G 11.4 3.9 3 Not significant Not significant 'One-way ANOVA p-value = 0.0013; Bonferroni post-hoc test Ab versus WT: p < 0.05 as indicated.'

[00482] A viability test was performed to study the effect of the combination of Fc-Fc E430G enhancing substitution with C1q-binding substitutions S267E / H268F / S324T (Moore et al., MAbs 2010) or the chimeric isotype variant IgG1 / IgG3113F (Natsume et al., Cancer Res. 2008) on the agonistic activity of anti-DR5 antibody IgG1-hDR5-01-G56T in DR5-positive BxPC-3 and COLO 205 cells. The viability test was performed as described in Example 3. The viability of the cultured cells was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 2.

[00483] Figure 5B / C shows that the combination of the enhancing Fc-Fc substitution E430G with the C1q-binding substitutions S267E / H268F / S324T resulted in the induction of killing efficacy for an anti-DR5 antibody IgG1-hDR5-01-G56T when tested as a single agent in an in vitro viability assay in adherent human pancreatic cancer cells BxPC-3 (Figure 5B) and colon cells COLO 205 (Figure 5C). When E430G was incorporated into the 113F variant of the chimeric IgG1 / IgG3 isotype of IgG1-hDR5-01-G56T, induction of killing efficacy was observed in COLO 205 (Figure 5C) and slightly in BxPC-3 where the Petition 870260062878, dated 06 / 26 / 2026, page 165 / 410 156 / 194 Agonistic activity was observed only at the highest antibody concentration tested (Figure 5B). However, the efficacy of these IgG1hDR5-01-G56T-S267E / H268F / S324T / E430G and IgG113F-hDR5-01-G56TE430G variants was significantly lower than for IgG1-hDR5-01-G56TK326W / E333S / E430G and IgG1-hDR5-01-G56T-K326A / E333A / P396L / E430G in both cell lines. As in the previous examples, the WT antibody and IgG1-hDR5-01-G56T-E430G showed no efficacy.

[00484] Together, these data showed that combining the enhancing Fc-Fc E430G substitution with the S267E / H268F / S324T substitutions resulted in strongly enhanced C1q binding and agonistic activity of the IgG1-hDR5-01-G56T-E430G anti-DR5 antibody with the E430G Fc-Fc enhancing substitution alone. The introduction of an E430G substitution in the IgG113F-hDR5-01-G56T format variant resulted in slightly enhanced C1q binding and agonistic activity of the antibody. Example 7: Summary of the effect of combining E430G with other Fc mutations and variants on the efficacy of agonistic anti-DR5 antibodies.

[00485] In previous examples, viability tests were described in which the effect on the agonistic activity of anti-DR5 antibody IgG1-hDR501-G56T was tested when the enhancing Fc-Fc E430G substitution was combined with another Fc region substitution or variants that were written to affect DR5 agonism or C1q binding. In this example, a summary is presented of all viability tests in adherent human pancreatic BxPC-3 cancer cells by representation and ranking of the percentage of viable cells after incubation for three days with 10 μg / mL of the indicated antibodies relative to WT IgG1-hDR5-01-G56T, which was shown to have no effects in Examples 2, 4, 5, and 6. Details of the viability tests in adherent BxPC-3 cells and the CellTiterGlo luminescent test are described in Example 2.

[00486] Figure 6 shows that the combination of substitution Petition 870260062878, dated 06 / 26 / 2026, page 166 / 410 157 / 194 enhancing Fc-Fc E430G with the C1q double-binding substitution K326W / E333S showed the most significant effect when compared with the WT IgG1-hDR5-01-G56T antibody after a three-day incubation period of adherent human BxPC-3 pancreatic cancer cells with 10 μg / mL of antibody in a complete culture medium containing heat-inactivated fetal calf serum. Also, combinations of E430G with K326A / E333A / P396L, E333A / P396L, and K326A / E333A resulted in significantly lower percentages of viable cells than the WT antibody.Other Fc variants that have been shown to enhance binding to C1q, such as chimeric IgG-113F S267E / H268F / S324T and IgG1 / IgG3, did not show significant induction of killing efficacy when combined with E430G in IgG1-hDR5-01-G56T in the experimental setup here with 10 μg / mL of antibody in adherent BxPC-3 cells, where IgG1-hDR5-01-G56T-E430G also did not result in the induction of killing efficacy when tested as a single agent. Example 8: Effect of C1q on the in vitro activity of agonistic anti-DR5 antibodies with an enhancing Fc-Fc substitution in combination with C1q-binding substitutions.

[00487] Previous examples suggested that enhanced binding to C1q contributes to improved agonist activity of the tested anti-DR5 antibodies containing the enhancing Fc-Fc E430G mutation. To test the effect of C1q, a viability assay was performed with IgG1-CONAK326A / E333A / P396L / E430G and IgG1-hDR5-01-G56T-K326W / E333S / E430G in WIL2-S SF cells in serum-free medium in the presence or absence of purified human C1q. WIL2-S SF cells were derived from WIL2-S B lymphoblasts (ATCC, CRL-8885) and adapted to grow under serum-free conditions in culture medium formulated by HyQ-ADCFMab (Perbio, catalog number SH30349) containing 50 U / mL Pen / Strep and 1 mM sodium pyruvate. WIL2-S SF cells in suspension were passed Petition 870260062878, dated 06 / 26 / 2026, page 167 / 410 158 / 194 cells were filtered through a cell filter, granulated by centrifugation for 5 minutes at 300xg, and resuspended in serum-free culture medium at a concentration of 0.5x10⁶ cells / mL. 100 μL of the single cell suspensions (50,000 cells per well) were cultured in 96-well flat-bottomed polystyrene plates (Greiner Bio-One, catalog number 655182). 25 μL of a series of serial dilution antibody preparations (final concentration range 0.0003 to 20.000 ng / mL in 4-fold dilutions) and 25 μL of purified C1q (Quidel, catalog number A400; final concentration 2.5 μg / mL) were added and incubated for 1 day at 37°C. As a negative and positive control, cells were incubated in medium without antibody or with 5 μM of staurosporine (Sigma Aldrich, catalog number S6942), respectively. Cell viability was determined by TO-PRO-3 staining.TO-PRO-3 is a cell-impermeable carbocyanine monomer dye that binds to double-stranded DNA. As such, TO-PRO-3 can be used as an indicator of cell death. All samples were transferred to 96-well U-bottom polystyrene plates (Greiner Bio-One, catalog number 650261) and centrifuged for 3 minutes at 300 xg before removing 70 μL of the supernatant. A mixture of 10 μL TO-PRO-3 (Invitrogen, catalog number T3605) + 20 μL TO-PRO-3 + 1980 μL PBS) was added before resuspending the cells 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).

[00488] Figure 7 shows that the addition of purified C1q to serum-free medium greatly enhanced the potency of both IgG1-CONAK326A / E333A / P396L / E430G (Figure 7A) and IgG1-hDR5-01-G56TK326W / E333S / E430G (Figure 7B) in WIL2-S SF cells. These data indicate that binding to C1q contributes to improved agonist activity of Petition 870260062878, dated 06 / 26 / 2026, p. 168 / 410 159 / 194 agonistic anti-DR5 antibodies containing the enhancing Fc-Fc E430G substitution in combination with C1q-binding K326A / E333A / P396L or K326W / E333S substitutions. Example 9: Effect of C1q on the in vitro agonistic activity of agonistic antiDR5 antibodies with an enhancing Fc-Fc substitution in combination with C1q-binding substitutions.

[00489] In Example 8, the effect of C1q on the efficacy of agonistic anti-DR5 antibodies was tested in a viability assay in WIL2S SF cells in serum-free medium with a series of antibody concentrations and a fixed concentration of C1q. In this example, the effect of a series of C1q concentrations was tested on the efficacy of IgG1-hDR5-01-G56T agonistic antibody variants with the Fc-Fc enhancing substitution (E430G) in combination with C1q-binding substitutions (K326A / E333S / P396L, K326W / E333S or K326A / E333A) in a viability assay in WIL2-S SF cells in serum-free medium. Viability assays were performed, essentially as described in Example 8, with a fixed antibody concentration of 2.5 μg / mL and a series of purified C1q concentrations in the final concentration range of 0.0002 to 2.5 μg / mL in 4-fold dilutions.

[00490] Figure 8 shows that the addition of purified C1q to serum-free medium enhanced the potency of anti-DR5 antibodies containing the Fc-Fc E430G enhancing substitution. All tested variants of IgG1-hDR5-01-G56T-E430G antibodies containing C1q-enhancing substitutions (K326A / E333S / P396L, K326W / E333S, or K326A / E333A) showed efficacy against WIL2-S SF cells in a C1q dose-dependent manner (Figure 8A). IgG1-hDR5-01-G56TK326W / E333S / E430G showed the highest efficacy of all tested antibodies and achieved maximum kill at a C1q concentration range starting from 0.16 μg / mL. These data indicate that C1q binding contributes to activity. Petition 870260062878, dated 06 / 26 / 2026, page 169 / 410 160 / 194 best of agonistic anti-DR5 antibodies containing the enhancing Fc-Fc E430G substitution in combination with the C1q-binding substitutions K326A / E333A / P396L, K326W / E333S or K326A / E333A. Also, the combination of antibody targeting the dual epitope IgG1-hDR5-01-G56TE430G + IgG1-hDR5-05-E430G showed a dose-dependent increase in C1q efficacy to kill WIL2S-SF cells in serum-free medium, reaching maximum kill at around 0.16 μg / mL C1q (Figure 8B). Example 10: Neutralizing effect of C1q on the in vitro agonistic activity of agonistic anti-DR5 antibodies with the enhancing Fc-Fc substitution in combination with C1q-binding substitutions.

[00491] The C1q requirement for the efficacy of anti-DR5 agonist IgG1-hDR5-01-G56T-K326W / E333S / E430G containing the enhancing Fc-Fc E430G substitution in combination with the K326W / E333S substitution for C1q binding was tested using an anti-C1q neutralizing antibody directed against the C1q globular head region, in a viability assay on WIL2-S SF cells in serum-free medium containing purified C1q. Similarly, the C1q neutralization effect was also tested in the same settings for the combination of dual-epitope-targeted antibody IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G. The viability test was performed essentially as described in Example 8. Briefly, WIL2-S SF cells were resuspended in serum-free culture medium at a concentration of 0.67 x 10⁶ cells / mL. 75 μL of the single cell suspensions (50.Cells (000 cells per well) were cultured in serum-free culture medium in 96-well flat-bottom polystyrene plates. Then, 25 μL of anti-DR5 antibody sample (final concentration of 2.5 μg / mL), 25 μL of purified C1q (final concentration of 0.01 μg / mL), and 25 μL of anti-C1q antibody sample (Sanquin, CLB / C1q-85 CAT # MW1828; final concentration of 10 μg / mL) were added and incubated for 1 day. Petition 870260062878, dated 06 / 26 / 2026, pp. 170 / 410 161 / 194 at 37°C. Cell viability was determined by TO-PRO-3 staining as described in Example 8.

[00492] The effect of adding purified C1q to serum-free medium to enhance the potency of anti-DR5 IgG1-hDR5-01-G56TK326W / E333S / E430G antibody as described in Example 9 was confirmed in this experiment (Figure 9A). Furthermore, this potency was diminished when the binding to supplemented C1q to the anti-DR5 antibody was neutralized by the presence of an excess of anti-C1q antibody (Figure 9A). These data illustrate that C1q binding is required for optimal activity of agonistic anti-DR5 antibodies containing the enhancing Fc-Fc E430G substitution in combination with the C1q-binding K326W / E333S substitution. C1q-dependent efficacy in killing WIL2S-SF cells was also confirmed for the antibody-targeting combination of the dual epitope IgG1-hDR501-G56T-E430G + IgG1-hDR5-05-E430G, showing enhanced efficacy when C1q was added to the serum-free medium, and neutralization of this effect by the presence of an excess of anti-C1q antibody (Figure 9B). Example 11: Solution-phase complement activation assay for antibodies with an enhancing Fc-Fc substitution in combination with C1q-binding substitutions.

[00493] Target-binding complement activation by antibody variants was determined by quantification of C4d, a marker for classical complement pathway activation, after antibodies were incubated in normal human serum (NHS). A three-step ELISA procedure was performed using the MicroVue C4d Enzyme Immuno Assay (Quidel, catalog number A0008) containing (1) a microtest plate coated with a mouse monoclonal antibody that specifically binds to C4d-containing activation fragments of human C4, (2) an HRP-conjugated goat anti-human C4d antibody, and (3) a chromogenic substrate. Internal controls and standards were supplemented with the kit and used as Petition 870260062878, dated 06 / 26 / 2026, page 171 / 410 162 / 194 described by the manufacturer's instructions. As a positive control, heat-bonded gamma globulin was prepared as follows. 1 mL aliquots in 1.5 mL vials of IVIG solution (60 mg / mL; Sanquin, catalog number 04H04H443A) were heated for 20 min at 63°C. The vials were pooled and diluted to 20 mg / mL with PBS and filtered through 0.22 μm pore surfactant-free cellulose acetate (SFCA) membrane syringe filters (Corning, catalog number 431219). ~0.2 mL aliquots were stored at 4°C. For antibody samples, 50 μL samples of a 100 μg / mL antibody preparation in 90% normal human serum (NHS, Sanquin M0008AC) were incubated in 96-well U-bottom polypropylene plates (Greiner Bio-One; catalog number 650261) for 1 hour at 37°C.Next, 5 μL of these samples were diluted 90x with Sample diluent, and 100 μL of the diluted samples were incubated per well for 30 minutes at room temperature while agitating on coated strips that had been pre-washed three times with 250 μL of washing solution. Then, the wells were washed five times with 250 μL of washing solution before incubating 50 μL of C4d conjugate per well for 30 minutes at RT while agitating. The wells were washed five times with 250 μL of washing solution before incubating 100 μL of substrate per well for 30 minutes at RT while agitating. The reactions were stopped by adding 50 μL of stopping solution per well, and the color intensity was measured spectrophotometrically at 405 nm using a BioTek EL808 Microplate Reader (BioSPX).

[00494] Positive control samples showed clearly enhanced C4d levels compared with negative control samples (Figure 10). In contrast, no clear enhancement of C4d levels was observed for all tested IgG1-hDR5-01-G56T antibody variants containing the enhancing Fc-Fc E430G substitution in combination with Petition 870260062878, dated 06 / 26 / 2026, page 172 / 410 163 / 194 C1q-binding substitutions K326W / E333S, K326A / E333A, or K326A / E333A / P396L were produced when incubated in NHS in the absence of target cells (Figure 10), while C4d was produced when HAGG positive controls, representing random immune complexes, and IgG1-CONA-RGY, representing fluid-phase IgG1 hexamers, were incubated in NHS. These data indicate that IgG1-hDR5-01-G56T antibody variants containing the enhancing Fc-Fc E430Gs substitution in combination with C1q-binding substitutions K326W / E333S, K326A / E333A, or K326A / E333A / P396L do not show hexamerization and target-independent complement activation in the solution phase. Example 12: Effect of combining E430G and K326W, E333S or K326W / E333S on C1q binding and efficacy of agonistic anti-DR5 antibodies.

[00495] A C1q binding ELISA was performed to evaluate the effect of introducing K326W, E333S, or K326W / E333S substitutions on C1q binding to IgG-CONA-C49W variants with or without the E430G substitution. IgG2F8-I253D / K322A was used as a negative control for C1q binding. The ELISA experiment was performed in 96-well plates coated with 1 pg / mL antibody that were tested for binding of different concentrations of purified C1q (range 0.010 - 30 μg / mL in 3-fold dilutions) as described in Example 4. Absorbance was measured at 405 nm and logarithm-transformed data were analyzed by setting variable-slope sigmoidal dose-response curves using GraphPad Prism software.Figure 11A,B shows that the introduction of the K326W, E333S, or K326W / E333S substitutions all resulted in increased C1q binding to a randomly immobilized antibody for both the anti-DR5 antibody IgG1-CONA-C49W and its variant IgG1-CONA-C49W-E430G, with the interaction enhancing Fc-Fc E430G and mutation enhancing hexamerization. IgG1-CONA-C49W-K326W / E333S / E430G showed the highest. Petition 870260062878, dated 06 / 26 / 2026, p. 173 / 410 164 / 194 apparent binding affinity to C1q of all antibody variants tested.

[00496] Binding of purified antibody variants to WIL2-S SF cell suspension was analyzed by flow cytometry. Cells were collected, counted, washed in PBS, and resuspended at 3.33 x 10⁶ cells / mL in culture medium. 30 pL cells (1 x 10⁵ cells per well) were pipetted into 96-well plates. Samples of 50 pL of antibody titration series (range 0.001 - 2.5 pg / mL final antibody concentrations in 3-fold dilutions) were added and incubated for 15 minutes at 37°C. Subsequently, 20 pL of purified C1q (2.5 pg / mL final concentration) were added and incubated for 45 minutes at 4°C. Next, 100 pL of FACS buffer (PBS + 0.1% (w / v) bovine serum albumin (BSA) + 0.02% (w / v) sodium azide) were added before washing the cells twice with 150 pL of FACS buffer.Washed cells were incubated for 30 minutes at 4°C with 50 pL of FITC-labeled rabbit anti-human C1q antibody (20 pg / mL final concentration; DAKO catalog number F0254). 100 pL of FACS buffer were added and cells were washed twice with FACS buffer. Cells were resuspended in 30 pL of FACS buffer and fluorescence was measured by flow cytometry using iQue Screener (IntelliCyt). Binding curves with a logarithmically transformed C1q concentration axis were analyzed using nonlinear regression analysis (sigmoidal dose-response with varying slope) using GraphPad Prism software. Figure 11C,D shows that introducing only the E333S or E430G substitution into anti-DR5 IgG1-CONA-C49W antibody had no effect on C1q binding to DR5-positive WIL2-S SF cells (Figure 11C).Introduction of the K326W mutation into anti-DR5 IgG1-CONA-C49W or IgG1-CONA-C49W-E430G antibodies resulted in increased C1q binding to WIL2-S SF opsonized cells. Petition 870260062878, dated 06 / 26 / 2026, p. 174 / 410 165 / 194 anti-DR5 antibody, consistent with the increased C1q binding observed for cells opsonized with IgG1-CONA-C49W-K326W / E333S / E430G (Figure 11C, D). Introduction of E333S into the anti-DR5 antibody IgG1-CONAC49W-E430G resulted in a modest increase in C1q binding to WIL2-S SF cells opsonized with antibody (Figure 11D). These flow cytometry data showing that IgG1-CONA-C49W-K326W / E333S / E430G bound to the cell showed the most avid C1q binding.

[00497] A viability test was performed to evaluate the effect of introducing K326W, E333S, or K326W / E333S into anti-DR5 IgG1CONA-C49W variants with or without the E430G substitution on DR5 agonist activity in WIL2-S SF cells. A one-day viability test was performed, essentially as described in Example 8. Briefly, 100 pL of cells in serum-free medium (50,000 cells / well) were pipetted into 96-well plates. 25 pL of purified C1q (final concentration 2.5 pg / mL) and 25 pL of antibody samples from a series of concentration dilutions (final concentration range 0.0003 - 20 pg / mL in 5-fold dilutions) were added and incubated at 37°C for 1 day. Cell viability was determined using the CellTiterGlo test as described in Example 2. Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer).Logarithmically transformed C1q concentration data were analyzed and plotted using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software. Figure 11E-G shows that introducing only the K326W, E333S, or E430G mutation into anti-DR5 IgG1-CONA-C49W antibody did not result in the induction of DR5 agonist activity in WIL2-S SF cells, while the K326W / E333S double mutation in IgG1-CONA-C49W resulted in the induction of DR5 agonist activity and partial death of WIL2-S SF cells (Figure 11E). The combination of the K326W mutation, the E333S mutation, or the K362W / E333S double mutation with the Fc-enhanced mutation. Petition 870260062878, dated 06 / 26 / 2026, page 175 / 410. 166 / 194 Fc E430G in the anti-DR5 IgG1-CONA-C49W antibody resulted in the induction of DR5 agonist activity, with K362W / E333S / E430G resulting in greater maximal cell death in WIL2-S SF cells (Figure 11F). Example 13: Effect of combining E430G and K326W, E333S or K326W / E333S on the C1q-de...

Claims

CLAIMS 1. Polypeptide, characterized in that it comprises an Fc region of an immunoglobulin and an antigen-binding region, wherein the Fc region comprises, a. an E430G substitution, and b. a K326W and an E333S substitution, wherein the positions correspond to human IgG1, in accordance with EU numbering, as set out in SEQ ID NO:

87.

2. Polypeptide according to claim 1, characterized in that it comprises one or more additional substitutions in the Fc region.

3. Polypeptide according to claim 2, characterized in that the additional substitution is selected from either K439E or S440K.

4. Polypeptide according to claim 2, characterized in that the additional substitution is selected from either F405L or K409R.

5. Polypeptide according to any one of claims 1 to 4, characterized in that the polypeptide is an antibody, monospecific antibody, bispecific antibody or multispecific antibody.

6. Polypeptide according to claim 5, characterized in that the polypeptide is a bispecific antibody comprising an Fc region comprising a first heavy chain and a first antigen-binding region, a second heavy chain and a second antigen-binding region, wherein a. said first heavy chain comprises an additional substitution at a position selected from the group consisting of K409, F405, T366, L368, K370, D399, Y407 Petition 870260062878, dated 06 / 26 / 2026, page 206 / 410 2 / 4 b. said second Fc heavy chain comprises an additional substitution at a position selected from the group consisting of K409, F405, T366, L368, K370, D399, Y407 c. wherein the additional substitution in said first heavy chain and in said second heavy chain is not in the same position.

7. Polypeptide according to claim 5 or 6, characterized in that a first heavy chain comprises a K409R or F405L substitution, a second heavy chain comprises a K409R or F405L substitution, wherein the substitution in said first Fc region and said second Fc region are not the same.

8. Polypeptide according to any one of claims 1 to 7, characterized in that the Fc region is a human IgG1 isotype.

9. Polypeptide according to any one of claims 1 to 8, characterized in that the polypeptide is a human antibody, humanized antibody or chimeric antibody.

10. Polypeptide according to any one of claims 1 to 9, characterized in that the antigen-binding region binds to a member of the tumor necrosis factor receptor (TNFR-SF) superfamily or G protein-coupled receptor (GPCR) superfamily.

11. Polypeptide according to any one of claims 1 to 10, characterized in that 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.

12. Method for increasing the in vitro agonist activity of a polypeptide as defined in any one of claims 1 to 11, the method characterized in that it comprises Petition 870260062878, dated 06 / 26 / 2026, p. 207 / 410 3 / 4 a. introducing an E430G substitution, and b. introducing a K326W and an E333S substitution, where the positions correspond to human IgG1, according to EU numbering, as set out in SEQ ID NO:

87.

13. Method for increasing the in vitro CDC activity of a polypeptide as defined in any one of claims 1 to 11, the method characterized in that it comprises a. introducing an E430G substitution, and b. introducing a K326W and an E333S substitution, wherein the position(s) correspond(s) to human IgG1, in accordance with EU numbering, as set out in SEQ ID NO:

87.

14. Method according to claim 12 or 13, characterized in that said method comprises introducing one or more additional substitutions in the Fc region.

15. Method according to any one of claims 12 to 14, characterized in that said method comprises introducing an additional substitution in the Fc region which is K439E or S440K.

16. Method according to any one of claims 12 to 15, characterized in that said method comprises introducing another substitution in the Fc region which is F405L or K409R.

17. Composition, characterized in that it comprises at least one polypeptide as defined in any one of claims 1 to 16.

18. Composition according to claim 17, characterized in that it comprises a first polypeptide and a second polypeptide, as defined in any one of claims 1 to 11.

19. Composition according to claims 17 to 18, characterized in that the composition is a pharmaceutical composition. Petition 870260062878, dated 06 / 26 / 2026, page 208 / 410 4 / 4 20. Polypeptide according to any one of claims 1 to 11 or a composition according to any one of claims 17 to 19, characterized in that they are for use as a medicament.

21. Polypeptide according to any one of claims 1 to 11, characterized in that it is for use in the treatment of cancer, autoimmune diseases, inflammatory diseases or infectious diseases.

22. Composition according to any one of claims 18 to 20, characterized in that it is for use in the treatment of cancer, autoimmune diseases, inflammatory diseases or infectious diseases.

23. Kit of parts, characterized in that it comprises a polypeptide as defined in any of the preceding claims, wherein said polypeptide is in one or more containers, such as small vials.

24. Kit of parts, characterized in that it comprises a composition as defined in any of the preceding claims, wherein said composition is in one or more containers, such as small bottles.

25. Use of a polypeptide or a composition as defined in any of claims 1 to 11 or 17 to 19, characterized in that it is for the manufacture of a medicament for the treatment of a disease.

26. Use according to claim 25, characterized in that the disease is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

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