Pharmaceutical composition, use of the pharmaceutical composition, kit in parts, and method for preparing a pharmaceutical composition.

Description

Pharmaceutical composition, use of the pharmaceutical composition, kit in parts, and method for preparing a pharmaceutical composition. FIELD OF THE INVENTION

[001] The present invention relates to pharmaceutical compositions of multispecific antibodies that bind to CD3 and CD30 and to the use of such pharmaceutical compositions. FUNDAMENTALS OF THE INVENTION

[002] CD30, also known as Ki-1 or TNFRSF8, is a 120 kD transmembrane glycoprotein receptor and part of the tumor necrosis factor (TNFR) receptor superfamily (Smith et al. (1994) Cell 76: 959-962). CD30 is a type I single-pass membrane protein with six cysteine-rich repeats in its extracellular domain (Durkop et al. (1992) Cell 68: 421-427). Furthermore, a soluble form of CD30 (sCD30) has been detected in the serum of patients with inflammatory diseases, including ulcerative colitis (UC) (Giacomelli et al. Clin Exp Immunol. 1998;111:532-5), and CD30-positive hematological malignancies (Josimovic-Alasevic et al. (1989) Eur J Immunol). sCD30 represents the cleavage product of the extracellular portion of CD30 by cell membrane-anchored metalloproteinases, such as TACE / ADAM17 and ADAM10 (Nagata et al., PNAS 2005; Hansen et al., FASEB, 2004).

[003] In normal tissues, CD30 expression is mainly restricted to activated T and B lymphocyte subgroups (Bowen et al. (1996) J Immunol. 156:442-9; Shanebeck et al. (1995) Eur J Immunol. 25:2147-53). CD30 expression has been detected in a variety of lymphoid neoplasms. Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) show high levels of CD30 expression. In particular, the vast majority of Reed-Sternberg (RS) cells, which are typically found in cHL, are CD30 positive (Frizzera et al. (1992) Semin. Diagn. Pathol. Petition 870250088813, dated 09 / 30 / 2025, page 251 / 496 / 205 9: 291-296).

[004] For example, the CD30-targeted antibody-drug conjugate brentuximab vedotin (BV; SGN-35) has been used for or suggested for use in the treatment of cancers such as hCL, ALCL, and CTCL (Younes et al. J Clin Oncol. 2012 Jun 20;30(18):2183-9; Pro et al. Blood. 2017 Dec 21;130(25):2709-2717; Shea et al. Curr Hematol Malig Rep. 2020 Feb;15(1):9-19). The tetravalent bispecific antibody CD30xCD16A, AFM13, is under development as an NK cell-mediated immunotherapy for hCL and CD30-positive lymphoma (Rothe et al. Blood. 2015 Jun 25;125(26):4024-31).

[005] In addition, CAR-T cell therapies targeted to CD30 antibodies are being developed for HLc and CD30-positive lymphoma. Other antibodies against CD30 have been described in WO2003059282 (Medarex), US8257706 (Seattle Genetics), US20100239571 (Seattle Genetics), WO2007040653 (US Government & Health), and WO20160177846 (Affimed).

[006] Pohl et al. (1993 Int. J. Cancer, 54: 820-827) describe the bispecific CD3xCD30 antibody, OKT-3 / HRS-3, which was generated by fusing hybridoma cells producing the monoclonal antibody against CD30, HRS-3, hybridoma cells producing the monoclonal antibody against CD3, OKT (hybridoma technology).

[007] WO2008119567 describes the generation and characterization of single-chain, bispecific molecules across species against CD30 and CD3.

[008] US20200095330 describes bispecific antibodies CD3xCD30, resulting from the chemical heteroconjugation of two anti-CD30 clones (designated 8D10 and 10C2) with anti-CD3 (Orthoclone OKT-3).

[009] However, none of these bispecific antibodies CD3xCD30 was tested in a clinical setting.

[0010] There is therefore a need for improved cancer therapy targeting CD30. There is an ongoing need for compounds Petition 870250088813, dated 09 / 30 / 2025, p. 252 / 496 / 205, directed towards CD30 antibodies that are effective, safe, have good manufacturability and / or a long shelf life. There is also a need for pharmaceutically acceptable antibody formulations for use in such therapy. SUMMARY OF THE INVENTION

[0011] The present invention relates to providing antibody pharmaceutical compositions involving T cells that bind to human and Cynomolgus monkey CD30 and CD3. A further object is to provide antibody pharmaceutical compositions whose formulations are stable over a wide range of antibody concentrations and / or temperatures. A further object is to provide antibody pharmaceutical compositions whose formulations are stable over a period of at least 3 months or even longer. The present invention further aims to provide antibody pharmaceutical formulations that are well tolerated by IV infusion as well as by subcutaneous administration. In addition, a bispecific antibody with a functionally inactive Fc main chain has been identified that is suitable for development into a pharmaceutical composition because of its excellent stability and solubility.

[0012] In one aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antibody comprising an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3; and a buffering agent, wherein the pH of the composition is from 4.0 to 8.0. Such pharmaceutical compositions have been found to provide surprisingly high antibody stability, such as thermal stability and storage stability, as well as a high degree of solubility.

[0013] In one embodiment, the pharmaceutical composition of the present invention comprises a multispecific antibody comprising: (i) a CD30 binding region comprising a first variable region of Petition 870250088813, dated 09 / 30 / 2025, page 253 / 496 / 205 heavy chain comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively.

[0014] In further aspects, the present invention relates to pharmaceutical compositions according to the present invention for use as a medicament, for example, in the treatment of cancer.

[0015] In yet another additional aspect, the present invention relates to a kit comprising a) the pharmaceutical composition described herein, b) a container for the pharmaceutical composition, and c) instructions for the use of said kit.

[0016] In a further aspect, the present invention relates to a method for preparing a pharmaceutical composition as defined herein, comprising the steps of mixing in water: a) a multispecific antibody, b) a buffering agent, optionally, c) a non-ionic excipient and, optionally, d) a surfactant; and adjusting the pH to 4.0-8.0. DESCRIPTION OF THE FIGURES

[0017] Figure 1A: Binding of bispecific CD3xCD30 antibodies and their bivalent monospecific equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of bsG1-huCD3FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxb12-FEAR, bsG1-b12FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR.

[0018] Figure 1B: Binding of bispecific CD3xCD30 antibodies and their monospecific bivalent equivalents against CD3 and CD30 to cells Petition 870250088813, dated 09 / 30 / 2025, pages 254 / 496 / 205 SU-DHL-1 or HDML-2. Dose-dependent binding of bsG1-huCD3FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30hAC10-FEAR.

[0019] Figure 1C: Binding of bispecific CD3xCD30 antibodies and their bivalent monospecific equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30HRS-3-FEAR.

[0020] Figure 1D: Binding of bispecific CD3xCD30 antibodies and their monospecific bivalent equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of BsIgG1-huCD3FEALxCD30-HeFi-I-FEAR and bsG1-huCD3-FEALxb12-FEAR.

[0021] Figure 1E: Binding of bispecific CD3xCD30 antibodies and their bivalent monospecific equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of bsIgG1-huCD3FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30T405-FEAR.

[0022] Figure 1F: Binding of bispecific CD3xCD30 antibodies and their bivalent monospecific equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of bsIgG1-huCD3FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30T105-FEAR.

[0023] Figure 1G: Binding of bispecific CD3xCD30 antibodies and their bivalent monospecific equivalents against CD3 and CD30 to SU-DHL-1 or HDML-2 cells. Dose-dependent binding of bsIgG1-huCD3FEALxCD30-T408-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30T408-FEAR.

[0024] Figure 1H: Binding of bispecific CD3xCD30 antibodies and their monospecific bivalent equivalents against CD3 and CD30 to cells Petition 870250088813, dated 09 / 30 / 2025, pages 255 / 496 / 205 SU-DHL-1 or HDLM-2. Dose-dependent binding of bsIgG1-huCD3FEALxCD30-T215-FEAR, bsG1-huCD3-FEALxb12-FEAR and IgG1-CD30T215-FEAR to SU-DHL-1 cells (left panels) or HDLM-2 cells (right panels).

[0025] Figure 1I: Binding of bispecific CD3xCD30 antibodies and monospecific antibodies against CD30 to SU-DHL-1 (left panels) or HDLM-2 (right panels) cells at a concentration of 1.11 pg / mL. The antibody clones used for the CD30 arm are indicated on the x-axis. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0026] Figure 2A: Binding of bsG1-huCD3xCD30-MDX060 to HL and LAGC cell lines. The binding of bsG1-huCD3xCD30MDX060 to HDLM-2 (HL) cells was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060 and the monospecific antibodies IgG1-CD30-MDX060, IgG1-huCD3, and IgG1-12 were included as controls. All antibodies contained the FEAL and / or FERR mutations for Fc silencing and DuoBody® technology in the Fc domain as indicated. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0027] Figure 2B: Binding of bsG1-huCD3xCD30-MDX060 to HL and LAGC cell lines. The binding of bsG1-huCD3xCD30MDX060 to L-428 (HL) was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060 and the monospecific antibodies IgG1-CD30-MDX060, IgG1-huCD3 and IgG1-12 were included as controls. All antibodies contained the FEAL and / or FERR mutations for Fc silencing and DuoBody® technology in the Fc domain as indicated. The data shown are intensity values ​​of Petition 870250088813, dated 09 / 30 / 2025, page 256 / 496 / 205 mean fluorescence (MFI), as determined by flow cytometry, from a representative experiment.

[0028] Figure 2C: Binding of bsG1-huCD3xCD30-MDX060 to HL and LAGC cell lines. The binding of bsG1-huCD3xCD30MDX060 to DEL (LAGC) was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060 and the monospecific antibodies IgG1-CD30-MDX060, IgG1-huCD3, and IgG1-12 were included as controls. All antibodies contained the FEAL and / or FERR mutations for Fc silencing and DuoBody® technology in the Fc domain as indicated. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0029] Figure 2D: Binding of bsG1-huCD3xCD30-MDX060 to HL and LAGC cell lines. The binding of bsG1-huCD3xCD30MDX060 to KI-JK (LAGC) was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060 and the monospecific antibodies IgG1-CD30-MDX060, IgG1-huCD3, and IgG1-12 were included as controls. All antibodies contained the FEAL and / or FERR mutations for Fc silencing and DuoBody® technology in the Fc domain as indicated. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0030] Figure 3A: Induction of in vitro cytotoxicity by bispecific CD3xCD30 antibodies in SU-DHL-1 or HDLM-2 cells. Bispecific CD3xCD30 antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The antibodies Petition 870250088813, dated 09 / 30 / 2025, page 257 / 496 / 205. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0031] Figure 3B: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1CD30-hAC10-FEAR. The bsG1-huCD3-FEALxb12-FEAR antibody was included as a control in all experiments. The data shown are percentages of viable cells; The data for each graph were obtained from a representative experiment.

[0032] Figure 3C: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1CD30-HRS-3-FEAR. The bsG1-huCD3-FEALxb12-FEAR antibody was included as a control in all experiments. The data shown are percentages of viable cells; The data for each graph were obtained from Petition 870250088813, dated 09 / 30 / 2025, page 258 / 496 / 205 a representative experiment.

[0033] 3D Figure: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi-I-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are percentages of viable cells; The data for each graph were obtained from a representative experiment.

[0034] Figure 3E: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1CD30-T405-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are percentages of viable cells; The data for each graph were obtained from a representative experiment.

[0035] Figure 3F: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDML-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, cells Petition 870250088813, dated 09 / 30 / 2025, page 259 / 496 / 205 SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1CD30-T105-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0036] Figure 3G: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1CD30-T408-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are percentages of viable cells; The data for each graph were obtained from a representative experiment.

[0037] Figure 3H: In vitro cytotoxicity induction by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 (left panels) or HDLM-2 (right panels) as target cells, and T cells (CD3-positive ADCC type IV effector cells; Clean Cells, Montaigu, France) as effector cells. The following antibodies were tested: bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included. Petition 870250088813, dated 09 / 30 / 2025, page 260 / 496 / 205 as a control in all experiments. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0038] Figure 4A: Induction of T cell-mediated cytotoxicity and T cell proliferation in vitro by CD3xCD30 bispecific antibodies in various LAGC and LH cell lines. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using different LAGC and LH cell lines as target cells and purified T cells. The CD3xCD30 bispecific antibodies contained the huCD3-FEAL Fab arm or the huCD3-H101G-FEAL variant, which has lower affinity for CD3, and the CD30-specific MDX060-FEAR Fab arm. IgG1huCD3 and IgG1-b12 were included as controls. Data shown are percentages of viable cells; data for each graph were obtained from a representative experiment.

[0039] Figure 4B: Induction of T cell-mediated cytotoxicity and T cell proliferation in vitro by CD3xCD30 bispecific antibodies in various LAGC and LH cell lines. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using different LAGC and LH cell lines, such as type IV effector ADCC cells. The CD3xCD30 bispecific antibodies contained the huCD3FEAL Fab arm or the huCD3-H101G-FEAL variant, which has lower affinity for CD3, and the CD30-specific MDX060-FEAR Fab arm. IgG1-huCD3 and IgG1-b12 were included as controls. Data shown are percentages of viable cells; data for each graph were obtained from a representative experiment.

[0040] Figure 4C: Induction of T cell-mediated cytotoxicity and T cell proliferation in vitro by CD3xCD30 bispecific antibodies in various LAGC and HL cell lines. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using Petition 870250088813, dated 09 / 30 / 2025, page 261 / 496 / 205 different cell lines of LAGC and LH, as effector cells. The bispecific CD3xCD30 antibodies contained the huCD3-FEAL Fab arm or the huCD3-H101G-FEAL variant, which has lower affinity for CD3, and the bsG1-b12-FEALxCD30-MDX060-FEAR arm and IgG1-CD30MDX060-FEAR was included as a control. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0041] Figure 4D: The number of CFSE-positive cells was evaluated as a measure of absolute T cell counts in cytotoxicity assays, using HDLM-2 cells (left panel) or NCEB-1 cells (right panel) as target cells.

[0042] Figure 5A: Binding of bispecific CD3xCD30 antibodies to complete human and Cynomolgus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent antibodies against CD30 to wild-type Expi293F cells. Cells were incubated with increasing concentrations of the following antibodies: IgG1-CD30-MDX060FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101GFEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR. Data are presented as mean fluorescence intensity (MFI) values, as determined by flow cytometry of two technical replicates.

[0043] Figure 5B: Binding of bispecific CD3xCD30 antibodies to human and Cynomolgus monkey whole CD30 transfected into Expi293F cells. Binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30. The cells were incubated with increasing concentrations of the following antibodies: IgG1-CD30-MDX060-FEAR, bsG1-huCD3FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101G-FEALxCD30MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR and bsG1-huCD3 Petition 870250088813, dated 09 / 30 / 2025, page 262 / 496 / 205 FEALxb12-FEAR. Data are presented as mean fluorescence intensity (MFI) values, as determined by flow cytometry using two technical replicates.

[0044] Figure 5C: Binding of bispecific CD3xCD30 antibodies to human and Cynomolgus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent antibodies against CD30 to Cynomolgus monkey CD30. Cells were incubated with increasing concentrations of the following antibodies: IgG1-CD30-MDX060FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101GFEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR. Data are presented as mean fluorescence intensity (MFI) values, as determined by flow cytometry of two technical replicates.

[0045] Figure 4D: Binding of IgG1-CD30-MDX060FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3FEALxb12-FEAR antibodies to human T cells or Cynomolgus monkey T cells. Data are presented as mean fluorescence intensity (MFI) values, as determined by flow cytometry from a representative experiment.

[0046] Figure 6A: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsG1huCD3-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, of Petition 870250088813, dated 09 / 30 / 2025, page 263 / 496 / 205 a representative experiment.

[0047] Figure 6B: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsG1huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0048] Figure 6C: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsG1huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0049] Figure 6D: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsIgG1, huCD3-FEALxCD30-HeFi-I-FEAR, and IgG1-CD30-HeFi-I-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0050] Figure 6E: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsIgG1huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0051] Figure 6F: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsIgG1huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0052] Figure 6G: Binding of bispecific CD3xCD30 antibodies to transfected human and Rhesus monkey complete CD30 cells Petition 870250088813, dated 09 / 30 / 2025, pages 265 / 496 / 205 Expi293F. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with whole human CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsIgG1huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0053] Figure 6H: Binding of bispecific CD3xCD30 antibodies to human and Rhesus monkey whole CD30 transfected into Expi293F cells. The binding of monovalent and bivalent antibodies against CD30 to Expi293F cells transiently transfected with human whole CD30 (left panels) or Rhesus monkey CD30 (right panels) was evaluated by flow cytometry. The following antibodies were evaluated: bsIgG1huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in the experiment. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0054] Figure 7A: Thermostability of antibodies with different non-activating mutations, as determined by differential scanning fluorimetry (DSF). The stability of the protein conformation at increasing temperatures was evaluated in duplicate by DSF. The melting curves of the following antibody are shown: IgG1-CD30-MDX060-FEAR at pH 7.4.

[0055] Figure 7B: Thermostability of antibodies with different non-activating mutations, as determined by differential scanning fluorimetry (DSF). The stability of the protein conformation at increasing temperatures was evaluated in duplicate by DSF. The curves of Petition 870250088813, dated 09 / 30 / 2025, page 266 / 496 / 205 fusion of the following antibody: IgG1-CD30-MDX060-FERR at pH 7.4.

[0056] Figure 7C: Thermostability of antibodies with different non-activating mutations, as determined by differential scanning fluorimetry (DSF). The stability of the protein conformation at increasing temperatures was evaluated in duplicate by DSF. The melting curves of the following antibody are shown: IgG1-huCD3-FEAL at pH 7.4.

[0057] Figure 7D: Thermostability of antibodies with different non-activating mutations, as determined by differential scanning fluorimetry (DSF). The stability of the protein conformation at increasing temperatures was evaluated in duplicate by DSF. The melting curves of the following antibody are shown: BsG1-huCD3-FEALxCD30-MDX060-FERR at pH 7.4.

[0058] Figure 8A: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LH cell lines. The binding of bsG1-huCD3-FEALxCD30MDX060-FERR to LH cell lines, L-540, was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3-FEALxb12FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0059] Figure 8B: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LH cell lines. The binding of bsG1-huCD3-FEALxCD30MDX060-FERR to KM-H2 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, of Petition 870250088813, dated 09 / 30 / 2025, page 267 / 496 / 205 a representative experiment.

[0060] Figure 8C: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LH cell lines. The binding of bsG1-huCD3-FEALxCD30MDX060-FERR to L-1236 cell lines (C) was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3-FEALxb12FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0061] Figure 9A: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LAGC cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the LAGC and SUP-M2 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0062] Figure 9B: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LAGC cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to LAGC and DL-40 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0063] Figure 9C: Binding of bsG1-huCD3-FEALxCD30-MDX060 Petition 870250088813, dated 09 / 30 / 2025, pages 268 / 496 / 205 FERR binding to LAGC cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to LAGC and KARPAS-299 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3FEAL, and IgG1-12-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0064] Figure 9D: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LAGC cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the LAGC and L-82 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0065] Figure 9E: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to LAGC cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the LAGC and SR-786 cell lines was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0066] Figure 10A: Binding of bsG1-huCD3-FEALxCD30MDX060-FERR to NHL cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the SUP-T1 (TLL) cell line was evaluated. Petition 870250088813, dated 09 / 30 / 2025, page 269 / 496 / 205 by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0067] Figure 10B: Binding of bsG1-huCD3-FEALxCD30-MDX060FERR to NHL cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the JVM-2 (MCL) cell line was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0068] Figure 10C: Figure 10B: Binding of bsG1-huCD3FEALxCD30-MDX060-FERR to NHL cell lines. The binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to the HH (LCCT) cell line was evaluated by flow cytometry. The bispecific antibodies bsG1huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3FEAL and IgG1-12-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0069] Figure 10D: Binding of bsG1-huCD3-FEALxCD30MDX060-FERR to NHL cell lines. The binding of bsG1-huCD3FEALxCD30-MDX060-FERR to the NCEB-1 (MCL) cell line was evaluated by flow cytometry. The bispecific antibodies bsG1-huCD3FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and the antibodies Petition 870250088813, dated 09 / 30 / 2025, page 270 / 496 / 205. Monospecific IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG112-FEAL were included as controls. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0070] Figure 11A: Binding of bsG1-huCD3-FEALxCD30MDX060-FERR to human full-color CD30 for HEK293 cells. The binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HEK293 cells transiently transfected with human full-color CD30 was evaluated by flow cytometry. bsG1-huCD3-FEALxb12-FERR was included as a negative control. The data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0071] Figure 11B: Binding of transfected Rhesus monkey to HEK293 cells. The binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to transiently transfected HEK293 cells with CD30 from Rhesus monkey (B) was evaluated by flow cytometry. bsG1-huCD3-FEALxb12-FERR was included as a negative control. Data shown are mean fluorescence intensity (MFI) values, as determined by flow cytometry, from a representative experiment.

[0072] Figure 12A: Simultaneous binding of bsG1-huCD3FEALxCD30-MDX060-FERR to T cells and tumor cells. The simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to tumor cells and fluorescence-labeled naive T cells was studied by flow cytometry. Double-positive events are shown as the percentage of total viable cells in the presence of 6*10-5a 10 pg / mL of bsG1-huCD3FEALxCD30-MDX060-FERR or the control antibodies bsG1-huCD3FEALxb12-FEAR, bsG1-b12-FEALxCD30-MDX060-FERR, or IgG1-b12FEAL. The percentage of double-positive events in samples incubated without antibody is indicated by the dashed line. Petition 870250088813, dated 09 / 30 / 2025, page 271 / 496 / 205

[0073] Figure 12B: Example of the gating strategy for double-positive cells in samples incubated with 0.12 μg / mL of bsG1-huCD3FEALxCD30-MDX060-FERR.

[0074] Figure 13A: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and T cells purified from leukocyte layers of healthy human donors as effector cells. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060FERR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30T105-FEAR, bsIgG1-huCD3-FEALxCD30-T405-FEAR, bsIgG1-huCD3FEALxCD30-T408-FEAR and bsIgG1-huCD3-FEALxCD30-T215-FEAR. IC50 values ​​for cytotoxicity of Karpas-299 cells by CD3xCD30 antibodies.

[0075] Figure 13B: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and T cells purified from leukocyte layers of healthy human donors as effector cells. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060FERR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30T105-FEAR, bsIgG1-huCD3-FEALxCD30-T405-FEAR, bsIgG1-huCD3FEALxCD30-T408-FEAR and bsIgG1-huCD3-FEALxCD30-T215-FEAR. Petition 870250088813, dated 09 / 30 / 2025, page 272 / 496 / 205 Percentage of maximum cytotoxicity of Karpas-299 cells by antibodies in test.

[0076] Figure 13C: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and T cells purified from leukocyte layers of healthy human donors as effector cells. In this assay, CD25 expression was evaluated in CD4+ cells as a measure of T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR and bsIgG1-huCD3FEALxCD30-T215-FEAR. EC50 values ​​for induction of CD25 expression in CD4+ T cells by CD3xCD30 antibodies.The data were obtained from two independent experiments performed with T cells obtained from 6 different donors. The statistical values ​​represent the result of a Wilcoxon signed-rank test between the indicated clone and bsG1-huCD3-FEALxCD30-MDX060FERR. NS: Not significant, *: p <0.05.

[0077] Figure 13D: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and purified T cells from healthy human donor buffy coats as effector cells. In this assay, CD25 expression was evaluated in CD8+ cells as a Petition 870250088813, dated 09 / 30 / 2025, page. 273 / 496 / 205 measured for T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR and bsIgG1-huCD3FEALxCD30-T215-FEAR. EC50 values ​​for induction of CD25 expression in CD8+ T cells by CD3xCD30 antibodies. Data were obtained from two independent experiments performed with T cells obtained from 6 different donors. Statistical values ​​represent the result of a Wilcoxon signed-rank test between the indicated clone and bsG1-huCD3-FEALxCD30-MDX060FERR. NS: Not significant, *: p <0.05.

[0078] Figure 13E: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and T cells purified from leukocyte layers of healthy human donors as effector cells. In this assay, PD-1 expression was evaluated in CD4+ cells as a measure of T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR and bsIgG1-huCD3FEALxCD30-T215-FEAR. EC50 values ​​for induction of PD-1 expression in CD4+ T cells by CD3xCD30 antibodies.The data were obtained from two independent experiments that were performed with T cells. Petition 870250088813, dated 09 / 30 / 2025, pp. 274 / 496 / 205 obtained from 6 different donors. The statistical values ​​represent the result of a Wilcoxon signed-rank test between the indicated clone and bsG1-huCD3-FEALxCD30-MDX060FERR. NS: Not significant, *: p <0.05.

[0079] Figure 13F: Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in an in vitro cytotoxicity assay using the CD30-positive tumor cell line Karpas-299 as target cells and T cells purified from leukocyte layers of healthy human donors as effector cells. In this assay, PD-1 expression was evaluated in CD8+ cells as a measure of T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR and bsIgG1-huCD3FEALxCD30-T215-FEAR. EC50 values ​​for induction of PD-1 expression in CD8+ T cells by CD3xCD30 antibodies.The data were obtained from two independent experiments performed with T cells obtained from 6 different donors. The statistical values ​​represent the result of a Wilcoxon signed-rank test between the indicated clone and bsG1-huCD3-FEALxCD30-MDX060FERR. NS: Not significant, *: p <0.05.

[0080] Figure 14A: In vitro T-cell mediated cytotoxicity of cell lines by bsG1-huCD3-FEALxCD30-MDX060-FERR. The dose-dependent T-cell mediated cytotoxicity of bsG1-huCD3FEALxCD30-MDX060-FERR was tested in vitro using the L-428 tumor cell line as target cells and purified T cells as Petition 870250088813, dated 09 / 30 / 2025, page 275 / 496 / 205 effector cells. IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR and IgG1b12-FEAL were included as controls. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0081] Figure 14B: In vitro T-cell mediated cytotoxicity of bsG1-huCD3-FEALxCD30-MDX060-FERR. Dose-dependent T-cell mediated cytotoxicity by bsG1-huCD3FEALxCD30-MDX060-FERR was tested in vitro using the KM-H2 tumor cell line as target cells and purified T cells as effector cells. IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, and IgG1b12-FEAL were included as controls. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0082] Figure 14C: In vitro T-cell mediated cytotoxicity of bsG1-huCD3-FEALxCD30-MDX060-FERR. Dose-dependent T-cell mediated cytotoxicity by bsG1-huCD3FEALxCD30-MDX060-FERR was tested in vitro using the SUP-M2 tumor cell line as target cells and purified T cells as effector cells. IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, and IgG1b12-FEAL were included as controls. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0083] Figure 14D: In vitro T-cell mediated cytotoxicity of bsG1-huCD3-FEALxCD30-MDX060-FERR cell lines. Dose-dependent T-cell mediated cytotoxicity of bsG1-huCD3FEALxCD30-MDX060-FERR was tested in vitro using the cell line of Petition 870250088813, dated 09 / 30 / 2025, page 276 / 496 / 205 KI-JK tumor cells as target cells and purified T cells as effector cells. IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, and IgG1b12-FEAL were included as controls. The data shown are percentages of viable cells; the data for each graph were obtained from a representative experiment.

[0084] Figure 15A: In vitro T cell proliferation by bsG1huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell proliferation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. CD4+ T cells with diluted Celltrace Violet staining were gated, and the expansion index, as a measure of T cell proliferation, was calculated using the FlowJo proliferation modeling tool.

[0085] Figure 15B: In vitro T cell proliferation by bsG1huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell proliferation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. CD8+ T cells with diluted Celltrace Violet staining were gated, and the expansion index, as a measure of T cell proliferation, was calculated using the FlowJo proliferation modeling tool.

[0086] Figure 15C: In vitro T cell proliferation by bsG1huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell proliferation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. CD4+ T cells with diluted Celltrace Violet staining were gated, and the expansion index, as a measure of T cell proliferation, was calculated using the FlowJo proliferation modeling tool.

[0087] Figure 15D: In vitro T cell proliferation by bsG1huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. A Petition 870250088813, dated 09 / 30 / 2025, page 277 / 496 / 205 T cell proliferation was evaluated in T cell-mediated cytotoxicity assays, using KI-JK as target cells. CD8+ T cells with diluted Celltrace Violet staining were gated and the expansion index, as a measure of T cell proliferation, was calculated using the FlowJo proliferation modeling tool.

[0088] Figure 16A: Expression of CD69, a marker of T cell activation, in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of CD69, a marker of T cell activation, was evaluated in CD4+ T cells.

[0089] Figure 16B: Expression of CD69, a marker of CD69 T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of CD69, a marker of T cell activation, was evaluated in CD8+ T cells.

[0090] Figure 16C: Expression of CD69, a marker of T cell activation, in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of CD69, a marker of T cell activation, was evaluated in CD4+ T cells.

[0091] Figure 16D: Expression of CD69, a marker of T cell activation, in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of CD69, a marker of T cell activation, was evaluated in CD8+ T cells. Petition 870250088813, dated 09 / 30 / 2025, pages 278 / 496 / 205

[0092] Figure 17A: Expression of CD25, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of CD25, a marker of T cell activation, was evaluated in CD4+ T cells.

[0093] Figure 17B: Expression of CD25, a marker of in vitro cell activation by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of CD25, a marker of T cell activation, was evaluated in CD8+ T cells.

[0094] Figure 17C: Expression of CD25, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of CD25, a marker of T cell activation, was evaluated in CD4+ T cells.

[0095] Figure 17D: Expression of CD25, a marker of in vitro cell activation by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of CD25, a marker of T cell activation, was evaluated in CD8+ T cells.

[0096] Figure 18A: Expression of PD-1, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of the T cell activation marker, PD-1, was evaluated in cells Petition 870250088813, dated 09 / 30 / 2025, page 279 / 496 / 205 T CD4+.

[0097] Figure 18B: Expression of PD-1, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the L-428 cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using L-428 as target cells. The expression of the T cell activation marker, PD-1, was evaluated in CD8+ T cells.

[0098] Figure 18C: Expression of PD-1, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of the T cell activation marker, PD-1, was evaluated in CD4+ T cells.

[0099] Figure 18D: Expression of PD-1, a marker of T cell activation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in the KI-JK cell line. T cell activation was evaluated in T cell-mediated cytotoxicity assays using KI-JK as target cells. The expression of the T cell activation marker, PD-1, was evaluated in CD8+ T cells.

[00100] Figure 19A: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The CD40 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00101] Figure 19B: In vitro production of Granzyme B by bsG1huCD3-FEALxCD30-MDX060-FERR. The concentration of Granzyme B was evaluated in supernatants that were collected during the experiments of Petition 870250088813, dated 09 / 30 / 2025, page 280 / 496 / 205, investigates T-cell mediated cytotoxicity in vitro using L-428 as target cells. The concentration of Granzyme B is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00102] Figure 19C: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-10 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00103] Figure 19D: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-12 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00104] Figure 19E: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-1e cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00105] Figure 19F: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-2 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00106] Figure 19G: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IFNγ cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-β12-FEAL.

[00107] Figure 19H: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-13 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00108] Figure 19I: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-4 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00109] Figure 19J: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-6 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with Petition 870250088813, dated 09 / 30 / 2025, page 282 / 496 / 205 different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00110] Figure 19K: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IL-8 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00111] Figure 19L: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The IP-10 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00112] Figure 19M: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The MCP-1 cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL.

[00113] Figure 19N: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The concentration of PDL-1 cytokine was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or Petition 870250088813, dated 09 / 30 / 2025, pages 283 / 496 / 205 of the IgG1-b12-FEAL control antibody.

[00114] Figure 19O: In vitro cytokine production by bsG1-huCD3FEALxCD30-MDX060-FERR. The TNFα cytokine concentration was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. The cytokine concentration is shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-β12-FEAL.

[00115] Figure 20A: T cell-mediated cytotoxicity and T cell proliferation in vitro by bsG1-huCD3-FEALxCD30-MDX060FERR in varying effector-to-target ratios. Dose-dependent T cell-mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060FERR was tested in vitro using L-428 tumor cells as target (T) cells and purified T cells as effector cells in E:T ratios of 1:1, 2:1, 4:1, or 8:1. bsG1-huCD3-FEALxb12-FERR was included as a control antibody.

[00116] Figure 20B: The data shown are percentages of viable cells, obtained from a representative experiment. The percentage of CD4+ T cells with diluted Celltrace Violet staining is shown as a measure of proliferating T cells.

[00117] Figure 20C: The data shown are percentages of viable cells, obtained from a representative experiment. The percentage of CD8+ T cells with diluted Celltrace Violet staining is shown as a measure of proliferating T cells.

[00118] Figure 21A: Kinetics of T cell-mediated cytotoxicity and T cell proliferation in vitro by bsG1-huCD3-FEALxCD30-MDX060FERR. The kinetics of T cell-mediated cytotoxicity by bsG1-huCD3FEALxCD30-MDX060-FERR were tested in vitro using L-428 tumor cells as target cells and purified T cells as effector cells in Petition 870250088813, dated 09 / 30 / 2025, pp. 284 / 496 / 205, an E:T ratio of 4:1. Cytotoxicity was assessed post-24 hours, 48 ​​hours, and 72 hours. bsG1-huCD3-FEALxb12-FERR was included as a control antibody.

[00119] Figure 21B: The data shown are percentages of viable cells, obtained from a representative experiment. CD4+ T cells with diluted Celltrace Violet staining were gated and the expansion index, as a measure of T cell proliferation in cytotoxicity assays, was calculated using the FlowJo proliferation modeling tool.

[00120] Figure 21C: The data shown are percentages of viable cells, obtained from a representative experiment. CD8+ T cells with diluted Celltrace Violet staining were gated and the expansion index, as a measure of T cell proliferation in cytotoxicity assays, was calculated using the FlowJo proliferation modeling tool.

[00121] Figure 22A: In vitro T-cell mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060-FERR and correlation with CD30 expression level. The correlation between maximum T-cell mediated death was evaluated in eight tumor cell lines. A Spearman rank correlation test (GraphPad Prism software) was used to assess the statistical significance of the degree of correlation between T-cell mediated cytotoxicity (maximum death or IC50) and CD30 expression.

[00122] Figure 22B: In vitro T-cell mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060-FERR and correlation with CD30 expression level. The correlation between IC50 concentrations of T-cell mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060-FERR versus CD30 expression levels was evaluated in eight tumor cell lines. A Spearman rank correlation test (GraphPad Prism software) was used to assess the statistical significance of the correlation coefficient. Petition 870250088813, dated 09 / 30 / 2025, pp. 285 / 496 / 205 between T-cell mediated cytotoxicity (maximum death or IC50) and CD30 expression.

[00123] Figure 23A: Fratricide of T cells activated by bsG1huCD3-FEALxCD30-MDX060-FERR. T cells isolated from healthy donors were stimulated with anti-CD3 (OKT-3) 1 pg / mL, anti-CD28 1 pg / mL and IL-15 0.025 pg / mL for 4 days. Upon confirmation of activation (upregulation of CD25), the T cells were incubated with increasing concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR, bsIgG1-b12FEALxCD30-MDX060-FERR, bsIgG1-huCD3-FEALxb12-FERR or IgG1b12-FEAL for 48 hours. The percentage of CD25+ cells in CD4+ or CD8+ T cells, 72 and 96 hours after stimulation with anti-CD3, anti-CD28 and IL-15, was determined by flow cytometry.

[00124] Figure 23B: Fratricide of T cells activated by bsG1huCD3-FEALxCD30-MDX060-FERR. T cells isolated from healthy donors were stimulated with anti-CD3 (OKT-3) 1 pg / mL, anti-CD28 1 pg / mL and IL-15 0.025 pg / mL for 4 days. T cell fratricide, induced by BsG1huCD3-FEALxCD30-MDX060-FERR, of activated CD30+ T cells was measured as the percentage of T cells in each condition relative to the number of viable T cells in the condition without the addition of any antibodies. The percentage of CD30+ cells in CD4+ or CD8+ T cells, 72 and 96 hours after stimulation with anti-CD3, anti-CD28 and IL-15, was determined by flow cytometry.

[00125] Figure 23C: T-cell fratricide is shown as the percentage of T-cell survival relative to the condition without the addition of any antibodies. Data for a representative T-cell donor are shown.

[00126] Figure 24A: Soluble CD30 in the supernatant of CD30+ cell cultures and its interference with the antitumor activity of BsG1huCD3-FEALxCD30-MDX060-FERR. The concentration of soluble CD30 Petition 870250088813, dated 09 / 30 / 2025, page 286 / 496 / 205 (sCD30) in the supernatant of different hematologic tumor cell lines, as measured by ELISA, is shown. The data shown are percentages of viable cells, obtained from a representative experiment.

[00127] Figure 24B: The correlation between sCD30 concentrations and the number of CD30 molecules on the cell surface, as determined by quantitative flow cytometry (Biocytex human IgG calibrator kit), in different hematologic tumor cell lines, was evaluated by Spearman's rank correlation test (GraphPad Prism software). The data shown are percentages of viable cells, obtained from a representative experiment.

[00128] Figure 24C: BsG1-huCD3-FEALxCD30-MDX060-FERR was tested in an in vitro cytotoxicity assay using the CD30-positive, DEL, LAGC tumor cell line as target cells, and T cells isolated from healthy donors as effector cells. The following control antibodies were included: BsG1-b12-FEALxCD30-MDX060FERR, IgG1-CD30-MDX060-FERR, BsG1-huCD3-FEALxb12-FERR, IgG1huCD30-FEAL, and IgG1-b12-FEAL. The data shown are percentages of viable cells, obtained from a representative experiment.

[00129] Figure 25A: Induction of T cell-mediated cytotoxicity and ex vivo T cell proliferation by bsG1-huCD3-FEALxCD30-MDX060FERR in L-428 tumor cells. bsG1-huCD3-FEALxCD30-MDX060FERR was tested in an ex vivo cytotoxicity assay using L-428 tumor cells as target cells and primary, patient-derived T cells as effector cells. Peripheral blood mononuclear cells (PBMCs) derived from patients with Hodgkin lymphoma (HL), acute myeloid leukemia (AML), and peripheral T-cell lymphoma (PTCL) were used as a source of T cells to assess CD30-dependent tumor cell death. IgG1-b12-FEAL was included as a control. Data shown are percentage of viable target cells. Petition 870250088813, dated 09 / 30 / 2025, pages 287 / 496 / 205

[00130] Figure 25B: T cell activation was assessed by upregulation of CD25 / CD69 markers in CD4+ / CD8+ T cells in the PBMC subset, indicated as a percentage of positive cells.

[00131] Figure 25C: T cell activation was assessed by upregulation of CD69 markers in CD4+ / CD8+ T cells in the PBMC subset, indicated as a percentage of positive cells.

[00132] Figure 25D: T cell activation was assessed by upregulation of PD-1 markers in CD4+ / CD8+ T cells in the PBMC subset, indicated as a percentage of positive cells.

[00133] Figure 26A: Plasma concentrations of BsG1-huCD3FEALxCD30-MDX060-FERR after intravenous injection in SCID mice. SCID mice received a single IV injection dose of 10 pg (0.5 mg / kg) or 100 pg (5 mg / kg) of BsG1-huCD3-FEALxCD30MDX060-FERR. Total human IgG concentration was determined by ELISA, and mean human IgG1 concentrations were plotted over time. The dashed line indicates the estimated clearance rate based on the standard volume of distribution of a regular unbound human IgG1 in mice.

[00134] Figure 26B: Plasma concentrations of BsG1-huCD3FEALxCD30-MDX060-FERR after intravenous injection in SCID mice. SCID mice received a single IV injection dose of 10 pg (0.5 mg / kg) or 100 pg (5 mg / kg) of BsG1-huCD3-FEALxCD30MDX060-FERR. The mean clearance rate was plotted for dose levels of 0.5 or 5 mg / kg. The dashed line indicates the estimated clearance rate based on the standard volume of distribution of a regular unbound human IgG1 in mice.

[00135] Figure 27A: Binding of C1q to membrane-bound bsG1-huCD3-FEALxCD30MDX060-FERR. Binding of C1q to activated human CD8+ T cells opsonized with bsG1-huCD3-FEALxCD30-MDX060 Petition 870250088813, dated 09 / 30 / 2025, pp. 288 / 496 / 205 FERR or NCEB-1 CD30+ cells were evaluated by flow cytometry using a FITC-labeled rabbit anti-C1q antibody. Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-huCD3-FEAL, or the positive control antibody IgG1-CD52-E430G to human CD8+ T cells stimulated with anti-CD3 / CD28 microspheres in the presence of normal human serum as a C1q source. The data shown represent the geometric mean fluorescence intensity (gMFI) ± SD of replicate wells from a representative experiment. The data shown are gMFI from a representative experiment.

[00136] Figure 27B: Binding of C1q to membrane-bound bsG1-huCD3-FEALxCD30MDX060-FERR. Binding of C1q to activated human CD8+ T cells opsonized with bsG1-huCD3-FEALxCD30-MDX060FERR or CD30+ NCEB-1 cells was assessed by flow cytometry using a FITC-labeled rabbit anti-C1q antibody. (B) Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-CD30-MDX060FERR, or the positive control antibody IgG1-7D8-E430G to CD30+ NCEB-1 cells in the presence of normal human serum as a C1q source. Data shown are gMFI from a representative experiment. DETAILED DESCRIPTION OF THE INVENTION Definitions

[00137] The term “antibody,” in this descriptive report, is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that is capable of specifically binding to an antigen under typical physiological conditions and / or specific tumor conditions with a significant half-life period, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least less than about 8 hours, at least about 12 hours, at least about 24 hours or more, at least Petition 870250088813, dated 09 / 30 / 2025, p. 289 / 496 / 205 approximately 48 hours or more, at least approximately 3, 4, 5, 6, 7 or more days, etc., or any other functionally relevant defined period (such as sufficient time to induce, promote, intensify and / or modulate a physiological response associated with antibody binding to antigen and / or sufficient time for the antibody to be internalized). An antibody comprises a binding region (or binding domain which may be used here, both having the same meaning) that can interact with an antigen, a binding region comprising variable regions of heavy and light chains of an immunoglobulin molecule or similar.Antibodies may comprise constant regions of antibodies (Abs) that can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation.

[00138] In the context of the present invention, the term “antibody” includes a monoclonal antibody (mAb), an antibody-like polypeptide, a chimeric antibody, a human antibody, a humanized antibody, as well as an “antibody fragment” or a “fragment thereof” that retains the ability to specifically bind to antigen (antigen-binding fragment) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant DNA technology. The term “antibody” includes bi-, tri-, or multispecific antibodies and / or antibodies with additional modifications, for example, antibody-drug conjugates and / or antibodies with modifications in the Fc domain of IgG. An antibody, as defined according to the present invention, may possess any isotype or have no isotype (for example, an scFv antibody), unless the present description is otherwise limited.

[00139] It has been shown that the antigen-binding function of an antibody can be performed by fragments of a complete antibody. Petition 870250088813, dated 09 / 30 / 2025, pages 290 / 496 / 205 Examples of linking fragments encompassed by the term “antibody” include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the variable light chain (VL) domain, variable heavy chain (VH) domain, constant light chain (CL) region and domains of the constant heavy chain 1 (CH1) region, or a monovalent antibody as described in WO 2007 / 059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544546 (1989)), which consists essentially of a VH domain and is also called a domain antibody (Holt et al; Trends Biotechnol-. 2003 Nov;21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther.2005 Jan;5(1):111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although encoded by separate genes, the two domains of the Fv fragment, VL and VH, can be joined using recombinant methods, by a synthetic linker that allows them to be prepared as a single protein chain in which the pair of VL and VH regions form monovalent molecules (known as single-chain antibodies or single-chain Fv (scFv)), see, for example, Revets et al; Expert Opin Biol Ther. 2005 Jan;5(1):111-24 and Bird et al., Science 242, 423426 (1988). Such single-chain antibodies are encompassed by the term antibody unless otherwise noted or clearly indicated by the context. Although they are generally included within the meaning of antibody, such fragments collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility.These and other useful antibody fragments in the context of the present invention are discussed in more detail below. Petition 870250088813, dated 09 / 30 / 2025, pages 291 / 496 / 205

[00140] An antibody, either as the final product or as an intermediate to generate, for example, a bispecific antibody through controlled Fab arm exchange (cFEA), can be produced in and collected from different expression or production systems in vitro or ex vivo, for example, from recombinantly modified host cells, from hybridomas or systems that make use of cell extracts in support of in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[00141] The term “immunoglobulin heavy chain” or “heavy chain of an immunoglobulin,” in this descriptive report, is intended to refer to one of the heavy chains of an immunoglobulin. A heavy chain is typically composed of a variable heavy chain region (here abbreviated as VH) and a constant heavy chain region (here abbreviated as CH) that defines the immunoglobulin isotype. The constant heavy chain region of an IgG is typically composed of three domains, CH1, CH2, and CH3. The term “immunoglobulin,” in this descriptive report, is intended to refer 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 has been well distinguished (see, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY)).(1989)). Within the immunoglobulin structure, the two heavy chains are interconnected by disulfide bonds in the so-called “hinge region”. Similarly to the heavy chains, each light chain is typically composed of several regions; a variable light chain region (here abbreviated as VL) and a constant light chain region. The constant light chain region is typically composed of one domain, CL. Furthermore, the VH and VL regions can be further subdivided into regions. Petition 870250088813, dated 09 / 30 / 2025, pp. 292 / 496 / 205 of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed 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 amine terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[00142] When used herein, the terms “half molecule,” “Fab arm,” and “arm” refer to a heavy-chain-light chain pair. When describing a bispecific antibody as comprising a half-molecule antibody “derived from” a first antibody and a half-molecule antibody “derived from” a second antibody, the term “derived from” indicates that the bispecific antibody was generated by recombination, by any known method, of said half molecules of each of said first and said second antibodies resulting in the bispecific antibody. In this context, “recombination” is not intended to be limited to any particular method and thus includes all methods for producing bispecific antibodies described herein, including, for example, recombination by half-molecule exchange, as well as recombination at the nucleic acid level and / or by co-expression of two half molecules in the same cells.

[00143] The terms “first” and “second,” when used herein in the context of an antibody or domain or region thereof, are intended simply for ease of reference and are not intended to indicate a particular relative location or similar.

[00144] The term “antigen-binding region” or “binding region,” in this descriptive report, refers to a region of an antibody that is capable of binding to the antigen. The antigen can be any molecule, such as a polypeptide, protein, polysaccharide, or a combination thereof. Petition 870250088813, dated 09 / 30 / 2025, pages 293 / 496 / 205 Antigens can, for example, be presented on a cell, bacterium, or virion. The terms “antigen” and “target” may, unless contradicted by the context, be used interchangeably in the context of the present invention. The terms “antigen-binding region” and “antigen-binding site” may, unless contradicted by the context, be used interchangeably in the context of the present invention.

[00145] The term “Kd” (M), in this descriptive report, refers to the equilibrium dissociation constant of a given antibody-antigen interaction, and is obtained by dividing kd by ka. Kd may also be referred to as “binding affinity”.

[00146] The term “kd” (s-1), in this descriptive report, refers to the dissociation rate constant of a given antibody-antigen interaction. This value is also referred to as the koff or off-rate value.

[00147] The term “ka” (M-1x s-1), in this descriptive report, refers to the association rate constant of a given antibody-antigen interaction. This value is also referred to as the kon or on-rate value.

[00148] The term “binding”, in this descriptive report, refers to the binding of an antibody to a predetermined antigen or target, typically with a binding affinity corresponding to a Kd equal to or less than 1E-6M, for example, equal to or less than 5E-7M, equal to or less than 1E-7M, equal to or less than 5E-8M, equal to or less than 1E-8M, equal to or less than 5E-9M, equal to or less than 1E-9M, equal to or less than 1E-10M or equal to or less than 1E-11M when determined by bilayer interferometry, using the antibody as the ligand and the antigen as the analyte, and binds to the predetermined antigen with an affinity corresponding to a Kd that is at least ten times smaller, such as at least 100 times smaller, for example, at least 1,000 times smaller, or at least 10,000 times smaller, for example, at least 100,000 times lower than its affinity for binding to a non-specific antigen Petition 870250088813, dated 09 / 30 / 2025, pp. 294 / 496 / 205 (e.g., BSA, casein) different from the predetermined antigen or a closely related antigen.

[00149] The term “freeze-thaw cycle”, in this descriptive report, describes a process of freezing the pharmaceutical composition at lower temperatures, such as -75°C, followed by thawing at room temperature.

[00150] The term “kd”, in this descriptive report, refers to the diffusion interaction parameter.

[00151] The term “B22”, in this descriptive report, refers to the second virial coefficient.

[00152] The term “stable,” in this descriptive report, refers to the ability of a pharmaceutical product to retain its physical stability and / or chemical stability and / or biological activity during storage. Thus, when stable, i.e., being a stable pharmaceutical composition, the pharmaceutical composition is suitable for pharmaceutical use even after storage for a given period of time. The quality of the antibody varies over time when exposed to factors such as temperature, agitation, and freeze-thaw cycles. A stable pharmaceutical composition retains or only insignificantly affects its physical, chemical, and biological properties throughout its shelf life. As an example, the percentage of change in physical, chemical, and / or biological properties from production to use would be limited.In one embodiment, the percentage of monomer, as measured by HP-SEC, would not decrease by more than 5%, such as 4%, such as 3%, such as 2%, when maintained at 5°C for at least 12 months. In a further embodiment, the percentage of intact IgG would not decrease by more than 10%, such as 5%, when maintained at 5°C for at least 12 months. In another embodiment, the acidic forms, as measured by icIEF, would not increase by more than 20%, such as more than 15%, such as more than 10%, when maintained at 5°C for at least 12 months. Petition 870250088813, dated 09 / 30 / 2025, pp. 295 / 496 / 205

[00153] The term “buffer” in this descriptive report indicates a pharmaceutically acceptable buffer. The term “buffer” encompasses those agents that maintain the pH value of a solution, for example, within an acceptable range and includes, among others, acetate, histidine, TRIS® (tris(hydroxymethyl)aminomethane), citrate, succinate, glycolate and the like. In general, the “buffer” in this descriptive report has a pKa and buffering capacity suitable for the pH range of about 4 to about 8, such as about 5 to about 6.5, such as about 5.5 to 6.

[00154] The term “non-ionic excipient” in this descriptive report indicates a pharmaceutically acceptable non-ionic excipient. As an example, the non-ionic excipient may be a sugar or a sugar alcohol. Sugars and polyols may also be referred to as monosaccharides, disaccharides, and polysaccharides. Examples include, but are not limited to, sucrose, glucose, dextrose, trehalose, mannitol, and sorbitol.

[00155] A “surfactant,” in this descriptive report, is a compound that is typically used in pharmaceutical formulations to prevent drug adsorption onto surfaces and / or aggregation. Additionally, surfactants reduce the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, an exemplary surfactant can significantly reduce surface tension when present in very low concentrations (e.g., 5% w / w or less, such as 3% w / w or less, such as 1% w / w or less). Surfactants are amphiphilic, meaning they are generally composed of hydrophilic and hydrophobic or lipophilic groups, thus being able to form micelles or similar self-assembled structures in aqueous solutions.Surfactants known for pharmaceutical use include glyceryl monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate, and tricaprylin (anionic surfactants); benzalkonium chloride, citrimide, cetylpyridinium chloride, and phospholipids. Petition 870250088813, dated 09 / 30 / 2025, pp. 296 / 496 / 205 (cationic surfactants); and alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene fatty acid esters, polyoxyethylene stearates, polyoxyl hydrostearate, polyoxyglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sucrose palmitate, sorbitan esters, sucrose stearate, tricaprilin and TPGS (non-ionic and zwitterionic surfactants).

[00156] A “diluent” of interest herein is one that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and useful for preparing dilutions of the pharmaceutical composition. Preferably, such dilutions of the composition of the invention dilute only the antibody concentration, but not the buffer and other potential components of the composition. Consequently, in a preferred embodiment, the diluent contains the same concentrations, for example, of the buffer as is present in the pharmaceutical composition of the invention. Additional exemplary diluents include sterile water, bacteriostatic water, water for injection (BWFI), a pH-buffered solution that is preferably an acetate or histidine buffer, sterile saline solution, Ringer's solution, or dextrose solution. In one embodiment, the diluent comprises or consists essentially of an acetate and sorbitol buffer.In another embodiment, the diluent comprises or consists essentially of a histidine and sucrose buffer.

[00157] The term “CD30” in this descriptive report refers to human Cluster of Differentiation 30 protein, also known as TNFRSF8 (tumor necrosis factor receptor superfamily member 8). CD30 is found in several species and thus the term “CD30” may not be limited to human CD30 unless contradicted by the context. The human CD30 sequence is established in SEQ ID NO:39.

[00158] The term “CD3”, in this descriptive report, refers to the protein Petition 870250088813, dated 09 / 30 / 2025, pp. 297 / 496 / 205 Human Cluster of Differentiation 3 (CD3) is part of the T cell coreceptor protein complex and is composed of four distinct chains. CD3 is found in various species, and thus the term "CD3" may not be limited to human CD3 unless contradicted by the context. In mammals, the complex contains a CD3y (gamma) chain (human CD3y chain UniProtKB / Swiss-Prot NoP09693, or monkey CD3y). UniProtKB / Swiss-Prot NoQ95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot NoP04234, or monkey CD3δ). UniProtKB / Swiss-Prot NoQ95LI8), two CD3ε (epsilon) chains (human CD3ε: UniProtKB / Swiss-Prot NoP07766, whose sequence is incorporated here as SEQ ID NO:42; Cynomolgus monkey CD3ε UniProtKB / Swiss-Prot NoQ95LI5; or Rhesus monkey CD3ε UniProtKB / Swiss-Prot NoG7NCB9), and one CD3Z (zeta) chain (human CD3Z UniProtKB / Swiss-Prot NoP20963, Cynomolgus monkey CD3Z UniProtKB / Swiss-Prot NoQ09TK0). These chains associate with a molecule known as the T cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex.

[00159] The term “antibody-binding region” refers to a region of the antigen, comprising the epitope to which the antibody binds. An antibody-binding region can be determined by epitope binning using bilayer interferometry, by alanine scanning, or by domain stacking assays (using antigen constructs in which regions of the antigen are exchanged for that of another species and determining whether the antibody still binds to the antigen or not). The amino acids within the antibody-binding region that are involved in the interaction with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and / or by crystallography of the antibody bound to its antigen. Petition 870250088813, dated 09 / 30 / 2025, pages 298 / 496 / 205

[00160] The term “epitope” means an antigenic determinant to which an antibody binds specifically. Epitopes generally consist of clusters on the surface of molecules, such as amino acids, sugar side chains, or a combination thereof, and generally have three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents or other agents that disrupt the three-dimensional structure of a protein or multimer thereof. The epitope may comprise amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the antibody when it is bound to the antigen.

[00161] The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb” or similar terms in this descriptive report refer to a preparation of antibody molecules of a single molecular composition that typically exhibits a single specificity and binding affinity for a given epitope. A monoclonal antibody can typically be produced by identical cells that are all clones of a single parent cell, such as hybridomas, stable cell lines, or similar. Consequently, the term “human monoclonal antibody” refers to antibodies that exhibit a single binding specificity and that possess variable and constant regions derived from human immunoglobulin germline sequences.Human monoclonal antibodies can be produced 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 and a human light chain transgene, fused into a single cell. Petition 870250088813, dated 09 / 30 / 2025, page 299 / 496 / 205 immortalized. Human monoclonal antibodies can be derived from human B cells or plasma cells. Monoclonal antibodies can also be produced by recombinant modified host cells, or systems that make use of cell extracts in support of in vitro transcription and / or translation of nucleic acid sequences that encode the antibody.

[00162] The term “isotype”, in this descriptive report, refers to the class of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotypes thereof, such as IgG1m(za) and IgG1m(f), that is encoded by genes in the heavy chain constant region. In addition, each heavy chain isotype may be combined with a kappa (κ) or lambda (λ) light chain.

[00163] The term “complete antibody,” as used herein, refers to an antibody (e.g., an original or variant antibody) comprising a pair of a heavy chain and a light chain or two pairs of heavy and light chains, each pair containing constant and variable heavy and light chain domains as normally found in a heavy and light chain pair of a wild-type antibody of that isotype. Thus, for example, a complete IgG1 antibody contains the VH, CH1, CH2, CH3, hinge, VL, and domains. In a complete variant antibody, the constant and variable heavy and light chain domains may contain, in particular, amino acid substitutions that modify and / or enhance functional properties of the antibody when compared to the original or wild-type complete antibody.A complete antibody according to the present invention can be produced by a method comprising the steps of (i) cloning CDR sequences into one or more suitable vectors comprising complete heavy and light chain sequences and (ii) expressing the suitable vectors obtained with the heavy and light chain sequences in suitable expression systems. This is within the knowledge of those skilled in the art. Petition 870250088813, dated 09 / 30 / 2025, page 300 / 496 / 205 to produce a complete antibody from CDR sequences or complete variable region sequences. Thus, a person skilled in the art knows how to generate a complete antibody according to the present invention.

[00164] The term “humanized antibody,” in this descriptive report, refers to a genetically modified non-human antibody containing human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology with human variable domains. This can be achieved by grafting complementarity-determining regions (CDRs) of non-human antibody, which together form the antigen-binding site, onto a homologous human acceptor framework region (FR) (see, among others, WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, it may be necessary to replace some of the framework residues of the parental antibody with some residues from the human framework region (i.e., the non-human antibody) (reverse mutations).Structural homology modeling can help identify amino acid residues in framework regions that are important for antibody binding properties. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally comprising one or more reverse amino acid mutations in the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily reverse mutations, can be applied to obtain a humanized antibody with preferred characteristics, such as particularly useful affinity and biochemical properties, for example, to include modifications that prevent deamidation and / or enhance fabrication. Furthermore, CDR and / or framework regions can be modified to enhance antigen affinity, for example, via affinity maturation procedures. Petition 870250088813, dated 09 / 30 / 2025, p. 301 / 496 / 205

[00165] “The term “human antibody” in this descriptive report refers to antibodies that have variable and constant regions derived from germline sequences of human immunoglobulins. Human antibodies may include amino acid residues not encoded by germline sequences of human immunoglobulins (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” in this descriptive report is not intended to include antibodies in which germline-derived CDR sequences from another mammalian species, such as a mouse, have been grafted onto human framework sequences.”The human monoclonal antibodies of the present invention can be produced by a variety of techniques, including conventional methodology for monoclonal antibodies, for example, the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred in principle, other techniques for monoclonal antibody production can be employed, for example, viral or oncogenic transformation of B lymphocytes or phage expression techniques using human antibody gene libraries. Human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system instead of the mouse system, such as an HCo12 mouse (see, for example, WO 03 / 059282) or similar.

[00166] The term “Fc region,” in this descriptive report, refers to a region comprising, from the N- to C-terminal ends of the two antibody heavy chain polypeptides, at least one hinge region, a CH2 region, and a CH3 region. Fc polypeptides are typically glycosylated. An antibody Fc region can mediate the binding of immunoglobulin to host tissues or factors, including various cells. Petition 870250088813, dated 09 / 30 / 2025, p. 302 / 496 / 205 of the immune system (such as effector cells) and components of the complement system. Fc regions also typically bind to FcRn and protein A.

[00167] The phrase “amino acid corresponding to the amino acid at position...” or similar, in this descriptive report, refers to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Unless stated otherwise or contradicted by the context, the amino acids in the constant region sequences are enumerated in this descriptive report according to the EU numbering index (described in Kabat, EA et al., 1991, Sequences of proteins of immunological interest, 5th edition - U.S. Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689).Thus, an amino acid or segment in one sequence that “matches” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program, such as ALIGN, ClustalW, or similar, typically in standard settings, and that has at least 50%, at least 80%, at least 90%, or at least 95% identity with a human IgG1 heavy chain. In the art, it is considered well known to align a sequence or segment within a sequence and thus determine the corresponding position in a sequence to an amino acid position according to the present invention.

[00168] The term “hinge region,” in this descriptive report, refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the I-numbering as established in Kabat, EA et al., Sequences of proteins of immunological interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991). However, the hinge region may Petition 870250088813, dated 09 / 30 / 2025, p. 303 / 496 / 205, may also be any of the other subtypes as described herein.

[00169] The term “CH1 region” or “CH1 domain”, in this descriptive report, refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the EU numbering as established in Kabat (ibid). However, the CH1 region may also be any of the other subtypes as described herein.

[00170] The term “CH2 region” or “CH2 domain”, in this descriptive report, refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering as established in Kabat (ibid). However, the CH2 region may also be any of the other subtypes as described herein.

[00171] The term “CH3 region” or “CH3 domain”, in this descriptive report, refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering as established in Kabat (ibid). However, the CH3 region may also be any of the other subtypes as described herein.

[00172] The term “Fc-mediated effector functions” in this descriptive report is intended to refer to functions that are a consequence of the binding of a polypeptide or antibody to its target or antigen on a cell membrane, where the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) binding to C1q, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor (FcgR) binding, (vi) antibody-dependent FcyR-mediated antigen cross-reaction, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) cytotoxicity Petition 870250088813, dated 09 / 30 / 2025, p. 304 / 496 / 205 complement-dependent cellular cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) antibody-mediated binding to the complement receptor of an opsonized antibody, (xi) opsonization and (xii) a combination of any of (i) to (xi).

[00173] The term “inertia,” “inert,” or “non-activating,” in this descriptive report, refers to an Fc region that is unable or has minimal ability to bind to any FcyR, induce Fc-mediated cross-linking of FcyRs, induce FcyR-mediated effector functions such as ADCC and ADCP, induce FcyR-mediated cross-linking of target antigens via two individual antibody Fc regions, and / or is unable to bind to C1q and induce complement-mediated effector functions such as CDC and CDCC. The inertness of an antibody Fc region can be tested using the antibody in a monospecific or bispecific format.

[00174] The term “monovalent antibody”, in the context of the present invention, refers to an antibody molecule that can interact with an antigen, with only one antigen-binding domain (e.g., a Fab arm). In the context of a multispecific antibody, such as a bispecific antibody, “monovalent antibody binding” refers to the binding of the multispecific antibody to an antigen with only one antigen-binding domain (e.g., a Fab arm).

[00175] The term “monospecific antibody”, in the context of the present invention, refers to an antibody with binding specificity to an antigen, an epitope only. The antibody may be a monospecific, monovalent antibody (i.e., carrying only one antigen-binding region), a monospecific, bivalent antibody (e.g., an antibody with two identical antigen-binding regions) or a monospecific, multivalent antibody (e.g., an antibody with more than two identical antigen-binding regions).

[00176] The term “multispecific antibody” refers to an antibody Petition 870250088813, dated 09 / 30 / 2025, page 305 / 496 / 205 with two or more antigen-binding domains that bind to two or more different epitopes. The term “bispecific antibody” refers to an antibody with two antigen-binding domains that bind to different epitopes, for example, two non-identical pairs of VH and VL regions, two non-identical Fab arms, or two Fab arms with two non-identical CDR regions. In the context of this invention, bispecific antibodies have specificity for two different epitopes, and multispecific antibodies have specificity for two or more different epitopes. Such epitopes may be on the same or different antigens or targets. If the epitopes are on different antigens, such antigens may be on the same cell or on different cell types or structures, such as extracellular matrix or vesicles and soluble protein.Multispecific and bispecific antibodies can therefore be capable of cross-linking with multiple antigens, for example, two different cells.

[00177] The term “bivalent antibody” refers to an antibody that has two antigen-binding regions, wherein said antigen-binding regions may be identical and bind to the same epitopes or may be non-identical and bind to different epitopes that may be on the same or different antigen(s). Consequently, a bivalent antibody may be a monospecific antibody or a bispecific antibody.

[00178] The terms “amino acid” and “amino acid residue” may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid. In the context of the present invention, amino acids can be classified based on their structure and chemical characteristics. Thus, the classes of amino acids can be reflected in one or both of the following tables. Petition 870250088813, dated 09 / 30 / 2025, page 306 / 496 / 205 Table 1 - Main classification based on the structure and general chemical characterization of the R group_____________________________________________ Amino Acid Class: Acidic residues D and E; Basic residues K, R, and H; Uncharged hydrophilic residues S, T, N, and Q; Uncharged aliphatic residues G, A, V, L, and I; Uncharged nonpolar residues C, M, and P; Aromatic residues F, Y, and W. Table 2 - Alternative physical and functional classifications of amino acid residues__________________________________________________________ Amino Acid Class Hydroxyl Group-Containing Residues S and T Aliphatic Residues I, L, V, and M Cycloalkenyl-Associated Residues F, H, W, and Y Hydrophobic Residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively Charged Residues D and E Polar Residues C, D, E, H, K, N, Q, R, S, and T Positively Charged Residues H, K, and R Small Residues A, C, D, G, N, P, S, T, and V Very Small Residues A, G, and S Residues Involved in Formation at a Time A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible Residues Q, T, K, S, G, P, D, E, and R

[00179] The substitution of one amino acid for another can be classified as conservative or non-conservative substitution. In the context of the present invention, a “conservative substitution” is a substitution of one amino acid for another amino acid with similar structural and / or chemical characteristics, such as the substitution of one amino acid residue for another amino acid residue of the same class as defined in either of the two tables above: for example, a conservative substitution could be the substitution of leucine for isoleucine, since both are hydrophobic, branched, aliphatic. Similarly, an example of a conservative substitution is the substitution of aspartic acid for glutamic acid, since both are small residues with a negative charge. Petition 870250088813, dated 09 / 30 / 2025, p. 307 / 496 / 205

[00180] In the context of the present invention, a substitution in an antibody is indicated as: original amino acid - substituted amino acid position number.

[00181] With reference to the well-recognized nomenclature for amino acids, the three-letter code or the one-letter code is used, including the codes “Xaa” or “X” to indicate any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. The term “natural,” in this descriptive report, refers to any of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine.

[00182] Consequently, the annotation “K409R” or “Lys409Arg” means that the antibody comprises a lysine substitution for arginine at amino acid position 409. The substitution of an amino acid at a given position for any other amino acid is referred to as: original amino acid - position; or for example, “K409”. For a modification in which the original amino acid(s) and / or substituted amino acid(s) may comprise more than one, but not all, amino acids, the more than one amino acid may be separated by “,” or “ / ”. For example, the substitution of lysine by arginine, alanine or phenylalanine at position 409 is: “Lys409Arg,Ala,Phe” or “Lys409Arg / Ala / Phe” or “K409R,A,F” or “K409R / A / F” or “K409 for R, A or F”. Such designation may be used interchangeably in the context of the present invention, but they have the same meaning and purpose.

[00183] In addition, the term “a substitution” covers a substitution in any one or more of the other nineteen naturally occurring amino acids, or in other amino acids, such as non-naturally occurring amino acids. For example, a K amino acid substitution at position 409 includes each of the Petition 870250088813, dated 09 / 30 / 2025, p. 308 / 496 / 205, refers to the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. These substitutions may also be designated K409A, K409C, etc., or K409A,C, etc., or K409A / C / etc. The same applies by analogy to any and all positions mentioned herein to specifically include any of these substitutions.

[00184] The term “host cell” in this descriptive report is intended to refer to a cell into which a nucleic acid, as an expression vector, has been introduced. It should be understood that such terms are intended to refer not only to the particular cell in question, but may also include the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may, in fact, not be identical to the parent cell, but are still encompassed by the term “host cell” in this descriptive report. Recombinant host cells (i.e., host cells used for the production of recombinant proteins) include, for example, transfectomes such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells and lymphocytes, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant and fungal cells.

[00185] The term “transfectome”, in this descriptive report, includes recombinant eukaryotic host cells that express the antibody or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant or fungal cells, including yeast cells.

[00186] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined along the length of the reference sequence, using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Petition 870250088813, dated 09 / 30 / 2025, page 309 / 496 / 205 Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). The Needle result marked as “longest identity” (obtained with the -nobrief option) is used as the percent identity and calculated as follows: (Identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[00187] Retention of similar residues can also, or alternatively, be measured by a similarity score, as determined using a BLAST program (e.g., BLAST 2.2.8 available through NCBI using default settings BLOSUM62, Gap Opening=11 and Extended Gap=1). Suitable variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95% or more (e.g., about 99%) similarity to the original or reference sequence. Table 3: Amino acid sequences SEQ ID NO: Reference Domain Sequence 1 IgG1-CD30-MDX060-FERR VH_CDR1 GGSFSAYY 2 IgG1-CD30-MDX060-FERR VH_CDR2 INHGGGT 3 IgG1-CD30-MDX060-FERR VH_CDR3 ASLTAY 4 IgG1-CD30-MDX060-FERR VL_CDR1 QGISSW 5 IgG1-CD30-MDX060-FERR VL_CDR2 AAS 6 IgG1-CD30-MDX060-FERR VL_CDR3 QQYDSYPIT 7 IgG1-huCD3-FEAL VH_CDR1 GFTFNTYA Petition 870250088813, dated 09 / 30 / 2025, page 310 / 496 / 205 8 IgG1-huCD3-FEAL VH_CDR2 IRSKYNNYAT 9 IgG1-huCD3-FEAL VH_CDR3 VRXGNFGNSYVSWFAY, em que X é H ou G 10 IgG1-huCD3-FEAL VL_CDR1 TGAVTTSNY 11 IgG1-huCD3-FEAL VL_CDR2 GTN 12 IgG1-huCD3-FEAL VL_CDR3 ALWYSNLWV 13 IgG1-CD30- MDX060-FERR VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYY wswirqppgkglewigdinhgggtnynpslksrv TISVDTSKNQFSLKLNSVTAADTAVYYCASLTAY WGQGSLVTVSS 14 IgG1-CD30- MDX060-FERR VL DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTW YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYDSYPITFGQGTRLEI K 15 IgG1-huCD3- FEAL VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAM NWVRQAPGKGLEWVARIRSKYNNYATYYADSVK drftisrddsksslylqmnnlktedtamyycvrx GNFGNSYVSWFAYWGQGTLVTVSS, em que X é H ou G 16 IgG1-huCD3- FEAL VL QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYA NWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSSLIG DKAALTITGAQADDESIYFCALWYSNLWVFGGGT KLTVL 17 IgG1-CD30- MDX060-FERR Cadeia pesada completa QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYY WSWIRQPPGKGLEWIGDINHGGGTNYNPSLKSRV TISVDTSKNQFSLKLNSVTAADTAVYYCASLTAY WGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPPCAPEFERGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL complete light chain DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTW YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYDSYPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 19 IgG1-huCD3- FEAL Complete heavy chain EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAM NWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRH GNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSH Petition 870250088813, dated 09 / 30 / 2025, page 311 / 496 / 205 EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG 20 IgG1-huCD3- FEAL Complete Light Chain DFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS 21 IgG1-CD30hAC10-FEAR Full heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYI TWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGR VTMTRDTSISTAYMELSRLRSDDTAVYYCANYGN YWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVF LFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 22 IgG1-CD30hAC10-FEAR Complete light chainDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDS YMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 23 IgG1-CD30-HRS3-FEAR Complete heavy chain QVQLQQSGAELARPGASVKMSCKASGYTFTTYTI HWVRQRPGHDLEWIGYINPSSGYSDYNQNFKGKT TLTADKSSNTAYMQLNSLTSEDSAVYYCARRADY GNYEYTWFAYWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEG GPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 24 IgG1-CD30-HRS3-FEAR Complete light chain DIVMTQSPKFMSTSVGDRVTVTCKASQNVGTNVA WFQQKPGQSPKVLIYSASYRYSGVPDRFTGSGSGT DFTLTISNVQSEDLAEYFCQQYHTYPLTFGGGTKL EINRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25 IgG1-CD30-HeFi- I-FEAR Complete Heavy Chain EVKLVESGGGLVQPGGSLRLSCATSGFTFSDYYM NWVRQPPGKALEWLGFIRNKANGYTTEFSASVM GRFTISRDDSQSILYLQMNTLRAEDSATYYCARDP PYGNPHYYAMDYWGQGTSVTVSSASTKGPSVFPL Petition 870250088813, dated 09 / 30 / 2025, page 312 / 496 / 205 APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEKPKSCDKTHTCPPCPAPE FEGGPSVFLFPKPKDTLMISRTPEVTCVVVAVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCCVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 26 IgG1-CD30-HeFi- I-FEAR Cadeia leve completa DIVLTQSPASLAVSLGQRATISCRASKSVSASGYN YMHWYQQKAGQPPKLLIHLASNLESGVPARFSGS GSGTDFTLNIHPVEEDASTYYCQHSGELPFTFGSG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 27 IgG1-CD30-T405FEAR Cadeia pesada completa QVQLQQIGAELVRPGASVKLSCKASGYTFNNYWI NWVKQRPGQGLEWIGNIYPSDSRNYNQKFKDKA TLTVDKPSSTAYMQLSSPTSEDASAVYCTLGSYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPKP KDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCCVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 28 IgG1-CD30-T405FEAR Full light chain DVVMTQTPLTLSVTIGQPASISCKSSQSLSDSDGKT YLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSG SGTDFTLKISRVEAEDLGVYYCWQGAHFPRTFGG GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 29 IgG1-CD30-T105-FEAR Full heavy chain QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGV SWIRQPSGKDLEWLAHIYWDDDKRYNPSLKSRLTI SKDTSSNQVFLKITSVDTADTATYYCARRADGLYF YLDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPPCAPEFEGGPSVFL FPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 30 IgG1-CD30-T105- FEAR Complete light chain DIVMTQSQKFMSTSVGDRVSVTCKASQNVNTNVA WYQQKPGQSPEALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 31 IgG1-CD30-T408- Heavy chain QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWI Petition 870250088813, dated 09 / 30 / 2025, page 313 / 496 / 205 Complete FEAR DKRVEPKSCDKTHTCPPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 32 IgG1-CD30-T408FEAR Full light chain DVVMTQTPLTLSVTIGQPASISCKSSQSLSDSDGKT YLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSG SGTDFTLKISRVEAEDLGVYYCWQGAHFPRTFGG GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 33 IgG1-CD30-T215- FEAR Full heavy chain QVTLKESGPGILQPSQTLSLTCSFSGFSLSVSGMGV SWIRQPSGKGLEWLAHIHWDDDRRSNPSLRSRLTI SKDTSNNQVFLKITSVDTADTATYYCARRPDLYG NYFFFDFWGQGTTLTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCCPPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD ​​IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 34 IgG1-CD30-T215- FEAR Cadeia leve completa DIVMTQSQKFMSTSVGDRVSVTCKASQNVFTNVA WYQQKLGQSPKPLIYSASYRYSGVPDRFTGSGSGT DFTLTISDVQSEDLAEYFCQQYNSYPVTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 35 IgG1huCD3H101GFEAL Cadeia pesada completa EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAM NWVRQAPGKGLEWVARIRSKYNNYATYADSVK DRFTISRDDSKSSLYLQMNNLKTAMYYCVRG GNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDTHTCPPCPAP EFEGGPSVFLFPKPKDTLMISRTPEVTCVVVAVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG 36IgG1-b12-FEAL Full heavy chain QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVI HWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDR VTFTADTSANTAYMELRSLRSADTAVYYCARVGP YSWDDSPQDNYYMDVWGKGTTVIVSSASTKGPS Petition 870250088813, dated 09 / 30 / 2025, page 314 / 496 / 205 VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 37 IgG1-b12-FEAL Cadeia leve completa EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAW YQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTD FTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLE RKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 38 IgG1-b12-FERR Cadeia pesada completa QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVI HWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDR VTFTADTSANTAYMELRSLRSADTAVYYCARVGP YSWDDSPQDNYYMDVWGKGTTVIVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEFERGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 39 CD30 humano FPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGL FPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVT CSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVN SCARCFFHSVCPAGMIVKFPGTAQKNTVCEPASPG VSPACASPENCKEPSSGTIPQAKPTPVSPATSSAST MPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPG LSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVS CSRDDLVEKTPCAWNSSRTCECRPGMICATSATNS CARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGT QPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIP TSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSS AFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVD SRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETC HSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVST EHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAG PAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLS VEEEGKEDPLPTAASGK 40 CD30 de macaco Cynomolgus FPQDRPFEDTCRGNPGHYYDKAVRRCCYRCPTGT AQKNTVCEPASPGVSPACASPENCKEPSSGTIPQA KPTPVSPATSNASTMPLRGGTRLAQEAASKLTRAP GSPSSVGRPSSDPGLSPTQPCPQGSGDCRKQCEPD YYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRIC ECRPGMICATSATNSCARCVPYPICAAETGTKPQDMAEKDTTFEAPPVGTQPDCSPTPENGEAPASTSPT LSSLVDSQASKTLPIPTSAPIALSSTGKPVLDAGPVL FWVILVLAVVVGSSTFLLCHRRACRKRIRQKLHLC YPVQTSRPKLELVDSRPRRSSTQFESGTFVTKPSAE ELGLMSLPPKETCRNVGAACPESLPLWDASPAGG Petition 870250088813, dated 09 / 30 / 2025, page 315 / 496 / 205 PSSPRDLPEPRVSTEHTNNRIEKIYIMKADTVIVGT VKAELPEGQGLAGPAEPELEEELEADHAPHYPEQE TEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 41 CD30 de macaco Rhesus FPQDRPFEDTCRGNPGHYYDKAVRRCCYRCPMGL FPTQQCPQRPADCRKQCEPDYYLDEAGRCTACVS CSRDDLVEKMPCAWNSSRVCECQPGMFCAVSVV NSCARCFFHSVCPAGMIVKFPGTAQKNTVCEPASP GVSPACASPENCKEPSSGTIPQAKPTPVSPATSNAS TMPLRGGTRLAQEAASKLTRAPGSPSSVGRPSSDP GLSPTQPCPQGSGDCRKQCEPDYYLDEAGRCTAC VSCSRDDLVEKTPCAWNSSRICECRPGMICATSAT NSCARCVPYPICAAETGTKPQDMAEKDTTFEAPPV GTQPDCSPTPENGEAPASTSPTLSSLVDSQASKTLP IPTSAPIALSSTGKPVLDAGPVLFWVILVLAVVVGS STFLLCHRRACRKRIRQKLHLCYPVQTSRPKLELV DSRPRRSSTQFESGTFVTKPSAEELGLMSLPPKETC RNVGAACPESLPLWDASPAGGPSSPRDLPEPRVST EHTNNRIEKIYIMKADTVIVGTVKAELPEGQGLAG PAEPELEEELEADHAPHYPEQETEPPLGSCSDVML SVEEEGKEDPLPTAASGK 42 CD3 humano (épsilon) QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEI LWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELE QSGYYVCYPRGSKPEDANFYLYLRARVCENCME MDVMSVATIVIVDICITGGLLLLVYYWSKNRKAK AKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIR KGQRDLYSGLNQRRI 43 CD3 de macaco CynomolgusQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEA QWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCY PRGSNPEDASHHLYLKARVCENCMEMDVMAVAT IVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAG AGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSG LNQRRI 44 Constant region of IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 45 Constant region of IgG1 with FER substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFERGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 46 Constant region of IgG1 with FEA substitution(bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPE VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS Petition 870250088813, dated 09 / 30 / 2025, page 316 / 496 / 205 NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 47 Constant region of IgG1 with F405L substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 48 Constant region of IgG1 with K409R substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 49 Constant region of IgG1 with FER substitution and with F405L substitution(bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFERGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 50 Constant region of IgG1 with FER substitution and with K409R substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFERGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 51 Constant region of IgG1 with FEA substitution and with F405L substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 52 Constant region of IgG1 with FEA substitution and with K409R substitution (bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPE Petition 870250088813, dated 09 / 30 / 2025, page 317 / 496 / 205 VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 53 constant region constant region of human kappa light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 54 constant region constant region of human lambda light chain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S 55 IgG1-CD30- MDX060-FEAR Complete heavy chain QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYY WSWIRQPPGKGLEWIGDINHGGGTNYNPSLKSRV TISVDTSKNQFSLKLNSVTAADTAVYYCASLTAY WGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG Additional aspects and embodiments of the invention Pharmaceutical compositions

[00188] In one aspect, the present invention relates to a pharmaceutical composition comprising: a) a multispecific antibody comprising an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3; and b) a buffering agent in which the pH of the composition is approximately 4.0 to approximately 8.0.

[00189] In one embodiment, the buffering agent is selected from the group consisting of acetate, histidine, TRIS® (tris(hydroxymethyl)aminomethane), citrate, succinate, glycolate, glutamate, and mixtures thereof. In a further embodiment, the buffering agent is histidine, acetate, and / or mixtures thereof. In a preferred embodiment, the buffering agent is acetate. The buffering agent that is acetate can be selected from the Petition 870250088813, dated 09 / 30 / 2025, page 318 / 496 / 205 group consisting of sodium acetate, potassium acetate, potassium acetate, their hydrates and mixtures thereof. In a preferred embodiment, the buffering agent is sodium acetate, such as sodium acetate trihydrate.

[00190] The concentration of the buffering agent is defined to maintain the pH. In one embodiment, the buffering agent is present at a concentration of about 5 to about 40 mM, such as about 10 to about 30 mM, such as about 15 to about 25 mM, such as about 18 to about 22 mM, preferably about 20 mM, such as about 17 mM.

[00191] The pH of the pharmaceutical composition is between about 4.0 and about 8.0. In one embodiment, the pH of the composition is from about 4.5 to about 6.5, as from about 5.0 to about 6.0, as from about 5.2 to about 5.7, as from about 5.4 to about 5.6, preferably about 5.5. In one embodiment, the pH of the composition is about 5.5. In a further embodiment, the pH of the composition is about 6.0.

[00192] The pH of the pharmaceutical composition can be adjusted with a pH-adjusting component to obtain the desired pH for the composition. Thus, in one embodiment, the pharmaceutical composition additionally comprises a pH-adjusting component. In a further embodiment, the pH-adjusting component is an acid. In yet another embodiment, the acid is HCl and / or acetic acid. In a preferred embodiment, the acid is glacial acetic acid.

[00193] The concentration of the pH-adjusting component would depend on several factors, such as the pH-adjusting component. However, in one embodiment, the pH-adjusting component is present at a concentration of about 0.1-30 mM, about 0.3-25 mM, about 0.5-20 mM, about 0.6-15 mM, about 1-10 mM, about 2-5 mM, or about 3 mM. In a preferred embodiment, the pharmaceutical composition comprises glacial acetic acid at 3 mM.

[00194] In one embodiment, the buffering agent is acetate in a Petition 870250088813, dated 09 / 30 / 2025, p. 319 / 496 / 205 concentration of about 5-40 mM, as about 10-30 mM, as about 20 mM, and the pH of the composition is about 5-6, as about 5.5. In a preferred embodiment, the buffering agent is acetate at a concentration of about 10-30 mM and the pH of the composition is about 5.5. In a further preferred embodiment, the buffering agent is acetate at a concentration of about 20 mM and the pH of the composition is about 5.5. In a further preferred embodiment, the composition comprises sodium acetate at about 17 mM and glacial acetic acid at about 3 mM, and the pH of the composition is about 5.5.

[00195] The pharmaceutical composition may advantageously comprise components in addition to a buffering agent and a multispecific antibody. Consequently, in a further embodiment, the pharmaceutical composition additionally comprises a non-ionic excipient.

[00196] The nonionic excipient may be as known to those skilled in the art. However, in a preferred embodiment, the nonionic excipient is a sugar or a sugar alcohol. In a further embodiment, the nonionic excipient is selected from sucrose, trehalose, mannitol, xylitol, sorbitol, and mixtures thereof. In a preferred embodiment, the nonionic excipient is selected from sorbitol, sucrose, trehalose, or mixtures thereof. In a more preferred embodiment, the nonionic excipient is sorbitol or trehalose. In a more preferred embodiment, the nonionic excipient is sorbitol.

[00197] The nonionic excipient is added at a concentration that would be acceptable for pharmaceutical use. In one embodiment, the nonionic excipient is present at a concentration of about 5 to about 450 mM, such as about 50 to about 400 mM, such as about 100 to about 350 mM, such as about 125 to about 300 mM, such as about 150 to about 275 mM, preferably about 250 mM. Petition 870250088813, dated 09 / 30 / 2025, p. 320 / 496 / 205

[00198] In a further embodiment, the nonionic excipient is present at a concentration of about 350 mM, about 340 mM, about 330 mM, about 320 mM, about 310 mM, about 300 mM, about 290 mM, about 280 mM, about 270 mM, about 260 mM, about 250 mM, about 240 mM, about 230 mM, about 220 mM, about 210 mM, about 200 mM, about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 90 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM or about 10 mM.

[00199] In one embodiment, the pharmaceutical composition comprises sorbitol at about 100-350 mM, as sorbitol at about 200-300 mM, as sorbitol at about 250 mM. Thus, in a further embodiment, the pharmaceutical composition comprises acetate, such as sodium acetate, and sorbitol and has a pH of about 5.5.

[00200] In yet another embodiment, the ratio of acetate buffer concentrations to sorbitol in the pharmaceutical composition is between 2:10 and 2:40, as between 2:15 and 2:35, as between 2:20 and 2:30, for example, 2:25.

[00201] The pharmaceutical composition may advantageously comprise further components. In one embodiment, the pharmaceutical composition additionally comprises a surfactant.

[00202] The surfactant could be a range of surfactants known for use in pharmaceutical compositions. In one embodiment, the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, Petition 870250088813, dated 09 / 30 / 2025, page 321 / 496 / 205 polyoxyethylene derivatives of castor oil, polyoxyethylene esters of fatty acids, polyoxyethylene stearates, polyoxyl hydrostearate, polyoxyglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sucrose palmitate of sorbitan esters, sucrose stearate, tricaprylin and TPGS, as well as mixtures thereof. In a preferred embodiment, the surfactant is a polysorbate. In a more preferred embodiment, the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80). In a more preferred embodiment, the surfactant is polysorbate 80 (PS80).

[00203] The surfactant is added at a concentration that would be acceptable for pharmaceutical use. In one embodiment, the surfactant is present at a concentration of about 0.01 to about 0.1% w / v, as of about 0.01 to about 0.09% w / v as of about 0.01 to about 0.06% w / v as of about 0.01 to about 0.05% w / v as of about 0.02% w / v, about 0.03% w / v, about 0.04% w / v or about 0.05% w / v, preferably about 0.02% w / v.

[00204] The general properties of the pharmaceutical composition, such as osmolality and viscosity, are affected by the components as well as the concentrations of the components in the pharmaceutical composition. In one embodiment, the osmolality of the pharmaceutical composition is less than about 600 mOsm / kg, such as less than about 550 mOsm / kg, such as less than about 500 mOsm / kg, such as less than about 450 mOsm / kg, such as less than about 400 mOsm / kg, such as less than about 350 mOsm / kg.

[00205] In an additional embodiment, the osmolality of the pharmaceutical composition is in the range of about 100-500 mOsm / kg, as in the range of about 200-400 mOsm / kg, as in the range of about 250-350 mOsm / kg.

[00206] In yet another embodiment, the osmolality of the pharmaceutical composition is approximately 250 mOsm / kg, approximately 260 mOsm / kg, approximately 270 mOsm / kg, approximately 280 mOsm / kg, approximately 290 mOsm / kg, approximately Petition 870250088813, dated 09 / 30 / 2025, p. 322 / 496 / 205 of 300 mOsm / kg, approximately 310 mOsm / kg, approximately 320 mOsm / kg, approximately 330 mOsm / kg, approximately 340 mOsm / kg or approximately 350 mOsm / kg.

[00207] In yet another embodiment, the viscosity of the pharmaceutical composition is less than about 30 cP, such as less than about 25 cP, such as less than about 20 cP, such as less than about 18 cP, such as less than about 16 cP, such as less than about 14 cP, such as less than about 12 cP, such as less than about 10 cP, such as less than about 9 cP, such as less than about 8 cP, such as less than about 7 cP, such as less than about 6 cP, such as less than about 5 cP, such as less than about 4 cP, such as less than about 3 cP, such as less than about 2 cP.

[00208] In a further embodiment, the viscosity of the pharmaceutical composition is in the range of about 1-30 cP, as in the range of about 125 cP, as in the range of about 1-20 cP, as in the range of about 1-18 cP, as in the range of about 1-15 cP, as in the range of about 1-12 cP, as about 1-10 cP, as about 1-9 cP, as about 1-8 cP, as about 1-7 cP, as about 1-6 cP, as about 1-5 cP, as about 1-4 cP, as about 1-3 cP.

[00209] In yet another embodiment, the viscosity of the pharmaceutical composition is approximately 2 cP, approximately 2.5 cP, approximately 3 cP, approximately 3.5 cP, approximately 4 cP, approximately 4.5 cP, approximately 5 cP, approximately 5.5 cP, approximately 6 cP, approximately 6.5 cP, approximately 7 cP, approximately 7.5 cP, approximately 8 cP, approximately 8.5 cP, approximately 9 cP, approximately 9.5 cP, approximately 10 cP, approximately 10.5 cP, approximately 11 cP, approximately 11.5 cP, approximately 12 cP, approximately 12.5 cP, approximately 13 cP, approximately 13.5 cP, approximately 14 cP, approximately 14.5 cP, approximately 15 cP, approximately 15.5 cP, about 16 cP, about 16.5 cP, about 17 cP, about 17.5 cP, about 18 cP, about 18.5 cP, about 19 cP, about 19.5 cP or about 20 cP.

[00210] The pharmaceutical composition comprises a multispecific antibody capable of binding to both CD30 and CD3. In one embodiment, the antibody concentration is approximately 0.5 to approximately 250 mg / ml, as approximately Petition 870250088813, dated 09 / 30 / 2025, page 323 / 496 / 205 from 1.0 to about 220 mg / ml, as from about 3 to about 190 mg / ml, as from about 5 to about 160 mg / ml, as from about 10 to about 130 mg / ml, or as from about 20 to about 120 mg / ml, as from about 30 to about 110 mg / ml, as from about 40 to about 100 mg / ml, as from about 50 to about 90 mg / ml, as from about 60 to about 80 mg / ml, or as from about 65 to about 75 mg / ml, as from about 70 mg / ml.

[00211] In a further embodiment, the antibody concentration is about 50 to about 250 mg / ml, or about 60 to about 240 mg / ml, or about 70 to about 220 mg / ml, or about 80 to about 210 mg / ml, or about 100 to about 200 mg / ml, or about 120 to about 190 mg / ml, or about 130 to about 180 mg / ml, or about 135 to about 165 mg / ml, or about 150 to about 190 mg / ml.

[00212] In yet another embodiment, the antibody concentration is approximately 10 mg / ml, approximately 12 mg / ml, approximately 14 mg / ml, approximately 16 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 22 mg / ml, approximately 24 mg / ml, approximately 26 mg / ml, approximately 28 mg / ml, approximately 30 mg / ml, approximately 32 mg / ml, approximately 34 mg / ml, approximately 36 mg / ml, approximately 38 mg / ml, approximately 40 mg / ml, approximately 42 mg / ml, approximately 44 mg / ml, approximately 46 mg / ml, approximately 48 mg / ml, approximately 50 mg / ml, approximately 52 mg / ml, approximately 54 mg / ml, approximately 56 mg / ml, approximately 58 mg / ml, approximately 60 mg / ml, about 62 mg / ml, about 64 mg / ml, about 66 mg / ml, about 68 mg / ml, about 70 mg / ml, about 72 mg / ml, about 74 mg / ml, about 76 mg / ml, about 78 mg / ml, about 80 mg / ml, about 82 mg / ml, about 84 mg / ml, about 86 mg / ml, about 88 mg / ml, about 90 mg / ml, about 92 mg / ml, about 94 mg / ml, about 96 mg / ml, about 98 mg / ml, about 100 mg / ml, about 102 mg / ml, about 104 mg / ml,approximately 106 mg / ml, approximately 108 mg / ml, approximately 110 mg / ml, approximately 112 mg / ml, approximately 114 mg / ml, approximately 116 mg / ml, approximately 118 mg / ml, approximately 120 mg / ml, approximately 122 mg / ml, approximately 124 mg / ml, approximately 126 mg / ml, approximately 128, Petition 870250088813, dated 09 / 30 / 2025, page. 324 / 496 / 205 mg / ml, about 130 mg / ml, about 132 mg / ml, about 134 mg / ml, about 136 mg / ml, about 138 mg / ml, about 140 mg / ml, about 142 mg / ml, about 144 mg / ml, about 146 mg / ml, about 148 mg / ml, about 150 mg / ml, about 152 mg / ml, about 154 mg / ml, about 156 mg / ml, about 158 ​​mg / ml, about 160 mg / ml, about 162 mg / ml, about 164 mg / ml, about 166 mg / ml, about 168 mg / ml, about 170 mg / ml, about 172 mg / ml, about 174 mg / ml, about 176 mg / ml, about 178 mg / ml, about 180 mg / ml, about 182 mg / ml, about 184 mg / ml, about 186 mg / ml, about 188 mg / ml, about 190 mg / ml, about 192 mg / ml, about 194 mg / ml, about 196 mg / ml, about 198 mg / ml or about 200 mg / ml.

[00213] The pharmaceutical composition is preferably a liquid composition. Thus, in one embodiment, the pharmaceutical composition is a liquid composition. In a further embodiment, the pharmaceutical composition is an aqueous composition.

[00214] The pharmaceutical composition is preferably a stable composition for pharmaceutical use even after storage for a given period of time. In one embodiment, the pharmaceutical composition is a stable pharmaceutical composition. A pharmaceutical composition that is stable would be interpreted as interchangeable with a stable pharmaceutical composition.

[00215] In one embodiment, the pharmaceutical composition is stable over a period of at least 2 months, such as at least 3 months, such as at least 6 months, such as at least 9 months, such as at least 12 months.

[00216] The pharmaceutical composition is additionally stable over a wide range of antibody concentrations from about 10 mg / ml to about 250 mg / ml, such as about 20 mg / ml to about 70 mg / ml or about 150 mg / ml or about 175 mg / ml or about 200 mg / ml. In one embodiment, the pharmaceutical composition is stable over a wide range of concentrations Petition 870250088813, dated 09 / 30 / 2025, p. 325 / 496 / 205 of the antibody from approximately 10 mg / ml to approximately 250 mg / ml, as from approximately 20 mg / ml to approximately 200 mg / ml, as from approximately 50 mg / ml to approximately 170 mg / ml.

[00217] In addition, the pharmaceutical composition is stable over a temperature range of about 2°C to about 25°C. In one embodiment, the pharmaceutical composition is stable over a temperature range of about 2°C to about 25°C.

[00218] It is surprising that a formulation would remain stable over such a wide range of antibody concentrations at temperatures ranging from about 2°C to about 25°C or even higher temperatures. The composition of the invention is stable for at least 3 months, such as at least 6 months, or even for at least 9 months or for at least 12 months when stored between 2°C and 8°C. The composition is preferably stable for pharmaceutical use for at least 2 months, such as at least 3 months, such as at least 4 months, such as at least 5 months, such as for at least 6 months, such as at least 9 months, or for at least 12 months at a storage temperature of about 2-8°C, such as about 5°C. In one embodiment, the pharmaceutical composition is stable for pharmaceutical use for 12 months at a storage temperature of about 2-8°C, such as about 5°C.

[00219] The pharmaceutical composition may be administered, for example, as a subcutaneous or intravenous composition. In one embodiment, the pharmaceutical composition is a subcutaneous composition and / or wherein the composition is for use in subcutaneous administration. In another embodiment, the pharmaceutical composition is an intravenous composition and / or wherein the composition is for use in intravenous administration.

[00220] The pharmaceutical composition, as described herein, can be prepared by mixing the components of the pharmaceutical composition in water. Thus, a further aspect of the present invention relates to a method for Petition 870250088813, dated 09 / 30 / 2025, page 326 / 496 / 205 to prepare a pharmaceutical composition, as defined herein, comprising the steps of mixing in water: a) a multispecific antibody, with approximately 0.5 to approximately 250 mg / ml of the antibody, b) a buffering agent, optionally, c) a non-ionic excipient and optionally, d) a surfactant; and adjust the pH to approximately 4.0-8.0.

[00221] In this descriptive report, pharmaceutical composition and pharmaceutical formulation are used interchangeably. Multispecific antibody formats

[00222] The present invention provides pharmaceutical compositions comprising multispecific antibodies capable of binding to CD30 and CD3. In one embodiment, the multispecific antibody comprises: (i) a CD30 binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively.

[00223] In one embodiment, the X in SEQ ID NO:9 is an H. In another embodiment, the X in SEQ ID NO:9 is a G.

[00224] In one embodiment, the first variable chain region Petition 870250088813, dated 09 / 30 / 2025, page 327 / 496 / 205 heavy chain is human or humanized. In another modality, the first variable region of the light chain is human or humanized. In one modality, the second variable region of the heavy chain is human or humanized. In another modality, the second variable region of the light chain is human or humanized. In another modality, the first variable region of the heavy chain and the first variable region of the light chain are human. In another modality, the first variable region of the heavy chain and the first variable region of the light chain are humanized. In another modality, the second variable region of the heavy chain and the second variable region of the light chain are human. In another modality, the second variable region of the heavy chain and the second variable region of the light chain are human.In another embodiment, the first variable heavy chain region and the first variable light chain region are human, and the second variable heavy chain region and the second variable light chain region are human. In another embodiment, the first variable heavy chain region and the first variable light chain region are human, and the second variable heavy chain region and the second variable light chain region are humanized.

[00225] As will be well known to those skilled in the art, each antigen-binding region of an antibody generally comprises a variable heavy chain (VH) region and a variable light chain (VL) region, and each of the variable regions comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively, and may comprise four framework sequences, FR1, FR2, FR3 and FR4, respectively. This structure is preferably also found in antibodies according to the present invention. In one embodiment, one, two, three or all four of said framework sequences are human framework sequences.

[00226] As described above, the pharmaceutical composition according to the invention comprises a multispecific antibody comprising an antigen-binding region capable of binding to human CD30, whose Petition 870250088813, dated 09 / 30 / 2025, page 328 / 496 / 205, sequence is established in SEQ ID NO:39. In particular, the antibody according to the invention is an antibody in which said antigen-binding region capable of binding to human CD30 is capable of binding to the extracellular domain of human CD30, such as a CD30 molecule expressed in a cell, more preferably a tumor cell.

[00227] As described, the pharmaceutical composition comprises an antibody comprising a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively.

[00228] The CDR1, CDR2, and CDR3 regions can be identified from the variable heavy and light chain regions using methods known in the art. The CDR regions of said variable heavy and light chain regions were annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[00229] In one embodiment, the antigen-binding region that binds to CD30 comprises: • a (first) variable heavy chain (VH) region comprising the sequence with SEQ ID NO:13, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence with SEQ ID NO:13; and • a (first) variable light chain (VL) region comprising the sequence with SEQ ID NO:14, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence with SEQ ID NO:14. Petition 870250088813, dated 09 / 30 / 2025, p. 329 / 496 / 205

[00230] In a further embodiment, the antibody used in the pharmaceutical composition according to the present invention comprises a variable heavy chain (VH) region of the antigen-binding region that binds to CD30 as defined herein, wherein the sequences comprise, in total, a maximum of 1, 2, 3, 4 or 5 amino acid substitutions when compared with SEQ ID NO:13.

[00231] In a further embodiment, the antibody used in the pharmaceutical composition according to the present invention comprises a variable light chain (VL) region of the antigen-binding region that binds to CD30 as defined herein, wherein the sequences comprise, in total, a maximum of 1, 2, 3, 4 or 5 amino acid substitutions when compared with SEQ ID NO:14.

[00232] In a further embodiment, the first variable heavy chain region comprises the sequence set forth in SEQ ID NO:13 and the first variable light chain region comprises the sequence set forth in SEQ ID NO:14.

[00233] Such antigen-binding regions that are capable of binding to human CD30 have been described, among others, in WO03059282 (Medarex), which is incorporated herein by reference.

[00234] The antibody used in the pharmaceutical composition, according to the present invention, can bind with a dissociation constant in the KD equilibrium between the antigen-binding region that binds to human CD30 and human CD30 that is in the range of 0.1 to 20 nM, for example, in the range of 0.5-5 nM, as in the range of 1.5-2 nM (monovalent binding). The so-called binding affinity can be determined by bilayer interferometry.

[00235] In one embodiment, the antibody used in the pharmaceutical composition of the invention can also bind to CD30 from Cynomolgus (SEQ ID NO:40).

[00236] As described above, the multispecific antibody used in Petition 870250088813, dated 09 / 30 / 2025, page 330 / 496 / 205. The pharmaceutical composition according to the invention comprises an antigen-binding region capable of binding to human CD3. Furthermore, the invention provides an antibody used in the pharmaceutical composition according to the invention capable of binding to human CD3ε (epsilon), as specified in SEQ ID NO:42. This antigen-binding region is capable of binding to human CD3ε (epsilon), as presented on a T cell, such as a human primary T cell.

[00237] As described, the antibody comprises a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2, and CDR3 sequences established at SEQ ID NO:7, 8, and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2, and CDR3 sequences established at SEQ ID NO:10, 11, and 12, respectively.

[00238] In one embodiment, the antigen-binding region that binds to CD3 comprises: • a (second) variable heavy chain (VH) region comprising the sequence of SEQ ID NO:15, or a sequence that has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:15; and, • a (second) variable light chain (VL) region comprising the sequence of SEQ ID NO:16, or a sequence that has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:16.

[00239] In a further embodiment, the antibody used in the pharmaceutical composition according to the invention comprises a variable heavy chain (VH) region of the antigen-binding region that binds to CD3 as defined herein, wherein the sequence comprises, in total, at most 1, 2, 3, 4, or 5 amino acid substitutions, when compared with Petition 870250088813, dated 09 / 30 / 2025, page 331 / 496 / 205 SEQ ID NO:15.

[00240] In a further embodiment, the antibody used in a pharmaceutical composition according to the invention comprises a variable light chain (VL) region of the antigen-binding region that binds to CD3 as defined herein, wherein the sequence comprises, in total, a maximum of 1, 2, 3, 4 or 5 amino acid substitutions, when compared with SEQ ID NO:16.

[00241] In a further embodiment, the second variable heavy chain region comprises the sequence set forth in SEQ ID NO:15 and the second variable light chain region comprises the sequence set forth in SEQ ID NO:16. In one embodiment, the X in SEQ ID NO:15 is an H. In another embodiment, the X in SEQ ID NO:15 is a G.

[00242] Such antigen-binding regions capable of binding to human CD3 have been described, among others, in WO2015 / 001085 (Genmab), which is incorporated herein by reference. Variants thereof, such as variants comprising a VH CDR3 region as set forth in SEQ ID NO:9 where X is G, which have a lower affinity for binding to human CD3 than the parental antibody where X is H, have been described in Example 2 of WO2017 / 009442 (Genmab), which is incorporated herein by reference.

[00243] The antibody used in a pharmaceutical composition according to the invention can bind with a dissociation constant in the KD equilibrium between the antigen-binding region that binds to human CD3 and human CD3 that is in the range of 5 to 30 nM, as well as between 10 and 20 nM (for monovalent binding).

[00244] In one embodiment, the antibody used in a pharmaceutical composition of the invention can also bind to CD3 from Cynomolgus monkeys (SEQ ID NO:43).

[00245] In an additional embodiment, the pharmaceutical composition Petition 870250088813, dated 09 / 30 / 2025, page 332 / 496 / 205, comprises a multispecific antibody comprising: (i) a CD30 binding region comprising a first variable heavy chain region comprising the sequence set out in SEQ ID NO:13 and a first variable light chain region comprising the sequence set out in SEQ ID NO:14 and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the sequence set out in SEQ ID NO:15 and a second variable light chain region comprising the sequence set out in SEQ ID NO:16. Antibody formats included in the pharmaceutical composition

[00246] The multispecific antibody used in a pharmaceutical composition of the invention may have two or more specificities, such as two or three or more specificities. Furthermore, the multispecific antibody may have more than one copy of the antigen-binding region for CD3 and / or CD30. For example, in one embodiment, the antibody has two antigen-binding regions capable of binding to CD3, such as two identical binding regions that bind to CD3. For example, in another embodiment, the antibody has two antigen-binding regions that bind to CD30, such as two identical binding regions that bind to CD30. An additional antigen-binding region may, for example, be present in the form of an scFv covalently linked to the constant region.

[00247] In a preferred embodiment, the pharmaceutical composition of the invention comprises a multispecific antibody that is a bispecific antibody. Many different formats and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97) and by Labrijn et al. (2019 Nat Rev Drug Discov 18(8) 585-608). A bispecific antibody according to the present invention may not be limited to any particular bispecific format or method for its production. Petition 870250088813, dated 09 / 30 / 2025, p. 333 / 496 / 205

[00248] Examples of bispecific antibody molecules that may be comprised in a pharmaceutical composition according to the present invention comprise (i) a single antibody having two arms comprising different antigen-binding regions; (ii) a single-chain antibody with specificity for two different targets, for example, through two scFvs linked in tandem by an extra peptide linker; (iii) a dual variable domain antibody (DVD-Ig), wherein each light chain and heavy chain contains two variable domains in tandem through a short peptide linker (Wu et al., Generation and Characterization of a Dual Variable Domain Immuneglobulin (DVD-Ig™) Molecule, in: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically linked bispecific fragment (Fab')2; (v) a Tandab, which is a fusion of two single-chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on “dimerization and docking domain” in protein kinase A, which, when applied to Fabs, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, for example, two scFvs fused to both ends of a human Fab arm; and (ix) a diabody.

[00249] Additional examples of different classes of bispecific antibodies include, among others, (i) IgG-like molecules with complementary CH3 domains to form the heterodimerization; (ii) dual-targeting recombinant IgG-like molecules, in which both sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which complete IgG antibodies are fused to fragments Petition 870250088813, dated 09 / 30 / 2025, page 334 / 496 / 205 Fab extra or parts of Fab fragments; (iv) Fc fusion molecules, in which single-chain Fv molecules or stabilized diabodies are fused to heavy chain constant domains, Fc regions or parts thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together, fused to heavy chain constant domains, Fc regions or parts thereof; and (vi) scFv and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused together or to another protein or carrier molecule fused to heavy chain constant domains, Fc regions or parts thereof.

[00250] Examples and IgG-like molecules with complementary CH3 domain molecules include, among others, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and electrostatically matching molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), and LUZY molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10,1074 / jbc.M112.397869.Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (seeDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and the DuoBody® molecules (Genmab A / S, WO2011131746). Petition 870250088813, dated 09 / 30 / 2025, p. 335 / 496 / 205

[00251] Examples of dual-targeting recombinant IgG-like molecules include, among others, dual-targeting (DT) Ig molecules (WO2009058383), two-in-one antibody (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), kappa / lambda body™ molecules (NovImmune, WO2012023053) and CovX-body (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17,501-506.).

[00252] Examples of IgG fusion molecules include, among others, dual variable domain (DVD)-Ig molecules (Abbott, US7,612,181), dual-domain double-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), bispecific IgG-like molecules (ILCMone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

[00253] Examples of Fc fusion molecules include, among others, scFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):117988), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al., AACR 100th Annual meeting 2009 (Abstract no. 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-based DART) molecules (MacroGenics, WO2008157379, WO2010080538) and double-Fab (scFv)2 molecules (National Research Center for Antibody Medicine - China). Petition 870250088813, dated 09 / 30 / 2025, page 336 / 496 / 205

[00254] Examples of bispecific Fab fusion antibodies include, among others, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86.), dual-action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dockand-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[00255] Examples of scFv, diabody, and domain-based antibodies include, among others, bispecific T Cell Engager (BiTE) type antibody molecules (Micromet, WO2005061547), tandem diabody (TandAb) molecules (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), DART molecules (MacroGenics, WO2008157379, WO2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-type antibodies (AIT, ReceptorLogics), fusion of human serum albumin and scFv (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual-targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual-targeting heavy chain-only domain antibodies.

[00256] In one embodiment, the pharmaceutical composition of the present invention comprises a bispecific antibody that is a diabody, a crossbody, or a bispecific antibody obtained through a controlled exchange of the Fab arm (as described in WO2011131746 (Genmab)).

[00257] In one embodiment, the pharmaceutical composition of the present invention comprises an antibody that is a bispecific DuoBody® molecule (Genmab A / S, WO2011131746).

[00258] The multispecific antibody, as well as the bispecific antibody, used in pharmaceutical compositions of the present invention may be of any isotype. Petition 870250088813, dated 09 / 30 / 2025, p. 337 / 496 / 205 Exemplary isotypes include, among others, any of the human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably, antibodies can be selected to be of the human IgG1 isotype, as shown in the examples. Either of the constant regions, kappa or lambda, of the human light chain, or both, can be used, for example, the sequences established in SEQ ID Nos: 53 and 54. For example, in one embodiment, the light chain involved in CD30 binding comprises a constant kappa region and the light chain involved in CD3 binding comprises a constant lambda region.

[00259] Thus, in a further embodiment, the antigen-binding region capable of binding to human CD30 is composed of a heavy chain and a light chain, said heavy chain comprising said VH region and a constant region of IgG1 heavy chain and said light chain comprising said VL region and a constant region of kappa light chain; and wherein said antigen-binding region capable of binding to human CD3 is composed of a heavy chain and a light chain, said heavy chain comprising said VH region and a constant region of IgG1 heavy chain and said light chain comprising said VL region and a constant region of lambda light chain. In yet another embodiment, one constant region of IgG1 heavy chain is as defined in SEQ ID NO. 51 and the other is as defined in SEQ ID NO. 50, and wherein said constant region of kappa light chain is as defined in SEQ ID NO.53 and the so-called constant region lambda of the light chain is as defined in SEQ ID NO. 54.

[00260] In one embodiment, both heavy chains of an antibody used in the pharmaceutical compositions of the present invention are of the IgG1 isotype. In another embodiment, the two heavy chains of a bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. Preferably, bispecific antibodies may be selected to be of the human IgG1 isotype, as shown in the examples. Optionally and Petition 870250088813, dated 09 / 30 / 2025, p. 338 / 496 / 205 preferably, the sequences of the heavy chain and the Fc region of the selected isotype may be modified, preferably in the hinge region, CH2 and / or CH3, to allow the generation of bispecific antibodies and / or introduce inertness.

[00261] In one embodiment, the multispecific antibody used in the pharmaceutical compositions of the present invention comprises an Fc region consisting of a first and a second Fc polypeptide.

[00262] In one embodiment, the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain, and the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain.

[00263] The first and second Fc polypeptides may each be of any isotype, including any human isotype, such as an IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotype, or a mixed isotype. Preferably, the Fc region is of an IgG1, IgG2, IgG3, IgG4 isotype, or a mixed isotype. In one embodiment, said Fc region is a human IgG1 Fc region.

[00264] In a further embodiment, the multispecific antibody is a complete antibody as defined herein.

[00265] The antibodies used in the pharmaceutical compositions according to the present invention may comprise modifications in the Fc region to render the antibody inert or a non-activating antibody. Consequently, in the antibodies described herein, one or both heavy chains may be modified so that the antibody induces Fc-mediated effector function to a lesser degree compared to an antibody that is identical except for not comprising said modifications. Fc-mediated effector function can be measured by determining Fc-mediated CD69 expression in T cells (i.e., CD69 expression resulting from antibody-mediated Fcy receptor-dependent cross-linking with CD3). Petition 870250088813, dated 09 / 30 / 2025, p. 339 / 496 / 205, for example, as described in WO2015001085), by binding to Fcy receptors, by binding to C1q, or by inducing Fc-mediated cross-linking of FcyRs. In particular, the heavy chain constant sequences can be modified so that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to that of a wild-type (unmodified) antibody, wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay, for example, by flow cytometry as described in Example 3 of WO2015001085. Modifications to the constant sequences of the heavy and light chains can also result in reduced binding of C1q to said antibody.Compared to an unmodified antibody, the reduction can be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and C1q binding can be determined by ELISA. Furthermore, the Fc region can be modified so that said antibody mediates reduced Fc-mediated T cell proliferation compared to an unmodified antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100%, for example, in a linear portion of the curve where said T cell proliferation is measured in a PBMC-based functional assay.

[00266] A wide range of different non-activating antibody formats have been developed, in which amino acid substitutions, and combinations thereof, have been introduced into the constant region of the heavy chain of an IgG1 isotype antibody to eliminate Fc-mediated effector functions (e.g., Chiu et al., Antibodies 2019 Dec; 8(4): 55; Liu et al., Antibodies, 2020 Nov 17;9(4):64; 29(10):457-66; Shields et al., J Biol Chem.

[00267] Examples of amino acid positions that can be Petition 870250088813, dated 09 / 30 / 2025, p. 340 / 496 / 205 modified, for example, in an IgG1 isotype antibody, include the L234 and L235 positions. In one embodiment, the first and / or second Fc polypeptide comprise an amino acid substitution corresponding to the amino acid at the L234 and / or L235 positions in a human IgG1 heavy chain, wherein the substitutions are preferably for F and E, respectively, wherein the amino acid positions are as defined by the I numbering.

[00268] It is understood that, in addition to modifications at the L234 and L235 amino acid positions, additional positions may be modified. Thus, in a further embodiment, the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and the first and / or second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position G236 in a human IgG1 heavy chain, wherein the substitution is preferably for R.

[00269] In another embodiment, the first and second Fc polypeptides comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and the first and second Fc polypeptides further comprise an amino acid substitution corresponding to the amino acid at position G236 in a human IgG1 heavy chain, wherein the substitution is preferably for R.

[00270] In another embodiment, the first and second Fc polypeptides comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and the first and / or second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position D265 in a human IgG1 heavy chain, wherein the substitution Petition 870250088813, dated 09 / 30 / 2025, p. 341 / 496 / 205 is preferably for A.

[00271] In another embodiment, the first and second Fc polypeptides comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and the first and second Fc polypeptides further comprise an amino acid substitution corresponding to the amino acid at position D265 in a human IgG1 heavy chain, wherein the substitution is preferably for A.

[00272] In another embodiment, one of the first and second Fc polypeptides comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the other Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235E and D265 for F, E and A, respectively.

[00273] In a further embodiment, the first Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the second Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235E and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the Eu numbering.

[00274] For example, constant regions with such substitutions in the Fc region are provided, among others, in SEQ ID NO. 45, 46, 49, 50, 51 and 52, which can be compared with SEQ ID NO. 44, which does not have such substitution(s). In one embodiment, the antibody of the present invention comprises a sequence selected from the group consisting of SEQ ID NO: 45, 46, 49, 50, 51 and 52.

[00275] In one embodiment, the multispecific or bispecific antibody used in the pharmaceutical compositions of the present invention Petition 870250088813, dated 09 / 30 / 2025, pp. 342 / 496 / 205, comprises an Fc region comprising a first and a second CH3 region that are different and are such that the heterodimeric interaction between said first and said second CH3 region is stronger than each of the homodimeric interactions of said first and said second CH3 region. Further details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference. Stable heterodimeric antibodies can be obtained in high yield, for example, by so-called Fab arm exchange, as provided in WO 2008 / 119353 and WO 2011 / 131746, based on two initial homodimeric antibodies containing only a few asymmetric mutations in the CH3 regions.

[00276] Consequently, in one embodiment, in the first Fc polypeptide, at least one of the amino acids at positions corresponding to a position selected from the group consisting of: T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain has been substituted and, in the second Fc polypeptide, at least one of the amino acids at positions corresponding to a position selected from the group consisting of: T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain has been substituted, and wherein said substitutions in the first and second Fc polypeptides are not at the same positions, wherein the amino acid positions are as defined by the I numbering. For example, constant regions containing such substitutions in the Fc region are provided, among others, in SEQ ID NO. 47, 48, 49, 50, 51 and 52, which can be compared with SEQ ID NO. 44, which does not have such a replacement.In one embodiment, the antibody of the present invention comprises a sequence selected from the group consisting of SEQ ID NO: 47, 48, 49, 50, 51 and 52.

[00277] In specific embodiments, the present invention provides a pharmaceutical composition comprising an antibody, wherein the Petition 870250088813, dated 09 / 30 / 2025, page 343 / 496 / 205 The amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in said first Fc polypeptide, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in said second Fc polypeptide, or vice versa. Preferably, the amino acid at the position corresponding to F405 is L in the first Fc polypeptide and the amino acid at the position corresponding to K409 is R in the second Fc polypeptide, or vice versa.

[00278] In a further embodiment, the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

[00279] Thus, in one embodiment, one of the first and second Fc polypeptides comprises amino acid substitutions corresponding to the amino acids at positions L234, L235, G236 and F405 for F, E, R and L, respectively, and the other Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235E, D265 and K409 for F, E, A and R, respectively.

[00280] In another embodiment, one of the first and second Fc polypeptides comprises substitutions of the amino acids corresponding to the amino acids at positions L234, L235, G236 and K409 for F, E, R and R, respectively, and the other Fc polypeptide comprises substitutions of the amino acids corresponding to the amino acids at positions L234, L235E, D265 and F405 for F, E, A and L, respectively.

[00281] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a multispecific antibody comprising: (i) a CD30 binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences set out in SEQ ID NO:1, 2 and 3, Petition 870250088813, dated 09 / 30 / 2025, pp. 344 / 496 / 205 respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively; wherein the multispecific antibody is a bispecific antibody and comprises an Fc region composed of a first and a second Fc polypeptide; wherein the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain, and wherein the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain; wherein the first Fc polypeptide comprises substitutions of the amino acids corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the second Fc polypeptide comprises substitutions of the amino acids corresponding to the amino acids at positions L234, L235 and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the Eu numbering; and wherein the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and wherein the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

[00282] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a multispecific antibody comprising: (i) a CD30 binding region comprising a Petition 870250088813, dated 09 / 30 / 2025, page 345 / 496 / 205 first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively; wherein the multispecific antibody is a bispecific antibody and comprises an Fc region composed of a first and a second Fc polypeptide; wherein the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain, and wherein the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain; wherein the first Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and D265 for F, E and A, respectively, and the second Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the Eu numbering; and wherein the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and wherein the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

[00283] In one embodiment, the antibody used in pharmaceutical compositions of the present invention comprises or consists of the sequences Petition 870250088813, dated 09 / 30 / 2025, p. 346 / 496 / 205 of heavy chain sequences established in SEQ ID NO:17 and 19 and in the light chain sequences established in SEQ ID NO:18 and 20.

[00284] In a further embodiment, the antibody used in pharmaceutical compositions of the present invention comprises or consists of the heavy chain sequences set forth in SEQ ID NO:17 and 19 and the light chain sequences set forth in SEQ ID NO:18 and 20, and said antibody is a bispecific antibody.

[00285] In one embodiment, the antibody used in pharmaceutical compositions of the present invention comprises or consists of the heavy chain sequences set forth in SEQ ID NO:17 and 35 and the light chain sequences set forth in SEQ ID NO:18 and 20.

[00286] In one embodiment, the antibody used in pharmaceutical compositions of the present invention comprises or consists of the heavy chain sequences set forth in SEQ ID NO:55 and 19 and the light chain sequences set forth in SEQ ID NO:18 and 20.

[00287] In one embodiment, the antibody used in pharmaceutical compositions of the present invention comprises or consists of the heavy chain sequences set forth in SEQ ID NO:55 and 35 and the light chain sequences set forth in SEQ ID NO:18 and 20.

[00288] In yet another embodiment, the antibody used in pharmaceutical compositions of the present invention is bsG1-huCD3FEALxCD30-MDX060-FEAR or bsG1-huCD3-FEALxCD30-MDX060FERR. In yet another embodiment, the antibody used in pharmaceutical compositions of the present invention is bsG1-huCD3FEALxCD30-MDX060-FERR. In yet another embodiment, the multispecific antibody is bsIgG1-huCD3-FEALxCD30-MDX0060-FERR, or a biosimilar thereof.

[00289] The constant region sequences listed in SEQ ID Nos: 44-52 and 55 do not include a C-terminal (K) lysine. However, in Petition 870250088813, dated 09 / 30 / 2025, page 347 / 496 / 205 natural sequences found in humans from which these Fc regions are derived, such C-terminal lysine may be present as part of the open reading frame. During cell culture production of recombinant antibodies, this terminal lysine may be cleaved and removed by proteolysis by endogenous carboxypeptidase(s), resulting in a constant region that has the same sequence, but without the C-terminal lysine. For antibody production purposes, the DNA encoding this terminal lysine may be omitted from the sequence so that such antibodies are produced without the lysine. Omitting the C-terminal lysine from the antibody-coding sequence may increase antibody homogeneity with respect to the presence of C-terminal lysine.Antibodies produced from nucleic acid sequences that encode or do not encode a terminal lysine are substantially identical in sequence and function, since the degree of processing of the C-terminal lysine is typically high when, for example, antibodies produced in CHO-based production systems are used (Dick, LW et al. Biotechnol. Bioeng. 2008;100: 1132-1143). Consequently, it is understood that antibodies used in pharmaceutical compositions according to the present invention can be generated without encoding or having a C-terminal lysine as listed herein. For production purposes, antibodies can therefore be generated without having a C-terminal lysine.

[00290] In alternative embodiments, the multispecific antibody used in pharmaceutical compositions according to the present invention is not a classic complete antibody comprising an Fc region. For example, in one embodiment, (i) the CD30-binding region and / or the CD3-binding region is a Fab, (ii) the CD30-binding region and / or the CD3-binding region is a scFv, Petition 870250088813, dated 09 / 30 / 2025, page 348 / 496 / 205 (iii) the CD30 binding region is Fab and the CD3 binding region is an scFv or (iv) the CD30 binding region is an scFv and the CD3 binding region is a Fab. T cell binding, cytotoxicity, and activation

[00291] Pharmaceutical compositions comprising antibodies, such as bispecific antibodies, as described herein, which can bind to human CD3 and human CD30 can be advantageously used to target T cells to human CD30-expressing cancer cells, thereby inducing T cell-mediated killing of said cancer cells.

[00292] As stated, preferably, the antibody used in pharmaceutical compositions according to the present invention is inert and, in addition, the antibody comprises one or more of the following features: a) is capable of binding to human tumor cells expressing CD30, such as SU-DHL-1 cells, SUP-M2 cells, DL-40 cells, Karpas-299 cells, L-82 cells, SR-786 cells, L-540 cells, KMH2 cells, L-1236 cells, JVM-2 cells, HH cells, NCEB-1 cells and / or HDLM-2 cells, when tested using flow cytometry, for example, as described in the Examples herein, b) is capable of mediating concentration-dependent cytotoxicity of human tumor cells expressing CD30 using, for example, purified PBMCs, ADCC effector cells or T cells as effector cells, when analyzed as described in the Examples herein, (c) is capable of mediating concentration-dependent cytotoxicity of one or more tumor cell lines expressing human CD30 selected from the group consisting of: SUDHL-1 cells, L-428 cells, KM-H2 cells, SUP-M2 cells, KI-JK cells and HDLM-2 cells, using, for example, purified PBMCs or T cells, when analyzed as described in the Examples herein, Petition 870250088813, dated 09 / 30 / 2025, pp. 349 / 496 100 / 205 (d) It is capable of inducing T cell proliferation in vitro in the presence of human tumor cells expressing CD30; for example, when analyzed as described in the Examples herein, (e) is capable of activating T cells in vitro in the presence of one or more human tumor cell lines expressing CD30, selected from the group consisting of: SU-DHL-1 cells, L428 cells, KI-JK cells and HDLM-2 cells, when analyzed as described in the Examples herein, f) is capable of inducing a dose-dependent in vitro production of cytokines and Granzyme B by T cells, when analyzed as described in the Examples herein, (g) is capable of not resulting in T cell fratricide even though CD30 is expressed in a subpopulation of activated T cells, when analyzed as described in the Examples herein and / or h) is capable of inducing T cell-mediated cytotoxicity even in the presence of sCD30, when analyzed as described in the Examples herein.

[00293] In one embodiment, the antibody described herein induces T-cell mediated cytotoxicity with an EC50 concentration less than 0.050 pg / ml, such as less than 0.045 pg / ml, less than 0.040 pg / ml, less than 0.035 pg / ml, less than 0.030 pg / ml, less than 0.025 pg / ml, less than 0.020 pg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry.

[00294] In a further embodiment, the antibody described herein exhibits maximal lysis when inducing T-cell mediated cytotoxicity above 80%, as above 85%, as above 90%, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry. Petition 870250088813, dated 09 / 30 / 2025, pp. 350 / 496 101 / 205

[00295] In one embodiment, the antibody described herein induces activation of CD4+ T cells with an EC50 concentration of CD25 expression less than 0.05 pg / ml, such as less than 0.045 pg / ml, less than 0.04 pg / ml, less than 0.035 pg / ml, or less than 0.03 pg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry. In a further embodiment, the antibody described herein induces activation of CD8+ T cells with an EC50 concentration of CD25 expression of less than 0.005 pg / ml, as well as less than 0.001 pg / ml, as well as less than 0.0009 pg / ml, as well as less than 0.0008 pg / ml, as well as less than 0.0007 pg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry.

[00296] In one embodiment, the antibody described herein induces activation of CD4+ T cells with an EC50 concentration of CD69 expression less than 0.004 pg / ml, as less than 0.003 pg / ml, as less than 0.002 pg / ml, as less than 0.001 pg / ml, as less than 0.0009 pg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry. In a further embodiment, the antibody described herein induces activation of CD8+ T cells with an EC50 concentration of CD69 expression of less than 0.0035 pg / ml, as well as less than 0.0030 pg / ml, as well as less than 0.0025 pg / ml, as well as less than 0.0020 pg / ml, as well as less than 0.0015 pg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry.

[00297] In one embodiment, the antibody described herein induces activation of CD4+ T cells with an EC50 concentration of PD-1 expression less than 0.01 pg / ml, less than 0.008 pg / ml, less than 0.007 pg / ml, less than 0.006 pg / ml, less than 0.005 pg / ml, when analyzed as described in the Examples hereof, using L-428 tumor cells and Petition 870250088813, dated 09 / 30 / 2025, pp. 351 / 496 102 / 205 measured by flow cytometry. In a further embodiment, the antibody described herein induces activation of CD8+ T cells with an EC50 concentration of PD-1 expression less than 0.007 μg / ml, as less than 0.006 μg / ml, as less than 0.005 μg / ml, as less than 0.004 μg / ml, as less than 0.003 μg / ml, when analyzed as described in the Examples herein, using L-428 tumor cells and measured by flow cytometry.

[00298] The assays may be performed as commonly known to those skilled in the art and may optionally comprise an effector-to-target (E:T) cell ratio of 4:1 and / or, optionally, an incubation period of 72 hours. The assays could be performed, for example, by providing tumor cells, preferably labeled tumor cells, such as L-428 tumor cells; adding the antibody described herein, preferably in a serial dilution; providing T cells, preferably labeled T cells, in an effector-to-target E:T ratio of 4:1 and incubating for 72 hours; optionally, staining for relevant parameters such as CD4, CD8, CD25, CD69 and / or PD-1 and, finally, analyzing the cells with flow cytometry such as FACS. The % live cells and / or T cell proliferation / activation may be calculated from the data obtained by the analysis as commonly known to those skilled in the art. Production of antibodies used in pharmaceutical compositions of the present invention.

[00299] Traditional methods such as hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be employed in the preparation of multispecific, as well as bispecific, antibodies used in pharmaceutical compositions of the present invention. Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be Petition 870250088813, dated 09 / 30 / 2025, pp. 352 / 496 103 / 205 alone, for example, by affinity chromatography or similar methods.

[00300] As mentioned, strategies that favor the formation of a bispecific functional product when different antibody constructs are co-expressed can also be used, for example, the method described by Lindhofer et al. (1995 J Immunol 155:219). The fusion of rat and mouse hybridomas that produce different antibodies leads to a limited number of heterodimeric proteins because of the species-restricted preferential heavy / light chain pairing. Another strategy to promote heterodimer formation relative to homodimer formation is a "knob-into-hole" strategy in which a protrusion is introduced into a first heavy chain polypeptide and a corresponding cavity into a second heavy chain polypeptide, such that the protrusion can be positioned in the cavity at the interface of these two heavy chains to promote heterodimer formation and prevent homodimer formation."Protrusions" are constructed by replacing small amino acid chains at the interface of the first polypeptide with larger side chains. Compensatory "cavities" of identical or similar size to the protrusions are created at the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (US Patent 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies to favor heterodimer formation when different antibody domains co-express in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced by a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describes yet another strategy, in which differences between IgA and IgG CH3 domains are exploited to promote heterodimerization. Petition 870250088813, dated 09 / 30 / 2025, pp. 353 / 496 104 / 205

[00301] Another in vitro method for producing bispecific antibodies was described in WO2008119353 (Genmab), in which a bispecific antibody is formed by an exchange of “Fab arm” or “half molecule” (exchange of a heavy chain and attached light chain) between two monospecific IgG4 or IgG4-like antibodies when incubated under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may comprise different sequences.

[00302] A preferred method for preparing the bispecific CD3xCD30 antibodies used in pharmaceutical compositions of the present invention includes methods described in WO2011131746 and WO13060867 (Genmab) comprising the following steps: a) to provide a first antibody comprising an Fc region, said Fc region comprising a first CH3 region; (b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein the first antibody is an antibody against CD30 and the second antibody is an antibody against CD3, or vice versa; wherein the sequences of said first and said second CH3 regions are different and are such that the heterodimeric interaction between said first and said second CH3 regions is stronger than each of the homodimeric interactions of said first and said second CH3 regions; c) incubate said first antibody together with said second antibody under reducing conditions; and d) obtain the aforementioned bispecific CD3xCD30 antibody.

[00303] Similarly, a multispecific antibody, such as a bispecific one, used in pharmaceutical compositions according to the present invention, can be produced by the following method, comprising: a) to provide a first homodimeric antibody comprising the CD30-binding region, as described herein, and a Petition 870250088813, dated 09 / 30 / 2025, pp. 354 / 496 105 / 205 second homodimeric antibody comprising the CD3-binding region as described herein, wherein said antibodies comprise an Fc region and optionally contain additional features described herein, wherein the sequences of the first and second CH3 regions of the first and second antibodies are different and are such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; b) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo isomerization of the disulfide bonds; and c) to obtain a heterodimeric multispecific antibody of the present invention as described herein, comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody.

[00304] In one embodiment, said first antibody together with said second antibody is incubated under reducing conditions sufficient to allow the cysteines in the hinge region to undergo isomerization of the disulfide bonds, wherein the heterodimeric interaction between said first and said second antibody in the resulting heterodimeric antibody is such that no exchange of Fab arms occurs in 0.5 mM GSH after 24 hours at 37°C.

[00305] Without being limited to theory, in step c), the disulfide bonds in the hinge regions of the heavy chain of the parental antibodies are reduced and the resulting cysteines are then able to form disulfide bonds between heavy chains with cysteine ​​residues of another parental antibody molecule (originally with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, for example, a reducing agent selected from Petition 870250088813, dated 09 / 30 / 2025, pp. 355 / 496 106 / 205 from the group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-c-carboxyethyl)phosphine (TCEP), L-cysteine ​​and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-c-carboxyethyl)phosphine. In a preferred embodiment, the reducing agent is 2-mercaptoethylamine. In a further embodiment, step c) comprises restoring the conditions to become non-reducing or less reducing, for example, by removing a reducing agent, for example, by desalination.

[00306] In a further aspect, the multispecific antibody used in pharmaceutical compositions according to the present invention can be produced by a method comprising the following steps: a) to provide a first monospecific antibody against CD30 comprising (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and an amino acid substitution corresponding to the amino acid at position D265 for A in a human IgG1 heavy chain; and b) to provide a second monospecific antibody against CD3 comprising (i) a CD3-binding region comprising a second Petition 870250088813, dated 09 / 30 / 2025, pp. 356 / 496107 / 205 variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively, and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and the amino acid substitution corresponding to the amino acid at position D265 for A in a human IgG1 heavy chain, wherein the sequences of the first and second CH3 regions of the first and second antibody are different and are such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions;whereby preferably the amino acid at the position corresponding to F405 is L in the first CH3 region and the amino acid at the position corresponding to K409 is R in the second CH3 region, or vice versa; c) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo isomerization of the disulfide bonds; and d) obtain the multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody.

[00307] In a further aspect, the multispecific antibody used in pharmaceutical compositions according to the present invention can be produced by a method comprising the following steps: a) to provide a first monospecific antibody against CD30 Petition 870250088813, dated 09 / 30 / 2025, pp. 357 / 496 108 / 205 comprising (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and an amino acid substitution corresponding to the amino acid at position G236 for R in a human IgG1 heavy chain; and (b) provide a second monospecific antibody against CD3 comprising (i) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2, and CDR3 sequences established in SEQ ID NO: 7, 8, and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2, and CDR3 sequences established in SEQ ID NO: 10, 11, and 12, respectively, and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second Fc polypeptides comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and an amino acid substitution corresponding to the amino acid at position D265 for A in a human IgG1 heavy chain wherein the sequences of the first and second CH3 regions of the first and second antibodies are different and are such that the interaction Petition 870250088813, dated 09 / 30 / 2025, pp. 358 / 496 The heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; wherein preferably the amino acid at the position corresponding to K409 is R in the first CH3 region and the amino acid at the position corresponding to F405 is L in the second CH3 region. c) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo isomerization of the disulfide bonds; and d) obtain the multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody.

[00308] The present invention further relates to a multispecific antibody, used in the pharmaceutical compositions according to the present invention, which is obtained by a method as described herein.

[00309] In the method above, the steps of providing a first or second homodimeric antibody capable of binding to CD30 and / or CD3 may include the steps of: - to provide cells containing expression vectors to produce said antibody or antibodies; and - to allow the cells to produce said antibody or antibodies and, subsequently, - to obtain the said antibody or antibodies, thereby providing the said antibody or antibodies.

[00310] In one embodiment of this method, the said first and / or said second homodimeric antibody are complete antibodies.

[00311] The Fc regions of the first and second homodimeric antibodies can be of any isotype, including, but not limited to, IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of Petition 870250088813, dated 09 / 30 / 2025, pp. 359 / 496 110 / 205 In both, the said first and said second homodimeric antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of said homodimeric antibodies is of the IgG1 isotype and the other of the IgG4 isotype. In the last embodiment, the resulting bispecific antibody comprises an Fc region of an IgG1 and an Fc region of IgG4 and may therefore possess interesting intermediate properties with respect to the activation of effector functions.

[00312] In an additional embodiment, one of the initial homodimeric antibodies was modified so as not to bind to protein A, thus allowing the heterodimeric antibody to be separated from the initial homodimeric antibody by passing the product through a protein A column, removing the passing stream, and eluting the heterodimeric antibody from the protein A column to obtain a purified heterodimeric antibody composition.

[00313] As described above, the sequences of the first and second CH3 regions of the initial homodimeric antibodies may be different such that the heterodimeric interaction between said first and said second CH3 regions is stronger than each of the homodimeric interactions of said first and said second CH3 regions. Further details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein in their entirety by reference.

[00314] In particular, a stable bispecific CD3xCD30 antibody can be obtained in high yield using the above method of the present invention, based on two initial homodimeric antibodies that bind to CD30 and CD3, respectively, and contain only a few reasonably conservative, asymmetric mutations in the CH3 regions. Asymmetric mutations mean that the sequences of said first and said second CH3 regions contain amino acid substitutions at non-identical positions.

[00315] Multispecific antibodies, like bispecific antibodies, used Petition 870250088813, dated 09 / 30 / 2025, pp. 360 / 496 111 / 205 in the pharmaceutical compositions of the present invention can also be obtained by co-expression of constructs encoding the first and second polypeptide in a single cell.

[00316] Thus, in a further aspect, the present invention relates to a nucleic acid construct, or a combination of nucleic acid constructs, encoding a multispecific antibody used in the pharmaceutical compositions according to the present invention and to an expression vector, or a combination of expression vectors, comprising such nucleic acid construct(s).

[00317] Furthermore, the present invention relates to a recombinant host cell capable of producing the multispecific antibody used in the pharmaceutical compositions according to the present invention, wherein the host cell comprises one or more nucleic acid constructs encoding the multispecific antibody used in the pharmaceutical compositions according to the present invention.

[00318] Consequently, the present invention also relates to a method for producing a multispecific antibody used in pharmaceutical compositions according to the present invention, comprising: (i) cultivate a recombinant host cell of the present invention under conditions in which the antibody is produced and (ii) isolate the multispecific antibody produced from the culture.

[00319] In one embodiment, the said method comprises the following steps: a) to provide a first nucleic acid construct encoding a first polypeptide comprising a first Fc region and a first antigen-binding region of a first antibody heavy chain, said first Fc region comprising a first CH3 region, Petition 870250088813, dated 09 / 30 / 2025, pp. 361 / 496 112 / 205 (b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region and a second antigen-binding region of a heavy chain of the second antibody, said second Fc region comprising a second CH3 region, wherein the sequences of said first and said second CH3 regions are different and are such that the heterodimeric interaction between said first and said second CH3 regions is stronger than each of the homodimeric interactions of said first and said second CH3 regions, optionally wherein said first and said second nucleic acid constructs encode light chain sequences of said first and said second antibody, c) co-express said first and said second nucleic acid constructs in a host cell and d) obtain said heterodimeric protein from cell culture.

[00320] Preferably, the amino acid encoded at the position corresponding to F405 is L in the first CH3 region and the amino acid encoded at the position corresponding to K409 is R in the second CH3 region, or vice versa.

[00321] Suitable expression vectors, including promoters, enhancers, etc., and suitable host cells for antibody production are well known in the art. Examples of host cells include yeast cells, bacteria, and mammalian cells, such as Chinese hamster ovary (CHO) cells, or human cells, such as human embryonic kidney (HEK) cells.

[00322] The nucleic acid or one or more nucleic acids, as defined herein, may be RNA or DNA. The nucleic acid or one or more nucleic acids, as defined herein, may be for use in expression in mammalian cells. Consequently, the present invention provides a cell or cells comprising a nucleic acid or comprising one or Petition 870250088813, dated 09 / 30 / 2025, pp. 362 / 496 113 / 205 more nucleic acids, as defined herein.

[00323] The nucleic acid in the context of the present invention may be an expression vector, which may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include vectors derived from SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA and viral nucleic acid (RNA or DNA).In one embodiment, a nucleic acid encoding an antibody against CD30 or against CD3 is comprised within a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for example, Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compact nucleic acid vector (as described in, for example, US 6,077,835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC 19 / 18 or pUC 118 / 119, a minimally sized “midge” nucleic acid vector (as described in, for example, Schakowski et al., Mol Ther 3, 793 800 (2001)), or in a precipitated construct of a nucleic acid vector, such as a precipitated construct with CaPO4 (as described in, for example, WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)).Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[00324] In one embodiment, the vector is suitable for expression of antibody against CD30 and / or antibody against CD3 in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript vectors (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vectors (Novagen, Madison WI) and the like. Petition 870250088813, dated 09 / 30 / 2025, pp. 363 / 496 114 / 205

[00325] An expression vector may also, or alternatively, be a suitable vector for expression in a yeast system. Any suitable vector for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as factor alpha, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516 544 (1987)).

[00326] A nucleic acid and / or expression vector may also comprise a nucleic acid sequence encoding a secretion / localization sequence, which may target a polypeptide, such as a polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretion or signal leader peptides. The nucleic acid and / or expression vector may comprise any suitable elements that facilitate expression, i.e., the transcription and / or translation of the nucleic acid so that the (bispecific) antibody components are expressed. The nucleic acid and / or vector may be associated with any promoter, enhancer, and other elements that facilitate expression.Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer, as well as the RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, a replication origin for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a poly-linker). Nucleic acids may also comprise an inducible promoter, as opposed to a constitutive promoter, such as the CMV IE. Uses (medical)

[00327] The pharmaceutical composition can be administered by Petition 870250088813, dated 09 / 30 / 2025, pp. 364 / 496 115 / 205 any suitable route and method. In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[00328] The pharmaceutical composition according to the present invention is preferably for use as a medicament.

[00329] The pharmaceutical composition according to the present invention is preferably for use in a method for treating a disease.

[00330] In particular, the pharmaceutical composition of the present invention can be used for the treatment of various forms of cancer. In one aspect, the present invention relates to the use of the pharmaceutical composition described herein for the treatment of cancer. In a further aspect, the present invention relates to a method for treating a disease, wherein the method comprises administering a pharmaceutical composition as defined herein to an individual in need thereof. In yet another further aspect, the present invention relates to a method for treating cancer in an individual comprising administering to an individual in need thereof the pharmaceutical composition described herein for a time sufficient to treat the cancer.

[00331] In one aspect, the present invention provides a method for treating cancer in an individual, which method comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention. In a further embodiment, the present invention provides a method for treating a disorder involving cells expressing CD30 in an individual, which method comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

[00332] As stated, the suitable diseases that can be contemplated in methods and uses according to the present invention are cancer. The said Petition 870250088813, dated 09 / 30 / 2025, pp. 365 / 496 116 / 205 The most preferable cancer is distinguished by CD30 expression. CD30 expression in cancer can be easily determined using methods known in the art, such as PCR, immunostaining, or FACS analysis, i.e., detecting the expression of the CD30 transcript and / or protein. The antibodies described here that are capable of binding to human CD30 can be used, for example, in immunostaining and / or FACS analysis or similar methods.

[00333] In one aspect, the present invention relates to the pharmaceutical composition according to the present invention for use in the treatment of Hodgkin's lymphoma or anaplastic large cell lymphoma.

[00334] In a further aspect, the present invention relates to the pharmaceutical composition according to the present invention for use in the treatment of Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).

[00335] In one form, Hodgkin lymphoma is a classical Hodgkin lymphoma (cHL),

[00336] In one embodiment, non-Hodgkin lymphoma is either T-cell non-Hodgkin lymphoma (T-NHL) or B-cell non-Hodgkin lymphoma (B-NHL). In a further embodiment, non-Hodgkin lymphoma is T-cell non-Hodgkin lymphoma (T-NHL).

[00337] In an additional embodiment, T-cell non-Hodgkin lymphoma is either peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma (CTCL). In yet another embodiment, T-cell non-Hodgkin lymphoma (T-NHL) is anaplastic large cell lymphoma (ALCL). In yet another embodiment, peripheral T-cell lymphoma (PTCL) is anaplastic large cell lymphoma (ALCL).

[00338] In one embodiment, B-cell non-Hodgkin lymphoma (B-NHL) is mantle cell lymphoma (MCL).

[00339] In yet another additional form, Hodgkin lymphoma (HL) is relapsed and refractory Hodgkin lymphoma. In yet another form, Hodgkin lymphoma (HL) is CD30+ Hodgkin lymphoma. In Petition 870250088813, dated 09 / 30 / 2025, pp. 366 / 496 117 / 205 yet another modality, Hodgkin lymphoma (HL) is a recurrent and refractory CD30+ Hodgkin lymphoma. In yet another additional modality, Hodgkin lymphoma (HL) is a classic recurrent and refractory CD30+ Hodgkin lymphoma.

[00340] In yet another additional form, non-Hodgkin lymphoma (NHL) is relapsed and refractory non-Hodgkin lymphoma. In yet another form, non-Hodgkin lymphoma (NHL) is CD30+ non-Hodgkin lymphoma. In yet another form, non-Hodgkin lymphoma (NHL) is relapsed and refractory CD30+ non-Hodgkin lymphoma.

[00341] In a further embodiment, the pharmaceutical composition according to the present invention for use in the treatment of Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) is or was administered intravenously and / or subcutaneously, preferably subcutaneously.

[00342] In a further embodiment, a patient diagnosed with cancer may undergo an assessment of CD30 expression in cancer cells and, when CD30 is detected, which may be in the low to high range, such a patient may be selected for treatment with a pharmaceutical composition according to the present invention. However, it may not necessarily be a requirement to include such an assessment in selecting a patient for treatment.

[00343] The pharmaceutical composition of the present invention comprising multispecific antibodies has numerous in vitro and in vivo diagnostic and therapeutic uses involving the diagnosis and treatment of disorders involving cells expressing CD30. For example, multispecific antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to individuals, e.g., in vivo, to treat, prevent and / or diagnose a variety of disorders. In this descriptive report, the term “individual” is intended to include both human and non-human individuals. Petition 870250088813, dated 09 / 30 / 2025, pp. 367 / 496 118 / 205

[00344] In one aspect, the present invention relates to a diagnostic composition comprising a pharmaceutical composition according to any of the embodiments described herein.

[00345] In one embodiment, the diagnostic composition is a complementary diagnostic tool that is used to screen and select those patients who will benefit from treatment with the pharmaceutical composition. Kits

[00346] The present invention further provides a kit in parts comprising a pharmaceutical composition comprising an antibody as described above, as a kit for use as a complementary diagnostic tool / to identify within a patient population those patients likely to respond to treatment with an antibody as defined above, or to predict the efficacy or antitumor activity of said antibody when used in the treatment of a patient, wherein the kit comprises an antibody as defined above; and instructions for use of said kit. In one aspect, the kit in parts comprises a pharmaceutical composition described herein, a container for the pharmaceutical composition, and instructions for use of said kit. In a further embodiment, the kit in parts further comprises a diluent.

[00347] In one embodiment, the present invention provides a cancer diagnostic kit comprising a container for the pharmaceutical composition comprising a multispecific CD3xCD30 antibody and, optionally, one or more reagents for detecting the cross-reaction of cells expressing CD30 and cells expressing CD3. The reagents may include, for example, fluorescent markers, enzymatic markers or other detectable markers. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reagents, wherein the enzymatic reactions generate a product that can be Petition 870250088813, dated 09 / 30 / 2025, pp. 368 / 496 119 / 205 views.

[00348] In a further aspect, the present invention relates to a method for detecting whether cross-linking occurs between cells expressing CD30 and cells expressing CD3 in a sample derived from a patient, when administering a pharmaceutical composition according to any of the embodiments described herein comprising a multispecific antibody, comprising the steps of: (i) bring the sample into contact with a pharmaceutical composition, according to any of the embodiments described herein comprising the multispecific antibody, under conditions that permit the formation of a complex between said bispecific antibody and cells expressing CD30 and cells expressing CD3; and (ii) analyze whether a complex has been formed. Types of a pharmaceutical composition

[00349] In the embodiments below, multispecific antibody or antibody refers to a multispecific antibody comprising an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3.

[00350] In one embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) a buffering agent, c) optionally, a non-ionic excipient and d) optionally, a surfactant; wherein the pH of the composition is from approximately 5.0 to approximately 6.5.

[00351] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) acetate or histidine, c) sorbitol, trehalose or sucrose, d) a polysorbate; wherein the pH Petition 870250088813, dated 09 / 30 / 2025, pp. 369 / 496 The 120 / 205 composition is approximately 5.0 to approximately 6.5.

[00352] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) histidine, c) sucrose and d) a polysorbate; wherein the pH of the composition is approximately 6.0.

[00353] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) acetate, c) trehalose and d) a polysorbate; wherein the pH of the composition is approximately 5.5.

[00354] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) acetate, c) sorbitol and d) a polysorbate; wherein the pH of the composition is approximately 5.5.

[00355] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) histidine, c) sucrose and d) polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00356] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) acetate, c) sorbitol and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00357] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) Petition 870250088813, dated 09 / 30 / 2025, pp. 370 / 496 121 / 205 acetate, c) trehalose and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00358] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) histidine, c) sucrose and d) polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00359] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) acetate, c) sorbitol and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00360] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) acetate, c) trehalose and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00361] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) histidine, c) sucrose and d) polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00362] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) acetate, c) sorbitol and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00363] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: Petition 870250088813, dated 09 / 30 / 2025, pp. 371 / 496 122 / 205 a) approximately 150-190 mg / ml of a multispecific antibody, b) acetate, c) trehalose and d) polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00364] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, b) approximately 5 to 40 mM of acetate or histidine, c) approximately 100-350 mM of sorbitol, trehalose or sucrose, and d) approximately 0.01-0.1% w / v of polysorbate; wherein the pH of the composition is approximately 5.0 to approximately 6.5.

[00365] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) approximately 5-40 mM of acetate or histidine, preferably acetate, c) approximately 100-350 mM of sorbitol, trehalose or sucrose, preferably sorbitol, and d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is from approximately 5.5 to approximately 6.0.

[00366] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) approximately 5-40 mM of acetate or histidine, preferably acetate, c) approximately 100-350 mM of sorbitol, trehalose or sucrose, preferably sorbitol, and d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is from approximately 5.5 to approximately 6.0.

[00367] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) approximately 5-40 mM of acetate or histidine, preferably acetate, c) approximately 100-350 mM of sorbitol, trehalose or sucrose, preferably sorbitol, and d) Petition 870250088813, dated 09 / 30 / 2025, pp. 372 / 496 123 / 205 approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5 to approximately 6.0.

[00368] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 70 mg / ml of a multispecific antibody, b) approximately 20 mM acetate, c) approximately 250 mM sorbitol and d) approximately 0.02% w / v polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00369] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150 mg / ml of a multispecific antibody, b) approximately 20 mM acetate, c) approximately 250 mM sorbitol and d) approximately 0.02% w / v polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00370] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 170 mg / ml of a multispecific antibody, b) approximately 20 mM acetate, c) approximately 250 mM sorbitol and d) approximately 0.02% w / v polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00371] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 70 mg / ml of a multispecific antibody, b) approximately 17 mM of acetate, such as sodium acetate, c) approximately 250 mM of sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM of glacial acetic acid; where the pH of the composition is approximately 5.5.

[00372] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150 mg / ml of a multispecific antibody, b) approximately 20 mM of acetate, such as sodium acetate, c) approximately 250 mM of sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM of glacial acetic acid; where the pH of the composition is approximately 5.5. Petition 870250088813, dated 09 / 30 / 2025, pp. 373 / 496 124 / 205

[00373] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 170 mg / ml of a multispecific antibody, b) approximately 20 mM of acetate, such as sodium acetate, c) approximately 250 mM of sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM of glacial acetic acid; wherein the pH of the composition is approximately 5.5.

[00374] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00375] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of sorbitol and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00376] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00377] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH Petition 870250088813, dated 09 / 30 / 2025, pp. 374 / 496 125 / 205 of the composition is approximately 6.0.

[00378] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of sorbitol and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00379] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 135-165 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00380] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00381] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of sorbitol and, optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00382] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150-190 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose, and Petition 870250088813, dated 09 / 30 / 2025, pp. 375 / 496 126 / 205 optionally, d) approximately 0.01-0.05% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00383] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 70 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00384] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 70 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of sorbitol and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00385] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 70 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00386] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00387] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150 mg / ml of a multispecific antibody, b) Petition 870250088813, dated 09 / 30 / 2025, pp. 376 / 496 127 / 205 approximately 10-30 mM acetate, c) approximately 200-300 mM sorbitol and, optionally, d) approximately 0.01-0.03% w / v polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00388] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 150 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00389] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 170 mg / ml of a multispecific antibody, b) approximately 10-30 mM of histidine, c) approximately 200-300 mM of sucrose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 6.0.

[00390] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 170 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of sorbitol and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00391] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 170 mg / ml of a multispecific antibody, b) approximately 10-30 mM of acetate, c) approximately 200-300 mM of trehalose and, optionally, d) approximately 0.01-0.03% w / v of polysorbate 80; wherein the pH of the composition is approximately 5.5.

[00392] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: Petition 870250088813, dated 09 / 30 / 2025, pp. 377 / 496 128 / 205 a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, wherein the antibody comprises: (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively; optionally wherein X in SEQ ID NO: 9 is H; b) a buffering agent, such as histidine or acetate, c) optionally, a non-ionic excipient, such as sucrose, trehalose or sorbitol, and d) optionally, a surfactant such as a polysorbate; wherein the pH of the composition is from about 5.0 to about 6.5.

[00393] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, wherein the antibody comprises or consists of the heavy chain sequences established in SEQ ID NO:17 and 19 and the light chain sequences established in SEQ ID NO:18 and 20, wherein the multispecific antibody is a bispecific antibody, b) a buffering agent, such as histidine or acetate, Petition 870250088813, dated 09 / 30 / 2025, pp. 378 / 496 129 / 205 c) optionally, a non-ionic excipient, such as sucrose, trehalose or sorbitol, and d) optionally, a surfactant such as a polysorbate; wherein the pH of the composition is from about 5.0 to about 6.5.

[00394] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 0.5-250 mg / ml of a multispecific antibody, such as approximately 20-200 mg / ml of a multispecific antibody, wherein the antibody comprises: (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively; optionally wherein X in SEQ ID NO: 9 is H; b) acetate, such as about 10-30 mM acetate, c) optionally, sorbitol as approximately 200-300 mM of sorbitol and, d) optionally, polysorbate as approximately 0.01-0.05% w / v of polysorbate 80; where the pH of the composition is approximately 5.5.

[00395] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: Petition 870250088813, dated 09 / 30 / 2025, pp. 379 / 496 130 / 205 a) approximately 65-75 mg / ml of a multispecific antibody, such as approximately 70 mg / ml of a multispecific antibody, wherein the antibody comprises or consists of the heavy chain sequences established in SEQ ID NO:17 and 19 and the light chain sequences established in SEQ ID NO:18 and 20, wherein the multispecific antibody is a bispecific antibody, b) a buffering agent, such as histidine or acetate, c) optionally, a non-ionic excipient, such as sucrose, trehalose or sorbitol and, d) optionally, a surfactant such as a polysorbate; wherein the pH of the composition is from about 5.0 to about 6.5.

[00396] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 65-75 mg / ml of a multispecific antibody, such as approximately 70 mg / ml of a multispecific antibody, wherein the antibody comprises: (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively; optionally wherein X in SEQ ID NO: 9 is H; b) acetate, such as about 10-30 mM acetate, c) optionally, sorbitol at approximately 200-300 mM of Petition 870250088813, dated 09 / 30 / 2025, pp. 380 / 496 131 / 205 sorbitol and, d) optionally, polysorbate as approximately 0.01-0.05% w / v of polysorbate 80; where the pH of the composition is approximately 5.5.

[00397] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, wherein the antibody comprises or consists of the heavy chain sequences established in SEQ ID NO:17 and 19 and the light chain sequences established in SEQ ID NO:18 and 20, wherein the multispecific antibody is a bispecific antibody, b) about 10-30 mM acetate, c) around 200-300 mM sorbitol and d) approximately 0.01-0.05% w / v of polysorbate 80; where the pH of the composition is approximately 5.5.

[00398] In a further embodiment, the pharmaceutical composition described herein comprises or consists essentially of: a) approximately 60-80 mg / ml of a multispecific antibody, wherein the antibody comprises: (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2, and CDR3 sequences established in SEQ ID NO: 1, 2, and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2, and CDR3 sequences established in SEQ ID NO: 4, 5, and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2, and CDR3 sequences established in SEQ ID NO: 7, 8, and 9, respectively, and a second variable light chain region comprising the sequences of Petition 870250088813, dated 09 / 30 / 2025, pp. 381 / 496 132 / 205 CDR1, CDR2 and CDR3 established in SEQ ID NO:10, 11 and 12, respectively; optionally where X in SEQ ID NO: 9 is H; b) about 10-30 mM acetate, c) around 200-300 mM sorbitol and d) approximately 0.01-0.05% w / v of polysorbate 80; where the pH of the composition is approximately 5.5.

[00399] The present invention is further illustrated by the following examples, which should not be interpreted as limiting the scope of the present invention. Examples Example 1 - Generation of bispecific CD3xCD30 antibodies by 2-MEA-induced Fab arm switching

[00400] The following antibodies were used in the examples: Humanized anti-CD3 antibodies

[00401] IgG1-huCD3-H1L1 as described in Example 1 of WO2015 / 001085 (Genmab). IgG1-huCD3-H1L1 is referred to herein as 'IgG1huCD3'.

[00402] IgG1-huCD3-H1L1-H101G as described in Example 2 of WO2017 / 009442 (Genmab). IgG1-huCD3-H1L1-H101G is referred to herein as 'IgG1-huCD3-H101G'. Anti-CD30 antibodies

[00403] MDX-060, which is also referred to as HuMab 5F11, was described in WO2003 / 059282 (Medarex). hAC10 (or SGN-30) was described in US 8257706 and US20100239571 (Seattle Genetics). HRS-3 was described in WO2016 / 0177846 (Affimed). HeFi-I, T405, T105, T408, and T215 were described in WO 2007 / 040653 (United States Government and Department of Health). Antibody expression

[00404] Antibody sequences were cloned into vectors of Petition 870250088813, dated 09 / 30 / 2025, pp. 382 / 496 133 / 205 pcDNA3.3 expression (Invitrogen, USA) and expressed as IgG1,κ or IgG1,λ, with or without Fc silencing and / or DuoBody® technology with amino acid substitutions in the Fc domain (see below). All antibodies were produced under serum-free conditions by co-transfection of relevant heavy and light chain expression vectors into Expi293FTM cells (Thermo Fisher Scientific, USA; cat. no. A14527), using ExpiFectamine™293 (Thermo Fisher Scientific; cat. no. A14525), essentially as described by the manufacturer. Generation of bispecific antibodies

[00405] Bispecific antibodies were generated in vitro using the DuoBody® platform technology, i.e., controlled 2-MEA-induced Fab arm exchange (cFAE) as described in WO2011147986, WO2011131746 and WO2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS 2013, 110: 5145-50; Gramer et al., MAbs 2013, 5: 962-973).

[00406] To enable the generation of bispecific antibodies by this method, IgG1 molecules carrying a single mutation in the CH3 domain were generated: in one parental IgG1 antibody, the F405L mutation (i.e., anti-CD3 antibodies or control antibodies, HIV-1 gp120-specific), in another parental IgG1 antibody, the K409R mutation (i.e., anti-CD30 or control antibodies). In addition to these mutations, the parental IgG1 antibodies included substitutions that result in an Fc domain that is unable to interact with IgG Fc receptors (Fc gamma receptors) and / or complement factors such as C1q: L234F, L235E, D265A (FEA; US 2015 / 0337049) or L234F, L235E, G236R (FER).

[00407] The combination of Fc silencing and mutations with DuoBody® technology has been designated as follows: L234F, L235E, D265A and F405L: FEAL L234F, L235E, D265A and K409R: FEAR L234F, L235E, G236R and K409R: FERR Petition 870250088813, dated 09 / 30 / 2025, pp. 383 / 496 134 / 205

[00408] The heavy chain (HC) and light chain (LC) sequences of the parental antibodies are established in the following SEQ IDs: IgG1-huCD3-FEAL: SEQ ID NO:19 (HC) and SEQ ID NO:20 (LC).

[00409] IgG1-huCD3-H101G-FEAL: SEQ ID NO:35 (HC) and SEQ ID NO:20 (LC).

[00410] IgG1-CD30-MDX060-FERR: SEQ ID NO:17 (HC) and SEQ ID NO:18 (LC).

[00411] IgG1-CD30-MDX060-FEAR: SEQ ID NO:55 (HC) and SEQ ID NO:18 (LC).

[00412] IgG1-CD30-hAC10-FEAR: SEQ ID NO:21 (HC) and SEQ ID NO:22 (LC).

[00413] IgG1-CD30-HRS-3-FEAR: SEQ ID NO:23 (HC) and SEQ ID NO:24 (LC).

[00414] IgG1-CD30-HeFi-I-FEAR: SEQ ID NO:25 (HC) and SEQ ID NO:26 (LC).

[00415] IgG1-CD30-T405-FEAR: SEQ ID NO:27 (HC) and SEQ ID NO:28 (LC).

[00416] IgG1-CD30-T105-FEAR: SEQ ID NO:29 (HC) and SEQ ID NO:30 (LC).

[00417] IgG1-CD30-T408-FEAR: SEQ ID NO:31 (HC) and SEQ ID NO:32 (LC).

[00418] IgG1-CD30-T215-FEAR: SEQ ID NO:33 (HC) and SEQ ID NO:34 (LC).

[00419] To generate bispecific antibodies, two parental antibodies were mixed in equal molar ratios in PBS buffer (Phosphate-buffered saline; HPO42-8.7 mM, H2PO4-1.8 mM, Na+163.9 mM, Cl140.3 mM, pH 7.4). 2-mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM and the reaction mixture was incubated at 31°C. Petition 870250088813, dated 09 / 30 / 2025, pp. 384 / 496 135 / 205 for 5 hours. 2-MEA was removed by dialysis in PBS buffer using Slide-A-Lyzer cartridges cut to a molecular weight of 10 kDa (Thermo Fisher Scientific) according to the manufacturer's protocol. Samples were stored overnight at 4°C to allow reoxidation of disulfide bonds and formation of intact bispecific antibodies. cFAE efficacy was >95%, as assessed by electrospray ionization mass spectrometry (ESI-MS) as described by Gramer et al. (MAbs. 2013 Nov 1; 5(6): 962-973). Non-binding b12 control antibody

[00420] IgG1-b12 is an HIV-1 gp120 specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23) that is used in some of the examples as a non-binding negative control antibody. The heavy chain sequence and the light chain sequence are included in this document as SEQ ID NOs: 36 and 37, respectively (FEAL) or SEQ ID NOs: 38 and 37, respectively (FERR). Example 2 - CD30 expression in human cell lines of Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL) and TLL

[00421] CD30 surface expression levels were evaluated in a panel of HL, ALCL and TLL cell lines (Table 4) using quantitative flow cytometry (Human IgG Calibrator Kit, Biocytex, cat. noCP010). Cells (5 x 10⁴ cells / well) were incubated in 96-well round-bottom polystyrene plates (Greiner bio-one, cat. no. 650180) with 10 pg / mL of IgG1-CD30-MDX060-FERR in 50 μL of dye buffer (PBS [Lonza, cat. no. BE17-517Q] supplemented with 0.1% bovine serum albumin [BSA, fraction V, Roche, cat. no. 10735086001] and 0.02% NaN₂ [Sigma Aldrich, cat. no. 13412]) at 4°C for 30 minutes. In parallel, a standard curve was generated using a Human IgG Calibrator kit (Biocytex, cat. no. CP010), essentially according to the manufacturer's instructions. Calibration microspheres, containing a number Petition 870250088813, dated 09 / 30 / 2025, pp. 385 / 496 136 / 205 well-defined concentrations of human IgG monoclonal antibodies per microsphere were incubated with the same R-PE-conjugated secondary antibody (Jackson ImmunoResearch, UK; cat. no. 109-116-098; 1:500 dilution) at 4°C protected from light for 30 minutes. Cells and microspheres were washed in FACS buffer and analyzed by flow cytometry on a Celesta FACS flow cytometer (BD Biosciences, USA). The standard curve obtained with the Human IgG Calibrator kit was used to interpolate the number of IgG1-CD30-MDX060-FERR antibodies bound per cell (ABC) using GraphPad Prism software, representing an estimate of the number of CD30 molecules expressed on the cell surface.

[00422] Tables 4 and 5 show that CD30 expression above the lower limit of quantification (LLOQ) was observed in all cell lines except for SUP-T1. Table 4: Overview of cell lines and CD30 expression Cell Lineage Tumor Type Supplier Catalog CD30 Expression (ABCs) Median Range n L-540 LH ACC 72 DSMZ 145,723 61,779-271,304 3 L-428 LH ACC 197 DSMZ 120,725 102,787-133,437 3 HDLM-2 LH CRL-2965 ATCC 56,578 51,737-87,841 3 KM-H2 LH ACC 8 DSMZ 19,720 17,283-29,129 3 NCEB-1 LCM CRL-3005 ATCC 15,406 11,102-18,333 3 L-1236 LH ACC 530 DSMZ 4,548 4,054-4,838 3 L-82 LAGC ACC 597 DSMZ 127,616 70,495-172,019 3 SR-786 LAGC ACC 369 DSMZ 104,947 69,630-133,464 3 KI-JK LAGC ACC 695 DSMZ 124,669 77.610-139.598 72,529-80,697 2 HuT 78 LAGC TIB-161 ATCC 19,399 20,608-18,189 2 SUP-T1 TLL CRL-1942 ATCC <LLOQa - 3 Table 5: Overview of cell lines and average CD30 expression Cell lineage Tumor type Catalog Supplier CD30 expression (ABCs) Mean Range n DEL LAGC ACC 338 DSMZ 110,301 55,416- 153,484 3 Petition 870250088813, dated 09 / 30 / 2025, pp. 386 / 496 137 / 205 DL-40 LAGC JCRB1337 JCRB 98,174 60,392 - 156,229 3 HDLM-2 LH CRL-2965 ATCC 64,510 51,737-87,841 3 HH LCCT ACC 707 DSMZ 161,740 122,598 - 206,774 3 HuT 78 LAGC TIB-161 ATCC 19,399 20,608-18,189 2 JVM-2 LCM ACC 12 DSMZ 25,842 12,644-43,081 3 KARPAS-299 LAGC 06072604 PHE 91,426 77,730-106,058 3 KI-JK LAGC ACC 695 DSMZ 118,794 77,610- 139,598 3 KM-H2 LH ACC 8 DSMZ 22,044 17,283 -29,129 3 L-1236 LH ACC 530 DSMZ 4,480 4,054-4,838 3 L-428 LH ACC 197 DSMZ 117,743 102,787 - 133,437 3 L-540 LH ACC 72 DSMZ 159,602 61,779-271,304 3 L-82 LAGC ACC 597 DSMZ 127,616 70,495 - 172,019 3 NCEB-1 LCM CRL-3005 ATCC 14,947 11,102- 18,333 3 SR-786 LAGC ACC 369 DSMZ 104,497 69,630 - 133,464 3 SU-DHL-1 LAGC ACC 356 DSMZ 76,613 72,529-80,697 2 SUP-M2 LAGC ACC 509 DSMZ 66,129 37,047 - 93,777 3 SUP-T1 TLL CRL-1942 ATCC <LLOQa - 3 a<LLOQ, abaixo do limite inferior de quantificação(3300 ABCs) Example 3 - Binding of bispecific CD3xCD30 antibodies to human Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) cells such as anaplastic large cell lymphoma (ALCL)

[00423] The binding of bispecific CD3xCD30 antibodies to two human tumor cell lines expressing CD30, SU-DHL-1 (LAGC; ATCC, cat. noACC 356) and HDLM-2 (HL; ATCC, cat. noCRL-2965) was analyzed by flow cytometry.

[00424] Cells (3x10⁴ cells / well) were incubated in 96-well round-bottom polystyrene plates (Greiner bio-one, cat. no. 650180) with serial antibody dilutions (range 0.0046 to 10 μg / mL in 3-fold dilution steps) in 50 μL of dye buffer at 4°C for 30 minutes. After washing twice in dye buffer, cells were incubated with 50 μL of secondary antibody at 4°C for 30 minutes. The secondary antibody used was anti-human IgG conjugated to goat R-PE (Jackson ImmunoResearch, UK; cat. no. 109-116-098) diluted 1:400 in dye buffer. The cells were then washed twice in buffer. Petition 870250088813, dated 09 / 30 / 2025, pp. 387 / 496 138 / 205 dye, resuspended in 100 µL of dye buffer supplemented with TO-PRO-3 iodide (Thermo Fisher Scientific; cat. no. T3605; dilution 1:8000) and analyzed on a FACSCelesta flow cytometer (BD Biosciences, USA). Live cells were gated based on FSC / SSC and absence of TOPRO-3 staining. Binding curves were analyzed by nonlinear regression of logarithmically transformed data (sigmoid dose-response with variable slope, four parameters) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA). Results

[00425] Figure 1 shows dose-response curves of binding of the bispecific antibodies CD3xCD30 bsG1-huCD3-FEALxCD30-MDX060FEAR (A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (B), bsG1-huCD3FEALxCD30-HRS-3-FEAR (C), BsG1-huCD3-FEALxCD30-HeFi-I-FEAR (D), bsG1-huCD3-FEALxCD30-T405-FEAR (E), bsG1-huCD3FEALxCD30-T105-FEAR (F), BisIgG1-huCD3-FEALxCD30-T408-FEAR (G) and bsG1-huCD3-FEALxCD30-T215-FEAR(H) cells SU-DHL-1 (left panels) and HDLM-2 tumor cells (right panels).

[00426] At a concentration of 1.11 pg / mL, bsG1-huCD3FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR and bsG1-huCD3-FEALxCD30T105-FEAR exhibited similar binding compared to the binding of the parental monospecific, bivalent anti-CD30 antibodies IgG1-CD30MDX060-FEAR, IgG1-CD30-hAC10-FEAR, IgG1-CD30-HRS-3-FEAR and IgG1-CD30-T105-FEAR (Figure 1I).

[00427] In contrast, at a concentration of 1.11 pg / mL, the binding of bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR was lower than the binding of the parental monospecific, bivalent anti-CD30 antibodies IgG1-CD30T405-FEAR, IgG1-CD30-T408-FEAR, and IgG1-CD30-T215-FEAR to cells. Petition 870250088813, dated 09 / 30 / 2025, pp. 388 / 496 139 / 205 SU-DHL-1 and HDLM-2 cells (Figure 1I).

[00428] Overall, the binding of bsG1-huCD3-FEALxCD30-MDX060FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR and bsG1-huCD3-FEALxCD30-T105-FEAR at a concentration of 1.11 pg / mL was greater than the binding of bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1huCD3-FEALxCD30-T408-FEAR and bsG1-huCD3-FEALxCD30-T215-FEAR at the same concentration (Figure 1I).

[00429] The negative control antibody BsG1-huCD3-FEALxb12FEAR that was included in these experiments did not show binding to SU-DHL-1 cells and HDLM-2 cells, indicating that neither SU-DHL-1 nor HDLM-2 cells express CD3.

[00430] In conclusion, the T405, T408, and T215 clones of the antiCD30 antibody showed reduced binding in the monovalent form compared to the bivalent form, while the MDX060, hAC10, HRS-3, and T105 clones showed efficient binding to tumor cells expressing CD30 in both monovalent and bivalent forms.

[00431] Figure 2 shows dose-response curves of the binding of bsG1huCD3-FEALxCD30-MDX060-FERR to (A) HDLM-2 (LH), (B) L428 (LH), (C) DEL (LAGC), and (D) KI-JK (LAGC) cells. BsG1-huCD3FEALxCD30-MDX060-FERR exhibited similar maximum binding when compared to the monospecific, bivalent anti-CD30 parental antibody IgG1CD30-MDX060-FERR. The negative control antibodies bsG1-huCD3FEALxb12-FEAR, IgG1-huCD3-FEAL, and IgG1-b12-FEAL showed no binding to any of these cell lines, indicating that these cells do not express CD3 on the cell surface. These data confirmed the efficient binding of bsG1-huCD3-FEALxCD30-MDX060-FEER to LH and LAGC cell lines.

[00432] Table 6 shows EC50 values ​​for the bsG1 link Petition 870250088813, dated 09 / 30 / 2025, page 389 / 496 140 / 205 huCD3-FEALxCD30-MDX060-FEAR to HDLM-2, L-428, DEL and KIJK cells as evaluated in two different experiments. EC50 values ​​ranged from 0.05 to 0.30 pg / mL. Table 6: ECs0_ values ​​for the bsG1-huCD3xCD30-MDX060 link EC50 (gg / mL) Cell line N=1 N=2 HDLM-2 0.1450 0.1197 L-428 0.1539 0.0705 DEL 0.3018 0.0568 KI-JK 0.1602 0.0735

[00433] Figures 8, 9, and 10 show dose-response curves of bsG1-huCD3-FEALxCD30-MDX060-FERR binding to HL cell lines (Figure 8), LAGC cell lines (Figure 9), and NHL cell lines (Figure 10) expressing CD30. In all cell lines, BsG1-huCD3-FEALxCD30-MDX060-FERR exhibited similar maximum binding when compared to the monovalent control antibody BsG1-b12-FEALxCD30-MDX060-FERR. The monospecific, bivalent anti-CD30 parental antibody IgG1-CD30-MDX060-FERR showed dose-dependent binding to all cell lines, but showed lower maximum binding compared to BsG1-huCD3-FEALxCD30-MDX060FERR. The negative control antibodies bsG1-huCD3-FEALxb12-FEAR, IgG1-huCD3-FEAL, and IgG1-b12-FEAL showed no binding to any of these cell lines, indicating that these cells do not express CD3 on their cell surface.These data confirmed the efficient binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HL cell lines, T-cell NHL such as LAGC and CTCL; and B-cell NHL as a MCL cell line.

[00434] Table 7 shows the EC50 values ​​for the binding of bsG1huCD3-FEALxCD30-MDX060-FERR to HL, T-cell NHL, and B-cell NHL cell lines, as evaluated in 2 or 3 independent experiments. EC50 values ​​ranged from 0.12 to 0.35 μg / mL. Petition 870250088813, dated 09 / 30 / 2025, pp. 390 / 496 141 / 205 Table 7: EC50 values ​​for the bsG1-huCD3-FEALxCD30MDX060-FERR linkage Cell lineage CD30 binding Name Cancer type Average binding EC50 EC50 range of binding (Kg / mL) n tests L-540 LH 0.3229 0.2020 -0.4694 3 L-428 LH 0.1743 0.1742 -0.1744 2 KM-H2 LH 0.1587 0.1405 -0.1824 3 L-1236 LH 0.2019 0.1628 -0.2247 3 HDLM-2 LH 0.1674 0.1253 -0.2206 3 L-82 LAGC 0.1999 0.1845 -0.2153 2 SR-786 LAGC 0.3279 0.1735 -0.5499 3 KI-JK LAGC 0.1914 0.1431 -0.2582 3 SUP-M2 LAGC 0.1667 0.1336 -0.2225 3 DL-40 LAGC 0.2364 0.1698 -0.3472 3 DEL LAGC 0.2011 0.1430 -0.2674 3 KARPAS-299 LAGC 0.2015 0.1343 -0.2686 2 SUP-T1 TLL 0.3458 0.2572 - 0.4344 2 JVM-2 LCM 0.1257 0.1068 -0.1411 3 NCEB-1 LCM 0.1564 0.1180 -0.2000 3 HH LCCT 0.1951 0.1559 -0.2425 3 Example 4 - Induction of T cell-mediated cytotoxicity and T cell proliferation in vitro by CD3xCD30 bispecific antibodies

[00435] CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines as target cells and T cells as effector cells. As a source of T cells, CD3 type IV positive ADCC effector cells (Clean Cells, Montaigu, France) or purified T cells (as described in Example 5) were used to assess CD3-dependent tumor cell death.

[00436] SU-DHL-1 (LAGC), HuT78 (LAGC), HDLM-2 (LH), NCEB-1 (LCM), or L540 (LH) cells were seeded at a density of 10,000 cells / well in 96-well round-bottom polystyrene plates (Greiner bio-one, cat. no. 650180). The effector cells were Petition 870250088813, dated 09 / 30 / 2025, pp. 391 / 496 142 / 205 cells labeled with CFSE 0.5 μM (carboxyfluorescein succinimidyl ester; Cell Signalling Technology, Danvers, MA; cat. no. C34554) were incubated at 37°C for 20 minutes and added to tumor cells in an E:T ratio of 10:1 (ADCC effector cells) or 7:1 (purified T cells). Serial dilutions of the bispecific antibodies CD3xCD30, b12xCD30, or CD3xb12, or of the monospecific, bivalent anti-CD30 antibodies were added (final concentration ranging from 10 to 0.041 μg / mL; 3-fold dilutions) and the cells were incubated for 72 hours at 37°C. In some experiments, a variant of bsG1-huCD3-FEALxCD30-MDX060-FEAR with a CD3-binding arm containing the H101G mutation was used, which reduced the affinity for CD3 (WO2017 / 009442, Genmab). After washing twice in 100 μL of dye buffer, the cells were resuspended in dye buffer containing TO-PRO-3 iodide (Thermo Fisher Scientific; cat.noT3605; dilution 1:4000) and analyzed on a FACSCelesta flow cytometer (BD Biosciences, USA).

[00437] The viability of tumor cell samples treated with staurosporine 5 μM (Sigma-Aldrich, USA, cat. no. S6942) was set at 0% and the viability of untreated tumor cell samples was set at 100%.

[00438] The “percentage of viable cells” was calculated as follows: % viable cells = ([number of cells in the sample - number of target cells treated with staurosporine] / [number of untreated target cells - number of target cells treated with staurosporine]) x 100.

[00439] CFSE-positive cells were counted as a measure of absolute T cell numbers to assess T cell proliferation.

[00440] Dose-response curves were analyzed by nonlinear regression (sigmoid dose-response with variable slope) using the software Petition 870250088813, dated 09 / 30 / 2025, pp. 392 / 496 143 / 205 GraphPad Prism V8 (GraphPad Software, San Diego, CA, USA). Results

[00441] Bispecific CD3xCD30 antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines, SU-DHL-1 cells or HDLM-2 cells, as target cells, and type IV effector ADCC cells (Clean Cells, Montaigu, France) as effector cells.

[00442] Figure 3 shows that bsG1-huCD3-FEALxCD30-MDX060FEAR (A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (B), bsG1-huCD3FEALxCD30-HRS-3-FEAR (C), bsG1-huCD3-FEALxCD30-HeFi-I-FEAR (D), bsG1-huCD3-FEALxCD30-T405-FEAR (E), bsG1-huCD3FEALxCD30-T105-FEAR (F), bsG1-huCD3-FEALxCD30-T408-FEAR (G), and bsG1-huCD3-FEALxCD30-T215-FEAR (H) induced dose-dependent T-cell mediated cytotoxicity (shown as a decrease in % of cells viable) of SU-DHL-1 cells (left panels) or HDLM-2 cells (right panels).

[00443] The monospecific, bivalent anti-CD30 antibodies IgG1MDX060-FEAR (A), IgG1-CD30-hAC10-FEAR (B), IgG1-CD30-HRS-3FEAR (C), IgG1-CD30-HeFi-I-FEAR (D), IgG1-CD30-T405-FEAR (E), IgG1-CD30-T105-FEAR (F), IgG1-CD30-T408-FEAR (G) and IgG1-CD30T215-FEAR (H) did not induce T cell-mediated cytotoxicity. The control antibody bsG1-huCD3-FEALxb12-FEAR also did not induce T cell-mediated cytotoxicity of SU-DHL-1 or HDLM-2 cells.

[00444] In addition, bispecific CD3xCD30 antibodies were tested in an in vitro cytotoxicity assay with different LCM, LAGC, and LH cell lines as target cells, and purified T cells or type IV effector ADCC cells as effector cells. Figures 4A-B show the T cell-mediated cytotoxicity of SU-DHL-1, HuT78, or NCEB-1 cells induced by bsG1-huCD3-FEALxCD30-MDX060 Petition 870250088813, dated 09 / 30 / 2025, pp. 393 / 496 144 / 205 FEAR is more potent compared to a variant of this antibody with a reduced-affinity CD3-binding arm (bsG1-huCD3-H101GFEALxCD30-MDX060-FEAR). Similar T cell-mediated maximal cytotoxicity of HDLM-2 cells was induced by bsG1-huCD3FEALxCD30-MDX060-FEAR and bsG1-huCD3-H101G-FEALxCD30MDX060-FEAR (Figure 4B). Incubation with the bivalent monospecific antibodies IgG1-huCD3-FEAL or IgG1-b12-FEAR, which were included as controls, did not induce cytotoxicity in these cell lines. Potent and similar T-cell-mediated cytotoxicity of L540 cells was induced by bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR at the lowest concentration tested (0.014 pg / mL; Figure 4C). The control antibodies bsG1-b12FEALxCD30-MDX060-FEAR or IgG1-MDX060-FEAR did not induce T-cell-mediated cytotoxicity of L540 cells.

[00445] In conclusion, bsG1-huCD3-FEALxCD30-MDX060-FEAR potently induced cell death in several CD30-expressing MCL, LAGC, and HL tumor cell lines. BsG1-huCD3-FEALxCD30MDX060-FEAR containing an H at position 101 of the VH-CDR3 arm of the CD3 is more potent in causing cell death in CD30-expressing MCL and LAGC tumor cells compared to the variant with the lower-affinity CD3 arm containing G at position 101.

[00446] In cytotoxicity assays using HDLM-2 cells (left panel) or NCEB-1 cells (right panel) as target cells, the number of CFSE-positive cells was assessed as a measure of absolute T cell counts. Figure 4D shows that T cell-mediated cytotoxicity of HDLM-2 cells and NCEB-1 cells induced by bsG1-huCD3-FEALxCD30-MDX060-FEAR or bsG1-huCD3-H101GFEALxCD30-MDX060-FEAR (see Figure 4B) was associated with a dose-dependent increase in T cell count. In general, numbers Petition 870250088813, dated 09 / 30 / 2025, pages 394 / 496 145 / 205 similar T cell counts were observed after incubation with bsG1-huCD3FEALxCD30-MDX060-FEAR or bsG1-huCD3-H101G-FEALxCD30MDX060-FEAR in this assay. Reduced T cell counts were observed in co-cultures with NCEB-1 cells incubated with bsG1huCD3-FEALxCD30-MDX060-FEAR at concentrations above 1 pg / mL. The control antibody IgG1-b12-FEAL did not affect T cell counts in these experiments.

[00447] In conclusion, BsG1-huCD3-FEALxCD30-MDX060-FEAR induced T cell proliferation in the presence of several CD30-expressing LAGC, HL, and LCM tumor cell lines. Example 5 - Binding of bispecific CD3xCD30 antibodies to human, Cynomolgus monkey or Rhesus CD30 expressed in Expi293F cells

[00448] The binding of bispecific CD3xCD30 antibodies and monospecific, bivalent anti-CD30 antibodies to the plasma membrane of Expi293 cells transiently transfected with complete human CD30 or Cynomolgus monkey CD30 was analyzed by flow cytometry. Transient expression of human, Cynomolgus monkey, or Rhesus monkey CD30 in HEK-293F or HEK-293 cells

[00449] The following codon-optimized constructs for the expression of several complete CD30 variants were generated: human CD30 (Homo sapiens) (huCD30; Uniprot accession node P28908), Cynomolgus macaque CD30 (Macaca fascicularis) (mfCD30; Uniprot accession node A0A2K5VW07) (SEQ ID NO:40), and Rhesus macaque CD30 (Macaca mulatta) (mmCD30; Uniprot accession node A0A1D5RK03) (SEQ ID NO:41). The constructs contained restriction sites suitable for cloning and an optimal Kozak sequence (GCCGCCACC) (Kozak, M., Gene 1999;234(2):187-208). Complete human, Cynomolgus monkey, and Rhesus monkey CD30 constructs with optimized codons were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen) and Petition 870250088813, dated 09 / 30 / 2025, pages 395 / 496 146 / 205 expressed with the Expi293F expression platform (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. A14527) essentially as described by the manufacturer. In another set of experiments, either full human CD30 or full Cynomolgus monkey CD30 constructs were expressed in HEK-293 cells. Binding of bispecific CD3xCD30 antibodies to human, Cynomolgus monkey, or Rhesus monkey CD30 cells expressed on Expi293 cells

[00450] Cells (3x10⁴ cells / well) were incubated in 96-well round-bottom polystyrene plates (Greiner bio-one, cat. no. 650180) with serial antibody dilutions (ranging from 0.005 to 10 μg / mL in 3-fold dilution steps) in 100 μL of dye buffer at 4°C for 30 minutes. Experiments were performed in technical duplicate. After washing twice in dye buffer, cells were incubated in 50 μL of secondary antibody at 4°C for 30 minutes. As a secondary antibody, anti-human IgG conjugated to goat R-PE (Jackson ImmunoResearch, UK; cat. no. 109-116-098) diluted 1:400 in dye buffer was used. The cells were washed twice in dye buffer, resuspended in 30 μL of dye buffer containing 0.4% EDTA, and analyzed on an iQue Screener (Intellicyt Corporation, USA). Binding curves were analyzed using nonlinear regression (sigmoid dose-response with variable slope) with GraphPad Prism V9.0 software.0 (GraphPad Software, San Diego, CA, USA). Binding of bispecific CD3xCD30 antibodies to human, Cynomolgus monkey, or Rhesus monkey CD30 cells in HEK293 cells.

[00451] Cells (3x10⁴ cells / well) were incubated in 96-well round-bottom polystyrene plates (Thermo Scientific, cat. no. 163320) with serial antibody dilutions (ranging from 0.0002 to 50 pg / ml in 4-fold dilution steps) in 50 pL of dye buffer at 4°C for 30 minutes. Experiments were performed in technical duplicate. After Petition 870250088813, dated 09 / 30 / 2025, pages 396 / 496 147 / 205 cells were washed twice in dye buffer and incubated in 50 pL of secondary antibody at 4°C for 30 minutes. The secondary antibody used was anti-human IgG conjugated to goat R-PE (Jackson ImmunoResearch, UK; cat. no. 109-116-098) diluted 1:200 in dye buffer. Cells were washed twice in dye buffer, resuspended in 30 pL of dye buffer containing 0.4% EDTA and the viability marker ToPro-3 (Invitrogen, cat. no. T3605) diluted 1:10,000. Cells were analyzed on an iQue Screener (Intellicyt Corporation, USA). Binding curves were analyzed using nonlinear regression (sigmoid dose-response with varying slope) with GraphPad Prism V9.0.0 software (GraphPad Software, San Diego, CA, USA). Binding of bispecific CD3xCD30 antibodies to human T cells or Cynomolgus monkey PBMCs

[00452] Cynomolgus monkey PBMCs (Tebu-Bio, Netherlands; cat. no. PBMCMFA-10) or purified human T cells were seeded in 96-well polystyrene round-bottom plates. T cells were derived from human donor buffy coats (Sanquin, Amsterdam, Netherlands) and isolated with the RosetteSep human T cell enrichment cocktail (Stemcell Technologies, France, cat. no. 15061) according to the manufacturer's instructions. Cells (3 x 10⁴ cells / well) were incubated with serial dilutions (ranging from 0.0001 to 10 μg / mL in 3-fold dilution steps) of IgG1-CD30-MDX060-FEAR, bsG1huCD3-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR antibodies in 50 μL of dye buffer at 4°C for 30 minutes. After washing twice in dye buffer, the cells were incubated in 50 μL of goat anti-human IgG secondary antibody conjugated to R-PE at 4°C for 30 minutes (1:400 dilution).After washing twice in dye buffer, the T cells were stained for T cell markers, CD3 (1:100; Miltenyi biotec, clone 10D12, conjugated to APC), CD4 (1:50; eBioscience, clone OKT4, conjugated). Petition 870250088813, dated 09 / 30 / 2025, pp. 397 / 496 148 / 205 a APC-Cy7), CD8 (1:100; Biolegend, clone RPA-T8, conjugated to AF700) and T cell activation markers, CD69 (1:50; BD Biosciences, clone AB2439, conjugated to FITC), CD25 (1:50; eBioscience, clone BC96, conjugated to PE-Cy7) and CD279 / PD1 (1:50; BD Biosciences, clone AEH12.2H7, conjugated to BV605). Single samples stained with Ultracomp microspheres (5 pL; Invitrogen, cat. no. 01-2222-42) were used for flow cytometer compensation adjustments. After 30 minutes of incubation at 4°C, the cells were washed twice with dye buffer, resuspended in 100 µL of dye buffer, and analyzed using FACS Fortessa (BD Biosciences). The data were processed with FlowJo (BD Biosciences). Results

[00453] The bispecific antibodies bsG1-huCD3-FEALxCD30MDX060-FEAR, bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR, and bsG1-b12-FEALxCD30-MDX060-FEAR targeting CD30 did not bind to wild-type Expi293F cells (Figure 5A), but exhibited dose-dependent binding to Expi293F cells transfected with huCD30 (Figure 5B) or mfCD30 (Figure 5C). The binding of these bispecific antibodies was comparable to the binding of the monospecific, bivalent anti-CD30 antibody IgG1-CD30-MDX060-FEAR. As expected, the negative control antibody bsG1-huCD3-FEALxb12-FEAR showed no binding to wild-type Expi293F cells or cells transfected with huCD30 or mfCD30. Similarly, bsG1-huCD3-FEALxCD30-MDX060-FERR showed dose-dependent binding to HEK293 cells transfected with huCD30 (Figure 11A) or mfCD30 (Figure 11B), while the negative control antibody bsG1-huCD3-FEALxb12-FERR showed no binding to HEK293 cells transfected with huCD30 or mfCD30.This indicates that the CD30-targeted antibody MDX060 binds efficiently to human full CD30 and to Cynomolgus monkey CD30 expressed on HEK cells in a single dose. Petition 870250088813, dated 09 / 30 / 2025, pp. 398 / 496 149 / 205 bivalent format, as well as a monovalent one.

[00454] Figure 5D shows that the bispecific antibody CD3xCD30 bsG1-huCD3-FEALxCD30-MDX060-FEAR and the bispecific control antibody bsG1-huCD3-FEALxb12-FEAR bound efficiently to human and Cynomolgus monkey primary T cells. This shows that these CD3-targeted bispecific antibodies bind efficiently to endogenously expressed human and Cynomolgus monkey CD3 on T cells. The parental bivalent antibody IgG1-CD30-MDX060 did not bind to human or Cynomolgus monkey T cells, indicating that CD30 was not expressed on these cells.

[00455] The binding of the bispecific CD3xCD30 antibody panel and the parental monospecific anti-CD30 antibody to Expi293F cells transfected with huCD30 or mmCD30 was evaluated by flow cytometry. The bispecific CD3xCD30 antibodies bsG1-huCD3-FEALxCD30MDX060-FEAR (Figure 6A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (Figure 6B), bsG1-huCD3-FEALxCD30-HRS-3-FEAR (Figure 6C), bsG1huCD3-FEALxCD30-T405-FEAR (Figure 6E), bsG1-huCD3-FEALxCD30T105-FEAR (Figure 6F), bsG1-huCD3-FEALxCD30-T408-FEAR (Figure 6G), and bsG1-huCD3-FEALxCD30-T215-FEAR (Figure 6H) showed equal binding to cells expressing huCD30 or mmCD30. Similarly, clones of the parental monospecific anti-CD30 antibody showed equal binding to cells expressing huCD30 or mmCD30. In contrast, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR and the parental monospecific anti-CD30 antibody IgG1-CD30-HeFi-I_FEAR showed binding to huCD30, but not to mmCD30 (Figure 6D). Example 6 - Evaluation of the conformational stability of non-activating monospecific and bispecific antibody variants by DSF analysis

[00456] The protein stability characteristics of variants of the bivalent monospecific anti-CD30, anti-CD3 and bispecific antibodies Petition 870250088813, dated 09 / 30 / 2025, pages 399 / 496 150 / 205 IgG1 CD3xCD30 IgG1 cells harboring non-activating mutations in the constant region of the heavy chain were evaluated by differential scanning fluorimetry (DSF).

[00457] Samples of IgG1-CD30-MDX060-FEAR, IgG1-CD30MDX060-FERR, IgG1-huCD3-FEAL and BsG1-huCD3-FEALxCD30MDX060-FERR were formulated in PBS, pH 7.4, at a concentration of approximately 1 mg / mL.

[00458] To assess conformational stability, DSF was performed on a real-time PCR detection system, iQ5 Multicolor (Bio-Rad) capable of detecting changes in fluorescence intensity caused by the binding of the extrinsic dye Sypro-Orange (5000x concentrated in DMSO, Cat. no. S5692, Sigma-Aldrich) to hydrophobic regions exposed during IgG unfolding. Sypro-Orange was diluted 320 times in PBS, pH 7.4 (Hyclone GE Healthcare). A thermal melt curve can be derived by measuring the increasing fluorescence during the step-by-step controlled thermal denaturation of the analyzed IgG. Therefore, duplicate samples of 5 pL of antibody solution (1 mg / mL in PBS) were added to 20 pL of Sypro-Orange diluted in PBS, pH 7.4, in 96-well iQ PCR plates.Fluorescence was recorded at increasing temperatures ranging from 25°C to 95°C, in stepwise increments of 0.5°C and a duration of 15 seconds plus the time required to record the fluorescence of all wells. The data were analyzed using Bio-Rad CFX Manager 3.0 software, and the melting points were determined from the fluorescence versus temperature graphs using the software. Results

[00459] Figure 7 and Table 8 show that the melting temperature (Tm) of IgG1-CD30-MDX060-FERR is 69.0°C, which is higher than the Tm of IgG1-CD30-MDX060-FEAR at pH 7.4 (64.5°C). This indicates that IgG1CD30-MDX060-FERR exhibits greater conformational stability than Petition 870250088813, dated 09 / 30 / 2025, pages 400 / 496 151 / 205 IgG1-CD30-MDX060-FEAR suggests that IgG1-CD30-MDX060 containing a FER backbone exhibits greater conformational stability than IgG1-CD30-MDX060 containing a FEA backbone. The melting point of BsG1-huCD3-FEAL x CD30-MDX060-FERR was determined to be 64.5°C, which is between the Tm determined for the two parental antibodies IgG1-huCD3-FEAL (62.5°C) and IgG1-CD30-MDX060FERR (69.0°C). Table 8: Melting temperatures (Tm) of the indicated antibodies, determined by differential scanning fluorimetry (DSF)______________________________ Antibody Tm (°C) IgG1-CD30-MDX060-FEAR 64.5 IgG1-CD30-MDX060-FERR 69.0 IgG1-huCD3-FEAL 62.5 BsG1-huCD3-FEALxCD30-MDX060-FERR 64.5 Example 7 - Simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060FERR to T cells and tumor cells

[00460] The simultaneous binding of bsG1-huCD3-FEALxCD30MDX060-FERR to tumor cells and naive T cells was studied.

[00461] Frozen T cells isolated from healthy donors were thawed and labeled with Celltrace Violet 0.25 mM (Pacific Blue; Invitrogen, cat. noC34557A) at 37°C for 15 minutes. L-428 tumor cells were labeled with Celltrace FarRed (APC; Invitrogen cat. noC34564A) at 37°C for 15 minutes and added to T cells in a 1:1 E:T ratio. Serial dilutions of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibodies bsG1-huCD3-FEALxb12-FEAR, bsG1b12FEALxCD30MDX060-FERR, or IgG1-b12-FEAL were added (final concentration ranging from 6*10-5 to 10 pg / mL; 3-fold dilutions) and the cells were incubated for 2 hours at 4°C. After incubation, the viability marker 7-AAD (BD Bioscience, cat. no. 559925) was added (100x final dilution) and the cells were analyzed on a Celesta FACS flow cytometer (BD Biosciences). Petition 870250088813, dated 09 / 30 / 2025, pp. 401 / 496 152 / 205 Results

[00462] Figure 12 shows that bsG1-huCD3-FEALxCD30-MDX060FERR induces the formation of double-positive CD3+CD30+ events (cells expressing both CellTrace Far Red and CellTrace Violet in flow cytometry staining) as a measure of bsG1-huCD3-FEALxCD30-MDX060-FERR-mediated cross-linking of tumor cells to T cells (simultaneous linkage). The increase in double-positive events was antibody concentration-dependent and showed a bell-shaped curve. No increase in cross-linking of tumor cells and T cells was observed in samples incubated with the control antibodies bsG1-huCD3-FEALxb12FEAR, bsG1b12FEALxCD30MDX060-FERR or IgG1-b12-FEAL or in samples incubated without antibody (A). Figure 12 B shows the simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to tumor cells and naive T cells, as detected by the percentage of cells expressing both CellTrace Far Red and CellTrace Violet (B).

[00463] These data indicate that bsG1huCD3FEALxCD30-MDX060FERR can simultaneously bind and cross-link with CD30+ tumor cells and CD3+ T cells. Example 8 - Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies

[00464] The T cell-mediated cytotoxicity of Karpas-299 tumor cells associated with T cell activation by a panel of CD3xCD30 bispecific antibodies was evaluated. The following antibodies were evaluated: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxCD30HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR and bsG1huCD3-FEALxCD30-T215-FEAR. Petition 870250088813, dated 09 / 30 / 2025, pp. 402 / 496 153 / 205

[00465] T cells were obtained from leukocyte layers of healthy human donors (Sanquin, Amsterdam, Netherlands) and isolated with the RosetteSep™ human T cell enrichment cocktail (Stemcell Technologies, France, cat. no. 15061) according to the manufacturer's instructions. T cells were labeled with Celltrace Violet (Invitrogen, cat. no. C34557A; final concentration 5 μM) for 15 minutes at 37°C. In parallel, Karpas-299 tumor cells were labeled with Celltrace FarRed (Invitrogen, cat. no. C34564A; final concentration 2 μM) for 15 minutes at 37°C. After labeling, 5* the volume of ice-cold DBSI was added and incubated for 5 minutes at room temperature. The cells were pelleted, resuspended in the medium, and tumor cells were seeded into 96-well plates (Greiner-bio-one, Netherlands, cat. no. 655180) at a density of 50,000 cells / well.Serial dilutions of bispecific CD3xCD30 antibodies were added (final concentration ranging from 1.000 to 0.051 ng / mL; 3-fold dilutions) and the plates were incubated for 15 minutes at room temperature. T cells were added to the tumor cells in an effector-to-target (E:T) ratio of 4:1 and the plates were incubated for 72 hours at 37°C. After washing twice with 0.1% PBS / BSA / 0.02% azide (stain buffer), the cells were stained for CD4 (1:50; Biolegend, cat. no. 300521, conjugated to Pacific Blue), CD8 (1:100; BD Biosciences, conjugated to FITC) T cell markers and CD69 (1:50; Biolegend, cat. no. 310934, conjugated to BV650), CD25 (1:100; Invitrogen, cat. no. 25-0259-42, conjugated to PE-Cy7) and CD279 / PD-1 (1:50; Biolegend, cat. no. 329930, conjugated to BV605) T cell activation markers. Single samples stained with Ultracomp microspheres (5 μL; Invitrogen, cat. no. 01-2222-42) were included and used for flow cytometer compensation adjustments.After 30 minutes of incubation at 4°C, the plates were washed twice with staining buffer, and the cells were stained with 7-AAD (diluted 1:100 in staining buffer) for 10 minutes at 4°C. The cells were analyzed using... Petition 870250088813, dated 09 / 30 / 2025, pp. 403 / 496 154 / 205 of FACS Celesta (BD Biosciences). The data were processed with FlowJo (BD Biosciences).

[00466] Dose-response curves were generated using nonlinear regression analysis (sigmoid dose-response with variable slope) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA). Results

[00467] Figures 13A and B show that all CD3xCD30 antibodies induce T cell-mediated cytotoxicity of Karpas299 cells. Bispecific CD3xCD30 antibodies generated with the CD30 clone MDX060 (bsG1-huCD3-FEALxCD30-MDX060-FERR and bsG1huCD3-FEALxCD30-MDX060-FEAR) were more effective in causing the death of Karpas-299 cells compared to all other clones tested. Indeed, the MDX060-based bispecific CD3xCD30 antibodies showed significantly lower IC50 values ​​when compared to the bispecific CD3xCD30 antibodies generated with the CD30 clones HRS-3, HeFi-I, T105, T405, T408, or T215 (bsG1-huCD3FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxCD30-T405FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3FEALxCD30-T215-FEAR; Figure 13A).Furthermore, bsG1-huCD3FEALxCD30-MDX060-FERR and bsG1-huCD3-FEALxCD30-MDX060FEAR induced greater maximum cell death compared to the bispecific CD3xCD30 antibodies generated with the CD30 clones hAC10, HeFi-I, T405, T408, or T215 (bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T405FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3FEALxCD30-T215-FEAR; Figure 13B). Figures 13C and D show that bsG1-huCD3-FEALxCD30-MDX060-FERR and bsG1-huCD3-FEALxCD30MDX060-FEAR were more effective (lower EC50 values) than... Petition 870250088813, dated 09 / 30 / 2025, pp. 404 / 496 155 / 205 any other bispecific CD3xCD30 antibodies in inducing CD25 expression as a measure of T cell activation in CD4+ T cells (Figure 13C) or CD8+ T cells (Figure 13D). Similar results were observed for PD-1 expression (Figure 13E and F) and CD69 (data not shown). No difference was observed in T cell-mediated death or T cell activation between the two MDX030-based CD3xCD30 bispecific antibodies containing the FEAR or FERR mutations.

[00468] The bispecific antibody bsG1-huCD3xCD30-MX060 was also more effective in inducing T cell-mediated cytotoxicity of L-428 cells compared to other CD3xCD30 bispecific antibodies tested from the panel (data not shown).

[00469] The mean IC50 concentrations and maximum percentage of T cell-mediated cytotoxicity lysis and the EC50 concentrations of T cell activation (CD25 expression), induced by the CD3xCD30 bispecific antibody panel used in these experiments, are summarized in Table 9.

[00470] In conclusion, these data demonstrated that bsG1huCD3xCD30-MDX060 was more effective than any of the other bispecific CD3xCD30 antibodies evaluated in inducing T cell-mediated cytotoxicity. Table 9: IC50 concentrations and maximum percentage of T cell-mediated cytotoxicity of Karpas-299 cells and EC50 concentrations of T cell activation (CD25 expression) induced by a panel of CD3xCD30 bispecific antibodies. Antibody Cytotoxicity T cell activation (CD25 expression) T cell activation (PD-1 expression) CD4+ T cells CD8+ T cells CD4+ T cells CD8+ T cells IC50 (pg / mL) Maximum lysis (%) EC50 (pg / mL) EC50 (pg / mL) EC50 (pg / mL) EC50 (pg / mL) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Petition 870250088813, dated 09 / 30 / 2025, pp. 405 / 496 156 / 205 bsG1-huCD3-FEALxCD30- MDX060-FERR 0,029 (0,004) 52,328 (3,929) 0,023 (0,004) 0,020 (0,005) 0,025 (0,006) 0,023 (0,009) bsG1-huCD3-FEALxCD30- MDX060-FEAR 0,033 (0,004) 53,703 (5,949) 0,024 (0,004) 0,020 (0,005) 0,024 (0,006) 0,027 (0,009) bsG1-huCD3-FEALxCD30hAC10-FEAR 0,032 (0,006) 37,765 (5,340) 0,046 (0,011) 0,054 (0,021) 0,056 (0,021) 0,057 (0,031) bsG1-huCD3-FEALxCD30- HRS-3-FEAR 0,068 (0,011) 47,472 (6,539) 0,089 (0,031) 0,079 (0,031) 0,123 (0,046) 0,112 (0,047) bsG1-huCD3-FEALxCD30HeFi-I-FEAR 0,052 (0,011) 42,987 (6,603) 0,069 (0,013) 0,076 (0,016) 0,075 (0,024) 0,083 (0,023) bsG1-huCD3-FEALxCD30T105-FEAR 0,042 (0,007) 52,582 (5,344) 0,056 (0,010) 0,060 (0,017) 0,064 (0,021) 0,065 (0,024) bsG1-huCD3-FEALxCD30T405-FEAR 0,078 (0,023) 41,667 (4,560) 0,093 (0,027) 0,080 (0,056) 0,138 (0,054) 0,140 (0,126) bsG1-huCD3-FEALxCD30T408-FEAR 0,226 (0,112) 41,295 (5,482) 0,172 (0,046) 0,134 (0,077) 0,286 (0,104) 0,220 (0,126) bsG1-huCD3-FEALxCD30T215-FEAR 0,077 (0,032) 42,840 (6,464) 0,092 (0.036) 0.105 (0.063) 0.355 (0.476) 0.281 (0.308) Example 9 - Induction of T cell-mediated cytotoxicity, T cell proliferation and T cell activation in vitro by bsG1-huCD3-FEALxCD30MDX060-FERR

[00471] The T cell-mediated cytotoxicity of tumor cells and the associated proliferation and activation of T cells by bsG1-huCD3FEALxCD30-MDX060-FERR was evaluated in HL and LAGC cell lines.

[00472] T cells were obtained from leukocyte layers of healthy human donors (Sanquin, Amsterdam, Netherlands) and isolated with the RosetteSep™ human T cell enrichment cocktail (Stemcell Technologies, France, cat. no. 15061) according to the manufacturer's instructions. T cells were labeled with Celltrace Violet (Invitrogen, cat. no. C34557A; final concentration 5 μM) for 15 minutes at 37°C. In parallel, L-428, KI-JK, KM-H2, or SUP-M2 tumor cells were labeled with Celltrace FarRed (Invitrogen, cat. no. C34564A; final concentration 2 μM) for 15 minutes at 37°C. After labeling, 5* the volume of ice-cold DBSI was added and incubated for 5 minutes at room temperature. The cells formed pellets, were resuspended in the medium, and the tumor cells were... Petition 870250088813, dated 09 / 30 / 2025, pages 406 / 496 157 / 205 cells were seeded in 96-well plates (Greiner-bio-one, Netherlands, cat. no. 655180) at a density of 50,000 cells / well. Serial dilutions of bsG1-huCD3-FEALxCD30-MDX060-FERR or control antibodies IgG1huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30-MDX060FERR, bsG1-b12-FEALxCD30-MDX060-FERR, IgG1-b12-FEAL were added (final concentration ranging from 1,000 to 0.051 ng / mL; 3-fold dilutions) and the plates were incubated for 15 minutes at room temperature. T cells were added to tumor cells in an effector-to-target (E:T) ratio of 4:1, and the plates were incubated for 72 hours at 37°C. After washing twice with 0.1% PBS / BSA / 0.02% azide (stain buffer), the cells were stained for CD4 T cell markers (1:50; Biolegend, cat. no. 300521, conjugated to Pacific Blue), CD8 T cell markers (1:100; BD Biosciences, cat. no. 345772, conjugated to FITC), and CD69 T cell activation markers (1:50; Biolegend, cat.The following antibodies were used: α-AAD (1:100; Invitrogen, cat. no. 25-0259-42, conjugated to PE-Cy7) and β-AAD (1:50; Biolegend, cat. no. 329930, conjugated to BV605). Single samples stained with Ultracomp microspheres (5 pL; Invitrogen, cat. no. 01-2222-42) were included and used for flow cytometer compensation adjustments. After 30 minutes of incubation at 4°C, the plates were washed twice with dye buffer and the cells were stained with 7-AAD (diluted 1:100 in dye buffer) for 10 minutes at 4°C. The cells were analyzed using FACS Celesta (BD Biosciences) and the data were processed with FlowJo (BD Biosciences).

[00473] The percentage of live target cells was calculated using the following equation: % live target cells = (absolute number of live, isolated cells labeled with Celltrace FarRed in each condition / absolute number of live, isolated cells labeled with Celltrace FarRed in the condition containing only target cells and T cells without the addition of any other cells) Petition 870250088813, dated 09 / 30 / 2025, pp. 407 / 496 158 / 205 antibodies) x 100.

[00474] T cell proliferation was assessed by gating CD4+ or CD8+ T cells stained with diluted Celltrace Violet. The expansion index was calculated using the FlowJo proliferation modeling tool. Peak generation was automatically adjusted, and expansion index values ​​were calculated according to the following equation: Expansion index = Total number of cells / number of cells at the beginning of the culture = (G0 + G1 + G2 + G3 + G4 + G5 + G6) / (G0 + G1:2 + G2:4 + G3:8 + G4:16 + G5:32 + G6:64).

[00475] Gn = number of cells in the generation of n peaks (with n = 0 to 6).

[00476] Dose-response curves were generated using nonlinear regression analysis (sigmoid dose-response with variable slope) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA). Results

[00477] Figure 14 shows that bsG1-huCD3-FEALxCD30-MDX060FERR induced dose-dependent T cell-mediated cytotoxicity in L-428 (LH), KM-H2 (LH), SUP-M2 (LAGC) and KI-JK (LAGC) cell lines in vitro. The mean IC50 concentrations of T cell-mediated cytotoxicity induced by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cells are summarized in Table 10. No cytotoxicity was observed in cells incubated with the control antibodies IgG1huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30-MDX060FERR, bsG1-b12-FEALxCD30-MDX060-FERR, IgG1-b12-FEAL or in samples incubated without antibody.

[00478] Figures 15, 16, 17, and 18 show that T cell-mediated cytotoxicity of L-428 and KI-JK cells, induced by bsG1-huCD3FEALxCD30-MDX060-FERR, was associated with proliferation of CD4+ and CD8+ T cells (Figure 15) and expression of T cell activation markers CD69 (Figure 16), CD25 (Figure 17), and PD-1 (Figure 18). The concentrations Petition 870250088813, dated 09 / 30 / 2025, pp. 408 / 496 159 / 205 Average EC50 values ​​for T cell proliferation and activation induced by bsG1huCD3-FEALxCD30-MDX060-FERR in these experiments are summarized in Table 10.

[00479] Consequently, bsG1-huCD3-FEALxCD30-MDX060FERR induced dose-dependent T cell-mediated cytotoxicity in LH and LAGC cell lines in vitro, which was associated with T cell proliferation and activation; Table 10: EC50 concentrations of T cell-mediated cytotoxicity induced by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cells and associated proliferation and activation of CD4+ and CD8+ T cells. Tumor cell line L-428 KI-JK Cytotoxicity Mean IC50 (g / mL) 0.002 0.0015 IC50 - range (g / mL) 0.001216 - 0.003439 0.0008858 - 0.001904 T cell type CD4+ CD8+ CD4+ CD8+ T cell proliferation PI Mean EC50 (g / mL) 0.0051 0.00096 0.00606 0.00213 EC50 range (g / mL) 0.003727 - 0.006534 0.0002052 - 0.002255 0.00338 - 0.007501 0.001103 - 0.002715 Activation CD69 T cell EC50 mean CD69 (gg / mL) 0.00083 0.00121 0.00146 0.00171 EC50 range CD69 (gg / mL) 0.0001221 - 0.001647 3.252E-14 - 0.002421 0.0004487 - 0.002086 4.694E-11 - 0.005097 CD25 EC50 mean CD25 (gg / mL) 0.00261 0.00061 0.00599 0.00656 EC50 range CD25 (gg / mL) 0.002164 - 0.002898 0.000298 - 0.001006 0.004483 - 0.007791 0.001901 - 0.01562 PD-1 EC50 mean PD-1 (gg / mL) 0.00491 0.00257 0.0112 0.00696 EC50 range PD-1 (gg / mL) 0.004387 - 0.005433 0.001302 - 0.003412 0.009616 - 0.01222 0.005845 - 0.009151 n tests 3 3 Example 10 - Induction of cytokine production in vitro by bsG1-huCD3FEALxCD30-MDX060-FERR Petition 870250088813, dated 09 / 30 / 2025, pp. 409 / 496 160 / 205

[00480] The production of cytokines and granzyme B induced by bsG1huCD3-FEALxCD30-MDX060-FERR was evaluated in supernatants collected during an in vitro T cell-mediated cytotoxicity experiment using L-428 target cells and healthy donor T cells, as described in Example 9. The supernatants were stored at -20°C and thawed for analysis. The concentration of 14 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, IP-10, MCP1, PDL-1, TNFα) and granzyme B were measured by a custom microsphere-based multiplex immunoassay (Luminex) made by R&D systems. Results

[00481] Increased concentrations were observed primarily for granzyme B and the cytokines IFNγ, IL-13, and TNFα (>2000 pg / mL) in supernatants of co-cultures of L-428 cells and T cells in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR. A moderate increase was observed for the concentrations of the cytokines CD40, IL-10, IL-12, IL-1ε, IL-2, IL-4, IL-6, and IP-10, compared to the control antibody IgG1-b12FEAL. The levels of IL-8, MCP-1, and PDL1 were not modulated when compared to the control antibody IgG1-b12-FEAL (Figure 19).

[00482] Thus, T cell-mediated cytotoxicity and T cell activation by bsG1-huCD3-FEALxCD30-MDX060-FERR were associated with dose-dependent production of cytokines and granzyme B. Example 11 - Induction of T cell-mediated cytotoxicity in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR with purified T cells as effector cells in varying effector-to-target ratios.

[00483] To determine the optimal ratio of effector cells to target cells for T cell-mediated tumor cell killing in the presence of the bispecific antibody bsG1-huCD3-FEALxCD30-MDX060FERR, an in vitro cytotoxicity assay was performed with a cell line of Petition 870250088813, dated 09 / 30 / 2025, pp. 410 / 496 161 / 205 CD30-positive, L-428 tumor cells as target cells and purified T cells as effector cells in varying effector to target cell ratios (E:T).

[00484] T cell-mediated cytotoxicity was assessed essentially as described in Example 9, except that T cells were added to tumor cells in varying effector-to-target cell (E:T) ratios of 1:1, 2:1, 4:1, or 8:1. Results

[00485] Figure 20A shows that dose-dependent T-cell mediated cytotoxicity was induced by bsG1-huCD3-FEALxCD30-MDX060FERR at all E:T ratios, with maximum tumor cell death (less than 20% viable tumor cells) observed at E:T ratios of 4:1 and 8:1. In line with this, CD4+ and CD8+ T-cell proliferation was observed at all E:T ratios, most prominently at E:T ratios of 4:1 and 8:1 (Figure 20B-C). No specific T-cell mediated cytotoxicity or T-cell proliferation was induced by the control antibody bsG1-huCD3-FEALxb12-FERR at any of the E:T ratios tested.

[00486] Taken together, these data indicate that the T-cell mediated cytotoxicity of L-428 tumor cells, induced by bsG1-huCD3FEALxCD30-MDX060-FERR in vitro, was most effective at E:T ratios of 4:1 and 8:1. Example 12 - Kinetics of T cell-mediated cytotoxicity and T cell proliferation in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR

[00487] To evaluate the kinetics of T cell-mediated tumor cell death in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR, an in vitro cytotoxicity assay was performed at varying incubation periods with the CD30-positive tumor cell line, L-428, as target cells and purified T cells as effector cells. Petition 870250088813, dated 09 / 30 / 2025, pp. 411 / 496 162 / 205

[00488] T-cell mediated cytotoxicity was assessed essentially as described in Example 9, except that tumor cell cytotoxicity and T-cell proliferation were assessed after 24 hours, 48 ​​hours, and 72 hours. Results

[00489] Figure 21 shows that bsG1-huCD3-FEALxCD30-MDX060FERR induced dose-dependent T-cell mediated cytotoxicity after 48 hours and 72 hours, while no significant T-cell mediated cytotoxicity was observed after 24 hours. Dose-dependent proliferation of CD4+ and CD8+ T cells was induced by bsG1-huCD3-FEALxCD30MDX060-FERR after 72 hours, while no T-cell proliferation was observed after 24 hours or 48 hours (Figure 21B and C).

[00490] Taken together, these data show that bsG1-huCD3FEALxCD30-MDX060-FERR induced T cell-mediated cytotoxicity of tumor cells and T cell proliferation in a time-dependent manner. Example 13 - Correlation of CD30 expression level with T cell-mediated cytotoxicity induced by bsG1-huCD3-FEALxCD30-MDX060FERR in vitro

[00491] T cell-mediated killing induced by bsG1-huCD3FEALxCD30-MDX060-FERR of eight CD30-expressing tumor cell lines was determined in an in vitro cytotoxicity assay as described in Example 9, using an E:T ratio of 4:1. The following cell lines were used: L-428, KM-H2, DEL, KI-JK, KARPAS-299, SUP-M2, NCEB-1, and JVM-2. For these tumor cell lines, CD30 expression levels were assessed by quantitative flow cytometry as detailed in Example 2. Results

[00492] Figure 22 shows that bsG1-huCD3-FEALxCD30-MDX060 Petition 870250088813, dated 09 / 30 / 2025, pp. 412 / 496 163 / 205 FERR induced T-cell mediated cytotoxicity in all cell lines in vitro with maximum target cell death between 68% and 98%. Maximum T-cell mediated death of tumor cells by bsG1-huCD3FEALxCD30-MDX060-FERR correlated significantly with the CD30 expression level (Figure 22A) (Spearman r=0.8571; p-value=0.0107).

[00493] In Figure 22B, the EC50 of T cell-mediated death in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR for each cell line is plotted against CD30 expression levels, showing a negative, but not significant, trend (Spearman r=-0.6190; p-value=0.1150).

[00494] Thus, these data show a positive correlation between CD30 expression levels and maximum cytotoxicity induced by bsG1huCD3-FEALxCD30-MDX060-FERR mediated by T cells in vitro. Example 14 - Fratricide of T cells activated by BsG1-huCD3FEALxCD30-MDX060-FERR

[00495] BsG1-huCD3FEALxCD30-MDX060-FERR-induced T cell fratricide of activated CD30+ T cells was evaluated in vitro.

[00496] A 96-well plate (Greiner-bio-one, Netherlands, cat. no. 655180) was coated with 100 pL of a 1 pg / mL anti-human CD3 solution (clone OKT3, Invitrogen, cat. no. 16-0037-85) in PBS. The plate was incubated for 4 hours at 37°C. After removal of the antibody solution, the wells were washed with 100 pL of PBS. T cells were obtained from buffy layers of healthy human donors (Sanquin, Amsterdam, Netherlands) and isolated with the RosetteSep™ human T cell enrichment cocktail (Stemcell Technologies, France, cat. no. 15061) according to the manufacturer's instructions. The purified T cells were resuspended in T cell medium (Roswell Park Memorial Institute [RPMI]-medium 1640 with Petition 870250088813, dated 09 / 30 / 2025, pp. 413 / 496 164 / 205 HEPES 25 mM and L-glutamine (Lonza, cat. no. BE12-115F), supplemented with 10% heat-inactivated bovine donor serum with iron (DBSI; Gibco, cat. no. 20731-030) and penicillin / streptomycin (pen / strep; Lonza, cat. no. DE17-603E) at a concentration of 2 x 10⁶ cells / mL. 100 pL of T-cell suspension (containing 200,000 T cells) were added to each well of the anti-CD3 coated plate. In addition, 100 pL of T cell culture medium supplemented with anti-CD28 2 pg / mL (clone CD28.2, Invitrogen, cat. no. 160289-85) and IL-15 0.05 pg / mL (ThermoFisher, cat. no. PHC9151) were added to each well. The T cells were then incubated for 96 hours at 37°C.

[00497] After 96 hours, T cells were collected and resuspended in T cell medium at a concentration of 2x10⁶ cells / mL. Flow cytometry analysis was performed to measure the expression of CD30 and T cell activation markers. Briefly, an aliquot of cells was washed with 0.1% PBS / BSA / 0.02% azide (stain buffer), stained with 50 µl of 1000x diluted FVS510 viability stain (BD Biosciences, cat. no. 564406), and incubated at room temperature for 15 minutes. Then, the cells were washed with dye buffer and stained for CD30 (1:50; Biolegend, cat. no. 333906, PE-conjugated), CD4 T cell markers (1:50; Biolegend, cat. no. 300506, FITC-conjugated), CD8 (1:100; Biolegend, cat. no. 301028, AF700-conjugated) and CD69 T cell activation markers (1:50; Biolegend, cat. no. 310910, APC-conjugated), CD25 (1:100; Invitrogen, cat. no. 25-0259-42, PE-Cy7-conjugated) and CD279 / PD1 (1:50; Biolegend, cat. no. 329924, BV605-conjugated).Single samples stained with Ultracomp microspheres (5 pL; Invitrogen, cat. no. 01-2222-42) were included and used for flow cytometer compensation adjustments. After 30 minutes of incubation at 4°C, the plates were washed twice with dye buffer. Cells were analyzed using FACS Celesta (BD Biosciences) and the data were processed with FlowJo (BD Biosciences). Petition 870250088813, dated 09 / 30 / 2025, pp. 414 / 496 165 / 205

[00498] To evaluate whether BsG1-huCD3-FEALxCD30-MDX060-FERR can induce fratricide in activated T cells, stimulated T cells were seeded in a 96-well plate at a density of 200,000 cells / well. Subsequently, 50 pL of BsG1-huCD3-FEALxCD30MDX060-FERR, or of the control antibodies, i.e., bsG1-b12FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxb12-MDX060-FERR, or IgG1-b12 were added to each well (final concentration ranging from 0.003 to 3.3 pg / mL in 3-fold dilution steps in T cell medium). The plates were incubated at 37°C for 48 hours.

[00499] After washing twice with dye buffer, the cells were stained with the viability dye FVS510 and, consequently, stained for the T cell markers CD4 and CD8 and the T cell activation markers CD69, CD25, and CD279 / PD1. Flow cytometry analysis was performed using FACS Celesta (BD Biosciences) and the data were processed with F...

Claims

1. Pharmaceutical composition, characterized in that it comprises: a) a multispecific antibody comprising an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3; and b) a buffering agent wherein the pH of the composition is from 4.0 to 8.

0.

2. Pharmaceutical composition according to claim 1, characterized in that the buffering agent is selected from the group consisting of acetate, histidine, TRIS® (tris (hydroxymethyl) aminomethane), citrate, succinate, glycolate, glutamate and mixtures thereof.

3. Pharmaceutical composition according to claim 2, characterized in that the buffering agent is histidine, acetate and / or mixtures thereof.

4. Pharmaceutical composition according to claim 3, characterized in that the buffering agent is acetate.

5. Pharmaceutical composition according to claim 4, characterized in that the buffering agent is selected from the group consisting of sodium acetate, potassium acetate, sodium acetate, their hydrates and mixtures thereof.

6. Pharmaceutical composition according to claim 5, characterized in that the buffering agent is sodium acetate, such as sodium acetate trihydrate.

7. Pharmaceutical composition according to any of the preceding claims, characterized in that the buffering agent is present at a concentration of about 5 to about 40 mM, such as about 10 to about 30 mM, such as about 15 to about 25 mM, such as about 18 to about 22 mM, preferably about 20 mM, such as about 17 to about 18 mM.

8. Pharmaceutical composition according to any of the preceding claims, characterized in that the pH of the composition is from about 4.5 to about 6.5, or from about 5.0 to about 6.0, or from about 5.2 to about 5.7, or from about 5.4 to about 5.6, preferably about 5.

5.

9. Pharmaceutical composition according to any of the preceding claims, characterized in that the pH of the composition is approximately 5.

5.

10. Pharmaceutical composition according to any one of claims 1 to 8, characterized in that the pH of the composition is about 6.

0.

11. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition additionally comprises a pH adjustment component.

12. Pharmaceutical composition according to claim 11, characterized in that the pH adjusting component is an acid.

13. Pharmaceutical composition according to claim 12, characterized in that the acid is HCl and / or acetic acid, such as glacial acetic acid.

14. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition additionally comprises a non-ionic excipient.

15. Pharmaceutical composition according to claim 14, characterized in that the non-ionic excipient is a sugar or a sugar alcohol.

16. Pharmaceutical composition according to claim 15, characterized in that the non-ionic excipient is selected from sucrose, trehalose, mannitol, xylitol, sorbitol and mixtures thereof.

17. Pharmaceutical composition according to claim 16, characterized in that the non-ionic excipient is selected from sorbitol, sucrose, trehalose or mixtures thereof.

18. Pharmaceutical composition according to claim 17, characterized in that the non-ionic excipient is sorbitol or trehalose.

19. Pharmaceutical composition according to claim 18, characterized in that the non-ionic excipient is sorbitol.

20. Pharmaceutical composition according to any one of claims 14 to 19, characterized in that the non-ionic excipient is present at a concentration of about 5 to about 450 mM, such as about 50 to about 400 mM, such as about 100 to about 350 mM, such as about 125 to about 300 mM, such as about 150 to about 250 mM, preferably about 250 mM.

21. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition comprises acetate, such as sodium acetate, and sorbitol and has a pH of about 5.

5.

22. Pharmaceutical composition according to any of the preceding claims, characterized in that the osmolality of the pharmaceutical composition is less than about 600 mOsm / kg, such as less than about 550 mOsm / kg, such as less than about 500 mOsm / kg, such as less than about 450 mOsm / kg, such as less than about 400 mOsm / kg, such as less than about 350 mOsm / kg.

23. Pharmaceutical composition according to any one of claims 1 to 21, characterized in that the osmolality of the pharmaceutical composition is in the range of about 100-500 mOsm / kg, as in the range of about 200-400 mOsm / kg, as in the range of about 250-350 mOsm / kg. Petition 870250088813, dated 09 / 30 / 2025, page 193 / 496 4 / 18 24. Pharmaceutical composition according to any of the preceding claims, characterized in that the viscosity of the pharmaceutical composition is less than about 30 cP, such as less than about 25 cP, less than about 20 cP, less than about 18 cP, less than about 16 cP, less than about 14 cP, less than about 12 cP, less than about 10 cP, less than about 9 cP, less than about 8 cP, less than about 7 cP, less than about 6 cP, less than about 5 cP, less than about 4 cP, less than about 3 cP, less than about 2 cP.

25. Pharmaceutical composition according to any one of claims 1 to 23, characterized in that the viscosity of the pharmaceutical composition is in the range of about 1-30 cP, as in the range of about 1-25 cP, as in the range of about 1-20 cP, as in the range of about 1-18 cP, as in the range of about 1-15 cP, as in the range of about 112 cP, as about 1-10 cP, as about 1-9 cP, as about 1-8 cP, as about 1-7 cP, as about 1-6 cP, as about 1-5 cP, as about 1-4 cP, as about 1-3 cP.

26. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition additionally comprises a surfactant.

27. Pharmaceutical composition according to claim 26, characterized in that the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene derivatives of castor oil, polyoxyethylene esters of fatty acids, Petition 870250088813, dated 09 / 30 / 2025, p. 194 / 496 5 / 18 polyoxyethylene stearates, polyoxyl hydrostearate, polyoxyglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sucrose palmitate, sorbitan esters, sucrose stearate, tricaprilin and TPGS, as well as mixtures thereof.

28. Pharmaceutical composition according to claim 27, characterized in that the surfactant is a polysorbate.

29. Pharmaceutical composition according to claim 28, characterized in that the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80), preferably polysorbate 80 (PS80).

30. Pharmaceutical composition according to any one of claims 26 to 29, characterized in that the surfactant is present at a concentration of about 0.01 to about 0.1% w / v, such as about 0.01 to about 0.09% w / v, such as about 0.01 to about 0.06% w / v, such as about 0.01 to about 0.05% w / v, such as about 0.02% w / v, about 0.03% w / v, about 0.04% w / v or about 0.05% w / v, preferably about 0.02% w / v.

31. Pharmaceutical composition according to any of the preceding claims, characterized in that the antibody concentration is about 0.5 to about 250 mg / ml, about 1.0 to about 220 mg / ml, about 3 to about 190 mg / ml, about 5 to about 160 mg / ml, about 10 to about 130 mg / ml, about 20 to about 120 mg / ml, about 30 to about 110 mg / ml, about 40 to about 100 mg / ml, about 50 to about 90 mg / ml, about 60 to about 80 mg / ml, or about 65 to about 75 mg / ml, or about 20 to about 200 mg / ml.

32. Pharmaceutical composition according to any one of claims 1 to 31, characterized in that the antibody concentration is about 50 to about 250 mg / ml, or about 60 to about 240 mg / ml, or about 70 to about 220 mg / ml, or about 80 to about 210 mg / ml, or about 100 to about 200 mg / ml, or about 120 to about 190 mg / ml, or about 140 to about 180 mg / ml, or about 150 to about 170 mg / ml.

33. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is a liquid composition.

34. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is an aqueous composition.

35. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is a stable pharmaceutical composition.

36. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is stable over a period of at least 2 months, such as at least 3 months, such as at least 6 months, such as at least 9 months, such as at least 12 months.

37. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is stable over a wide range of antibody concentrations from about 10 mg / ml to about 250 mg / ml, as from about 20 mg / ml to about 200 mg / ml, as from about 50 mg / ml to about 170 mg / ml.

38. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is stable over a temperature range of about 2°C to about 25°C.

39. Pharmaceutical composition according to any of the preceding claims, characterized in that the composition is stable for at least 2 months, as well as at least 3 months, as well as at least 4 months, as well as at least 5 months, as well as at least 6 months, as well as at least 9 months or at least 12 months at a storage temperature of 2-8°C, as well as 5°C.

40. Pharmaceutical composition according to any of the preceding claims, characterized in that the pharmaceutical composition is stable for at least 12 months at a storage temperature of about 5°C.

41. Pharmaceutical composition according to any of the preceding claims, characterized in that it comprises or consists essentially of: a) about 0.5-250 mg / ml of a multispecific antibody, such as about 20-200 mg / ml of a multispecific antibody, b) a buffering agent, c) optionally, a non-ionic excipient, d) optionally, a surfactant wherein the pH of the composition is from about 5.0 to about 6.

5.

42. Pharmaceutical composition according to any of the preceding claims, characterized in that the composition is a subcutaneous composition and / or in that the composition is for use in subcutaneous administration.

43. Pharmaceutical composition according to any of the preceding claims, characterized in that the composition is an intravenous composition and / or in that the composition is for use in intravenous administration.

44. Pharmaceutical composition according to any of the preceding claims, characterized in that the multispecific antibody comprises: (i) a CD30 binding region comprising a first variable heavy chain region comprising the sequences of Petition 870250088813, dated 09 / 30 / 2025, page. 197 / 496 8 / 18 CDR1, CDR2 and CDR3 established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively.

45. Pharmaceutical composition according to claim 44, characterized in that the first variable heavy chain region and / or the first variable light chain region are human.

46. ​​Pharmaceutical composition according to claim 44 or 45, characterized in that the second variable heavy chain region and / or the second variable light chain region are humanized.

47. Pharmaceutical composition according to any one of claims 44 to 46, characterized in that X in SEQ ID NO:9 is an H.

48. Pharmaceutical composition according to any one of claims 44 to 47, characterized in that the first variable heavy chain region comprises the sequence set forth in SEQ ID NO:13 and the first variable light chain region comprises the sequence set forth in SEQ ID NO:

14.

49. Pharmaceutical composition according to any one of claims 44 to 48, characterized in that the second variable heavy chain region comprises the sequence set forth in SEQ ID NO:15 and the second variable light chain region comprises the sequence set forth in SEQ ID NO:

16. Petition 870250088813, dated 09 / 30 / 2025, p. 198 / 496 9 / 18 50. Pharmaceutical composition according to claim 49, characterized in that X in SEQ ID NO:15 is an H.

51. Pharmaceutical composition according to any one of claims 44 to 50, characterized in that the multispecific antibody is a bispecific antibody.

52. Pharmaceutical composition according to any one of claims 44 to 51, characterized in that the multispecific antibody comprises an Fc region consisting of a first and a second Fc polypeptide.

53. Pharmaceutical composition according to any one of claims 44 to 52, characterized in that the Fc region is an Fc region of IgG1, preferably a human IgG1 Fc region.

54. Pharmaceutical composition according to any one of claims 44 to 53, characterized in that the multispecific antibody is a complete antibody.

55. Pharmaceutical composition according to any one of claims 44 to 54, characterized in that the multispecific antibody comprises an inert Fc region.

56. Pharmaceutical composition according to any one of claims 44 to 55, characterized in that the first and / or second Fc polypeptide comprise an amino acid substitution corresponding to the amino acid at positions L234 and / or L235 in a human IgG1 heavy chain, wherein the substitutions are preferably to F and E, respectively, in which the amino acid positions are as defined by I numbering.

57. Pharmaceutical composition according to claim 55 or 56, characterized in that the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and in which Petition 870250088813, dated 09 / 30 / 2025, page 199 / 496 10 / 18 the first and / or second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position G236 in a human IgG1 heavy chain, wherein the substitution is preferably for R, in which the amino acid positions are as defined by the I numbering.

58. Pharmaceutical composition according to claim 57, characterized in that the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and wherein the first and second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position G236 in a human IgG1 heavy chain, wherein the substitution is preferably for R.

59. Pharmaceutical composition according to claim 55 or 56, characterized in that the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and wherein the first and / or second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position D265 in a human IgG1 heavy chain, wherein the substitution is preferably for A, wherein the amino acid positions are as defined by the numbering I.

60. Pharmaceutical composition according to claim 59, characterized in that the first and second Fc polypeptide comprise amino acid substitutions corresponding to the amino acids at positions L234 and L235 for F and E, respectively, and in that the first and second Fc polypeptide further comprise an amino acid substitution corresponding to the amino acid at position D265 in a human IgG1 heavy chain, wherein the substitution is preferably for A.

61. Pharmaceutical composition according to any one of claims 56 to 60, characterized in that one of the first and second Fc polypeptides comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the other Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235E and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the numbering I.

62. Pharmaceutical composition according to claim 61, characterized in that the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain and in that the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain.

63. Pharmaceutical composition according to claim 62, characterized in that the first Fc polypeptide comprises amino acid substitutions corresponding to amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the second Fc polypeptide comprises amino acid substitutions corresponding to amino acids at positions L234, L235E and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the numbering I.

64. Pharmaceutical composition according to any one of claims 44 to 63, characterized in that, in the first Fc polypeptide, at least one of the amino acids in the positions corresponding to a position selected from the group consisting of: T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain has been substituted and, in the second Fc polypeptide, at least one of the Petition 870250088813, dated 09 / 30 / 2025, p. 201 / 496 12 / 18 amino acids in the positions corresponding to a position selected from the group consisting of: T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain has been substituted and in which said substitutions in the first and second Fc polypeptide are not in the same positions, in which the amino acid positions are as defined by the I numbering.

65. Pharmaceutical composition according to claim 64, characterized in that the amino acid at the position corresponding to F405 is L in the first Fc polypeptide and that the amino acid at the position corresponding to K409 is R in the second Fc polypeptide, or vice versa.

66. Pharmaceutical composition according to claim 65, characterized in that the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and that the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

67. Pharmaceutical composition according to any one of claims 44 to 66, characterized in that the multispecific antibody comprises: (i) a CD30 binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3 binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively;Petition 870250088813, dated 09 / 30 / 2025, page 202 / 496 13 / 18 wherein the multispecific antibody is a bispecific antibody and comprises an Fc region consisting of a first and a second Fc polypeptide; wherein the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain and wherein the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain; wherein the first Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the second Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the numbering Eu;and wherein the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

68. Pharmaceutical composition according to any one of claims 44 to 67, characterized in that the multispecific antibody comprises or consists of the heavy chain sequences established in SEQ ID NO:17 and 19 and the light chain sequences established in SEQ ID NO:18 and 20.

69. Pharmaceutical composition according to any one of claims 44 to 68, characterized in that the multispecific antibody comprises or consists of the heavy chain sequences established in SEQ ID NO:17 and 19 and the light chain sequences established in SEQ ID NO:18 and 20, wherein the antibody is a bispecific antibody.

70. Pharmaceutical composition according to any of the preceding claims, characterized in that the multispecific antibody comprises a lambda (λ) light chain and a kappa (κ) light chain; for example, an antibody with a heavy chain and a lambda light chain comprising the binding region capable of binding to CD3, and a heavy chain and a kappa light chain comprising the binding region capable of binding to CD30.

71. Pharmaceutical composition according to any of the preceding claims, characterized in that the antigen-binding region capable of binding to human CD30 comprises a heavy chain and a light chain, said heavy chain comprising said VH region and a constant region of IgG1 heavy chain and said light chain comprising said VL region and a constant region of kappa light chain; and wherein said antigen-binding region capable of binding to human CD3 comprises a heavy chain and a light chain, said heavy chain comprising said VH region and a constant region of IgG1 heavy chain and said light chain comprising said VL region and a constant region of lambda light chain.

72. Pharmaceutical composition according to claim 71, characterized in that one constant region of the IgG1 heavy chain is as defined in SEQ ID NO. 51 and the other is as defined in SEQ ID NO. 50, and wherein said kappa constant region of the light chain is as defined in SEQ ID NO. 53 and said lambda constant region of the light chain is as defined in SEQ ID NO.

54.

73. Pharmaceutical composition according to any of the preceding claims, characterized in that the antibody comprising: (i) a CD30 binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region. Petition 870250088813, dated 09 / 30 / 2025, p. 204 / 496 15 / 18 comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10, 11 and 12, respectively, wherein X in SEQ ID NO: 9 is H;wherein the antibody is a bispecific antibody and comprises an Fc region consisting of a first and a second Fc polypeptide; wherein the first Fc polypeptide and the first variable heavy chain region are comprised within the same polypeptide chain and wherein the second Fc polypeptide and the second variable heavy chain region are comprised within the same polypeptide chain; wherein the first Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and G236 for F, E and R, respectively, and the second Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234, L235 and D265 for F, E and A, respectively, wherein the amino acid positions are as defined by the I numbering; and wherein the amino acid at the position corresponding to K409 is R in the first Fc polypeptide and wherein the amino acid at the position corresponding to F405 is L in the second Fc polypeptide.

74. Pharmaceutical composition according to any of the preceding claims, characterized in that the antibody is bsIgG1-huCD3-FEALxCD30-FERR, or a biosimilar thereof.

75. Pharmaceutical composition according to any of the preceding claims, characterized in that it comprises or consists essentially of: Petition 870250088813, dated 09 / 30 / 2025, p. 205 / 496 16 / 18 a) approximately 0.5-250 mg / ml of antibody, as well as approximately 20-200 mg / ml of antibody, wherein the antibody comprises: (i) a CD30-binding region comprising a first variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:1, 2 and 3, respectively, and a first variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:4, 5 and 6, respectively, and (ii) a CD3-binding region comprising a second variable heavy chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:7, 8 and 9, respectively, and a second variable light chain region comprising the CDR1, CDR2 and CDR3 sequences established in SEQ ID NO:10,11 and 12, respectively; optionally wherein X in SEQ ID NO: 9 is H; b) a buffering agent, such as histidine or acetate, c) optionally, a non-ionic excipient, such as sucrose, trehalose or sorbitol, d) optionally, a surfactant such as a polysorbate, wherein the pH of the composition is from about 5.0 to about 6.

5.

76. Pharmaceutical composition according to any one of claims 1 to 75, characterized in that it is for use as a medicament.

77. Pharmaceutical composition for use as a medicament according to claim 76, characterized in that it is for use in a method for treating a disease.

78. Pharmaceutical composition according to any one of claims 1 to 75, characterized in that it is for use in the treatment of cancer.

79. Pharmaceutical composition according to claim Petition 870250088813, dated 09 / 30 / 2025, page 206 / 496 17 / 18 78, characterized by being for use in the treatment of Hodgkin lymphoma, such as classic Hodgkin lymphoma or non-Hodgkin lymphoma (NHL).

80. Pharmaceutical composition for use according to claim 79, characterized in that the non-Hodgkin lymphoma is T-cell non-Hodgkin lymphoma (T-NHL).

81. Pharmaceutical composition for use according to claim 80, characterized in that LNH-T is cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL).

82. Pharmaceutical composition for use according to any one of claims 79 to 81, characterized in that it is anaplastic large cell lymphoma (ALCL).

83. Pharmaceutical composition for use according to claim 78, characterized in that the non-Hodgkin lymphoma is B-cell non-Hodgkin lymphoma (B-NHL).

84. Pharmaceutical composition for use according to any of claims 78 to 83, characterized in that it has been administered intravenously and / or subcutaneously, preferably subcutaneously.

85. Use of the pharmaceutical composition as defined in any one of claims 1 to 75, characterized by the fact that it is for the treatment of cancer.

86. Kit in parts, characterized in that it comprises: a. a pharmaceutical composition as defined in any of claims 1 to 75, b. a container for the pharmaceutical composition, and c. instructions for use of said kit.

87. Kit in parts according to claim 86, characterized in that said kit in parts additionally comprises Petition 870250088813, dated 09 / 30 / 2025, page 207 / 496 18 / 18 a diluent.

88. Method for preparing a pharmaceutical composition as defined in any one of claims 1 to 75, characterized in that it comprises the steps of mixing in water: a) a multispecific antibody, such as from about 0.5 to about 250 mg / ml of the antibody, b) a buffering agent, optionally, c) a non-ionic excipient and optionally, d) a surfactant; and adjusting the pH to about 4.0-8.0.

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