Pharmaceutical composition containing antibodies that bind to CD30 and CD3

Abstract

The present invention relates to a pharmaceutical composition comprising a multispecific antibody capable of binding to CD3 and CD30. The present invention further provides the use of the pharmaceutical composition for therapeutic and diagnostic procedures, particularly for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition of a multispecific antibody that binds to CD3 and CD30, and to the use of such a pharmaceutical composition. [Background technology]

[0002] CD30, also known as Ki-1 or TNFRSF8, is a 120kD transmembrane glycoprotein receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily (Smith et al. (1994) Cell 76:959-962). CD30 is a single-pass type I membrane protein with six cysteine-rich repeats in its extracellular domain (Durkop et al. (1992) Cell 68:421-427). Furthermore, the soluble form of CD30 (sCD30) has been detected in the serum of patients with inflammatory diseases, such as 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-fixed metalloproteinases such as TACE / ADAM17 and ADAM10 (Nagata et al., PNAS 2005; Hansen et al., FASEB, 2004).

[0003] In normal tissues, CD30 expression is mainly limited to a subset of activated T lymphocytes and B lymphocytes (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 various lymphoid neoplasms. Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) show high levels of CD30 expression. In particular, the majority of Reed-Sternberg (RS) cells, which are typically found in cHL, are CD30-positive (Frizzera et al. (1992) Semin. Diagn. Pathol. 9:291-296).

[0004] For example, brentuximab vedotin (BV;SGN-35), a CD30-targeted antibody-drug conjugate, is used or suggested for use in the treatment of cancers such as cHL, 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 quadrivalent bispecific CD30xCD16A antibody AFM13 is being developed as an NK cell-mediated immunotherapy for cHL and CD30-positive lymphoma (Rothe et al. Blood. 2015 Jun 25;125(26):4024-31).

[0005] Furthermore, CD30-targeted CAR-T cell therapies are being developed for cHL and CD30-positive lymphoma. Other CD30 antibodies are described in International Publication No. 2003059282 (Medarex), U.S. Patent No. 8257706 (Seattle Genetics), U.S. Patent No. 20100239571 (Seattle Genetics), International Publication No. 2007040653 (US Government & Health), and International Publication No. 20160177846 (Affimed).

[0006] Pohl et al. (1993 Int.J.Cancer, 54:820-827) described the CD3xCD30 bispecific antibody OKT-3 / HRS-3 generated by the fusion of CD30 monoclonal antibody HRS-3-producing hybridoma cells and CD3 monoclonal antibody OKT-3-producing hybridoma cells (hybrid hybridoma technology).

[0007] International Publication No. 2008119567 describes the generation and characterization of CD30 and CD3 cross-species specific bispecific single-stranded molecules.

[0008] U.S. Patent Application Publication No. 20200095330 describes a CD3xCD30 bispecific antibody resulting from the chemical heteroconjugation of two anti-CD30 clones (named 8D10 and 10C2) to anti-CD3 (Orthoclone OKT-3).

[0009] However, none of these CD3xCD30 bispecific antibodies have been tested in clinical practice.

[0010] Therefore, improved CD30-targeted cancer therapies are needed. There is a continued need for CD30-targeted compounds that are effective, safe, have good manufacturability, and / or have a long shelf life. Pharmacologically acceptable formulations of antibodies for use in such therapies are also needed. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2003059282 (Medarex) [Patent Document 2] U.S. Patent No. 8257706 [Patent Document 3] U.S. Patent No. 20100239571 [Patent Document 4] International Publication No. 2007040653 [Patent Document 5] International Publication No. 20160177846 [License 6] International Publication No. 2008119567 [License 7] U.S. Patent and Trademark Publication No. 20200095330 [Non-licensed literature]

[0012] [Non-licensed Document 1] Smith et al. (1994) Cell 76:959-962 [Non-licensed Document 2] Durkop et al. (1992) Cell 68:421-427 [Non-licensed Document 3] Giacomelli et al. Clin Exp Immunol.1998;111:532-5 [Non-licensed Document 4] Josimovic-Alasevic et al. (1989) Eur J Immunol [Non-licensed Document 5] Nagata et al., PNAS 2005; Hansen et al., FASEB, 2004 [Non-licensed Document 6] Bowen et al.(1996)J Immunol.156:442-9 [Non-licensed Document 7] Shanebeck et al. (1995) Eur J Immunol. 25:2147-53 [Non-licensed Document 8] Frizzera et al. (1992)Semin. Diagn. Pathol.9:291-296 [Non-licensed Document 9] Younes et al.J Clin Oncol.2012 Jun 20;30(18):2183-9 [Non-licensed Document 10] Pro et al.Blood.2017 Dec 21;130(25):2709-2717 [Non-Patent Document 11] Shea et al.Curr Hematol Malig Rep.2020 Feb;15(1):9-19 [Non-Patent Document 12] Rothe et al.Blood.2015 Jun 25;125(26):4024-31 [Non-Patent Document 13] Pohl et al.(1993 Int.J.Cancer,54:820-827) [Overview of the Initiative] [Problems that the invention aims to solve]

[0013] The object of the present invention is to provide a pharmaceutical composition of a T cell-engaging antibody that binds to human and cynomolgus monkey CD30 and CD3. A further object is to provide a pharmaceutical composition of an antibody in which the formulation is stable over a wide range of antibody concentrations and / or temperatures. A further object is to provide a pharmaceutical composition of an antibody in which the formulation is stable for at least 3 months or longer. A further object of the present invention is to provide a pharmaceutical formulation of an antibody that is well tolerable for IV injection and subcutaneous administration. Furthermore, a bispecific antibody having a functionally inactive Fc skeleton that is suitable for development into pharmaceutical compositions due to its excellent stability and solubility has been identified. [Means for solving the problem]

[0014] In one embodiment, the present invention relates to a pharmaceutical composition comprising a multispecific antibody having an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3, and a buffer, wherein the pH of the composition is 4.0 to 8.0. Such a pharmaceutical composition has been found to provide remarkably high antibody stability, such as thermal stability and storage stability, as well as high solubility.

[0015] In one embodiment, the pharmaceutical composition of the present invention comprises a multispecific antibody comprising (i) a CD30 binding region comprising a first heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) a CD3 binding region comprising a second heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

[0016] In a further embodiment, the present invention relates to a pharmaceutical composition for use as a pharmaceutical in the treatment of cancer, for example.

[0017] In a further embodiment, the present invention relates to a parts kit comprising a) a pharmaceutical composition described herein, b) a container for the pharmaceutical composition, and c) instructions for use of the kit.

[0018] In a further embodiment, the present invention relates to a method for preparing a pharmaceutical composition as defined herein, comprising the steps of: a) mixing a multispecific antibody, b) a buffer, optionally c) a nonionic excipient, and optionally d) a surfactant in water; and adjusting the pH to 4.0 to 8.0. [Brief explanation of the drawing]

[0019] [Figure 1-1]Bispecific CD3xCD30 antibodies against SU-DHL-1 or HDML-2 cells, and their monospecific bivalent CD3 and CD30 counterparts. (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxb12-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG 1-CD30-hAC10-FEAR, (C)bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-H RS-3-FEAR, (D)BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, and bsG1-huCD3-FEALxb12-FEAR, (E)bsIgG1-huCD3- FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408- (I) Dose-dependent binding of FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T408-FEAR, and (H) bsIgG1-huCD3-FEALxCD30-T215-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T215-FEAR to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel). (I) Binding of CD3xCD30 bispecific antibody and CD30 monospecific antibody to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel) at a concentration of 1.11 μg / mL. The antibody clone used in the CD30 arm is shown on the x-axis. The data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 1-2]Bispecific CD3xCD30 antibodies against SU-DHL-1 or HDML-2 cells, and their monospecific bivalent CD3 and CD30 counterparts. (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxb12-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG 1-CD30-hAC10-FEAR, (C)bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-H RS-3-FEAR, (D)BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, and bsG1-huCD3-FEALxb12-FEAR, (E)bsIgG1-huCD3- FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408- (I) Dose-dependent binding of FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T408-FEAR, and (H) bsIgG1-huCD3-FEALxCD30-T215-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T215-FEAR to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel). (I) Binding of CD3xCD30 bispecific antibody and CD30 monospecific antibody to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel) at a concentration of 1.11 μg / mL. The antibody clone used in the CD30 arm is shown on the x-axis. The data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 1-3]Bispecific CD3xCD30 antibodies against SU-DHL-1 or HDML-2 cells, and their monospecific bivalent CD3 and CD30 counterparts. (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxb12-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG 1-CD30-hAC10-FEAR, (C)bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-H RS-3-FEAR, (D)BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, and bsG1-huCD3-FEALxb12-FEAR, (E)bsIgG1-huCD3- FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408- (I) Dose-dependent binding of FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T408-FEAR, and (H) bsIgG1-huCD3-FEALxCD30-T215-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T215-FEAR to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel). (I) Binding of CD3xCD30 bispecific antibody and CD30 monospecific antibody to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel) at a concentration of 1.11 μg / mL. The antibody clone used in the CD30 arm is shown on the x-axis. The data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 2]Binding of bsG1-huCD3xCD30-MDX060 to HL and ALCL cell lines. Binding of bsG1-huCD3xCD30-MDX060 to (A) HDLM-2 (HL) cells, (B) L-428 (HL) cells, (C) DEL (ALCL) cells, or (D) KI-JK (ALCL) cells was evaluated by flow cytometry. Bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060, as well as monospecific antibodies IgG1-CD30-MDX060, IgG1-huCD3, and IgG1-12 were included as controls. All antibodies contained FEAL and / or FERR Fc silencing and DuoBody® technology mutations in their Fc domains, as shown. The data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 3-1]In vitro induction of cytotoxicity in SU-DHL-1 or HDLM-2 cells by CD3xCD30 bispecific antibodies. CD30-positive tumor cell lines SU-DHL-1 (left panel) or HDLM-2 (right panel) were used as target cells, and T cells (CD3-positive ADCC effector cells type IV; Clean Cells, Montaigu, France) were used as effector cells to test CD3xCD30 bispecific antibodies in an in vitro cytotoxicity assay. The following antibodies were tested: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi-I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are the percentage of viable cells. The data for each graph was obtained from one representative experiment. [Figure 3-2]In vitro induction of cytotoxicity in SU-DHL-1 or HDLM-2 cells by CD3xCD30 bispecific antibodies. CD30-positive tumor cell lines SU-DHL-1 (left panel) or HDLM-2 (right panel) were used as target cells, and T cells (CD3-positive ADCC effector cells type IV; Clean Cells, Montaigu, France) were used as effector cells to test CD3xCD30 bispecific antibodies in an in vitro cytotoxicity assay. The following antibodies were tested: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi-I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are the percentage of viable cells. The data for each graph was obtained from one representative experiment. [Figure 3-3]In vitro induction of cytotoxicity in SU-DHL-1 or HDLM-2 cells by CD3xCD30 bispecific antibodies. CD30-positive tumor cell lines SU-DHL-1 (left panel) or HDLM-2 (right panel) were used as target cells, and T cells (CD3-positive ADCC effector cells type IV; Clean Cells, Montaigu, France) were used as effector cells to test CD3xCD30 bispecific antibodies in an in vitro cytotoxicity assay. The following antibodies were tested: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi-I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. The data shown are the percentage of viable cells. The data for each graph was obtained from one representative experiment. [Figure 4-1]In vitro induction of T cell-mediated cytotoxicity and T cell proliferation in several ALCL and HL cell lines by CD3xCD30 bispecific antibodies. (A-C) CD3xCD30 bispecific antibodies were tested in in vitro cytotoxicity assays using different ALCL and HL cell lines as target cells and purified T cells (A, B) or ADCC effector cells type IV (C) or effector cells. CD3xCD30 bispecific antibodies included huCD3-FEAL Fab arms or huCD3-H101G-FEAL variants (with lower affinity for CD3), and CD30-specific MDX060-FEAR Fab arms. IgG1-huCD3 and IgG1-b12 (A, B) or bsG1-b12-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR (C) were included as controls. The data shown are the percentage of viable cells. The data for each graph was obtained from one representative experiment. (D) The number of CFSE-positive cells was evaluated as a measure of the absolute number of T cells in a cytotoxicity assay using HDLM-2 cells (left panel) or NCEB-1 cells (right panel) as target cells. [Figure 4-2]In vitro induction of T cell-mediated cytotoxicity and T cell proliferation in several ALCL and HL cell lines by CD3xCD30 bispecific antibodies. (A-C) CD3xCD30 bispecific antibodies were tested in in vitro cytotoxicity assays using different ALCL and HL cell lines as target cells and purified T cells (A, B) or ADCC effector cells type IV (C) or effector cells. CD3xCD30 bispecific antibodies included huCD3-FEAL Fab arms or huCD3-H101G-FEAL variants (with lower affinity for CD3), and CD30-specific MDX060-FEAR Fab arms. IgG1-huCD3 and IgG1-b12 (A, B) or bsG1-b12-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR (C) were included as controls. The data shown are the percentage of viable cells. The data for each graph was obtained from one representative experiment. (D) The number of CFSE-positive cells was evaluated as a measure of the absolute number of T cells in a cytotoxicity assay using HDLM-2 cells (left panel) or NCEB-1 cells (right panel) as target cells. [Figure 5-1]Conjugation of CD3xCD30 bispecific antibodies to full-length human and cynomolgus monkey CD30 transfected into Expi293F cells. (A-C) Conjugation of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with wild-type Expi293F cells (A) or full-length human CD30 (B) or cynomolgus monkey CD30 (C). Cells were incubated with the following antibodies at increasing concentrations: IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR. Data are presented as mean fluorescence intensity (MFI) values ​​determined by flow cytometry of two technically replicated experiments. (D) Binding of antibodies IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3-FEALxb12-FEAR to human T cells or cynomolgus monkey T cells. Data are presented as mean fluorescence intensity (MFI) values ​​determined by flow cytometry of one representative experiment. [Figure 5-2]Conjugation of CD3xCD30 bispecific antibodies to full-length human and cynomolgus monkey CD30 transfected into Expi293F cells. (A-C) Conjugation of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with wild-type Expi293F cells (A) or full-length human CD30 (B) or cynomolgus monkey CD30 (C). Cells were incubated with the following antibodies at increasing concentrations: IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR, bsG1-b12-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR. Data are presented as mean fluorescence intensity (MFI) values ​​determined by flow cytometry of two technically replicated experiments. (D) Binding of antibodies IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3-FEALxb12-FEAR to human T cells or cynomolgus monkey T cells. Data are presented as mean fluorescence intensity (MFI) values ​​determined by flow cytometry of one representative experiment. [Figure 6-1]Binding of CD3xCD30 bispecific antibodies to full-length human and rhesus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with full-length human CD30 (left panel) or full-length rhesus monkey CD30 (right panel) was evaluated by flow cytometry. The following antibodies were evaluated: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi -I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in all experiments. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 6-2]Binding of CD3xCD30 bispecific antibodies to full-length human and rhesus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with full-length human CD30 (left panel) or full-length rhesus monkey CD30 (right panel) was evaluated by flow cytometry. The following antibodies were evaluated: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi -I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in all experiments. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 6-3]Binding of CD3xCD30 bispecific antibodies to full-length human and rhesus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with full-length human CD30 (left panel) or full-length rhesus monkey CD30 (right panel) was evaluated by flow cytometry. The following antibodies were evaluated: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi -I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in all experiments. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 6-4]Binding of CD3xCD30 bispecific antibodies to full-length human and rhesus monkey CD30 transfected into Expi293F cells. Binding of monovalent and bivalent CD30 antibodies to Expi293F cells transiently transfected with full-length human CD30 (left panel) or full-length rhesus monkey CD30 (right panel) was evaluated by flow cytometry. The following antibodies were evaluated: (A) bsG1-huCD3-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR, (B) bsG1-huCD3-FEALxCD30-hAC10-FEAR and IgG1-CD30-hAC10-FEAR, (C) bsG1-huCD3-FEALxCD30-HRS-3-FEAR and IgG1-CD30-HRS-3-FEAR, (D) bsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR and IgG1-CD30-HeFi -I-FEAR, (E)bsIgG1-huCD3-FEALxCD30-T405-FEAR and IgG1-CD30-T405-FEAR, (F)bsIgG1-huCD3-FEALxCD30-T105-FEAR and IgG1-CD30-T105-FEAR, (G)bsIgG1-huCD3-FEALxCD30-T408-FEAR and IgG1-CD30-T408-FEAR, or (H)bsIgG1-huCD3-FEALxCD30-T215-FEAR and IgG1-CD30-T215-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included as a negative control in all experiments. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 7] Thermal stability of antibodies with different inactivating mutations determined by differential scanning fluorescence (DSF). Conformational protein stability at elevated temperatures was evaluated in a double-deck manner by DSF. The melting curves for the following antibodies are shown: (A) IgG1-CD30-MDX060-FEAR at pH 7.4, (B) IgG1-CD30-MDX060-FERR at pH 7.4, (C) IgG1-huCD3-FEAL at pH 7.4, and (D) BsG1-huCD3-FEALxCD30-MDX060-FERR at pH 7.4. [Figure 8] Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HL cell lines. Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HL cell lines L-540(A), KM-H2(B), and L-1236(C) was evaluated by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR, as well as monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 9] Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to ALCL cell lines. Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to ALCL cell lines SUP-M2(A), DL-40(B), KARPAS-299(C), L-82(D), and SR-786(E) was evaluated by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR, as well as monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 10]Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to NHL cell lines. The binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to (A)SUP-T1(TLL), (B)JVM-2(MCL), (C)HH(CTCL), and (D)NCEB-1(MCL) cell lines was evaluated by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR, along with monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. The data shown are the mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 11] Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to full-length human and rhesus monkey CD30 transfected into HEK293 cells. Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HEK293 cells transiently transfected with full-length human CD30 (A) or rhesus monkey CD30 (B) was evaluated by flow cytometry. bsG1-huCD3-FEALxb12-FERR was included as a negative control. The data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 12]Simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to T cells and tumor cells. Simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to fluorescently labeled tumor cells and naive T cells was studied by flow cytometry. (A) Double-positive events are shown as the percentage of overall viable cells in the presence of 6 × 10⁻⁵ to 10 μg / mL of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody bsG1-huCD3-FEALxb12-FEAR, bsG1-b12-FEALxCD30-MDX060-FERR, or IgG1-b12-FEAL. The percentage of double-positive events in samples incubated without antibody is shown by the dotted line. (B) An example of a gating strategy for double-positive cells in a sample incubated with 0.12 μg / mL of bsG1-huCD3-FEALxCD30-MDX060-FERR. [Figure 13-1]In vitro induction of T cell-mediated cytotoxicity and T cell activation by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in in vitro cytotoxicity assays using the CD30-positive tumor cell line Karpas-299 as the target cell and T cells purified from the buffy coat of healthy human donors as effector cells. In these assays, CD25 expression was evaluated in CD4+ and CD8+ cells as a measure of T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30-T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR, and bsIgG1-huCD3-FEALxCD30-T215-FEAR. (A) IC50 values ​​for cytotoxicity of Karpas-299 cells by CD3xCD30 antibodies. (B) Percentage of maximum cytotoxicity in Karpas-299 cells by the test antibody. (C-F) EC50 values ​​for induction of CD25 (C-D) or PD-1 (E-F) expression in CD4+ (C, E) or CD8+ (D, F) T cells by CD3xCD30 antibody. Data were obtained from two independent experiments using T cells from six different donors. Statistical values ​​represent the results of Wilcoxon's paired signed-rank test between the indicated clones and bsG1-huCD3-FEALxCD30-MDX060-FERR. NS: No significant difference, *: p<0.05. [Figure 13-2]In vitro induction of T cell-mediated cytotoxicity and T cell activation by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in in vitro cytotoxicity assays using the CD30-positive tumor cell line Karpas-299 as the target cell and T cells purified from the buffy coat of healthy human donors as effector cells. In these assays, CD25 expression was evaluated in CD4+ and CD8+ cells as a measure of T cell activation. The following antibodies were tested: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsIgG1-huCD3-FEALxCD30-T105-FEAR, bsIgG1-huCD3-FEALxCD30-T405-FEAR, bsIgG1-huCD3-FEALxCD30-T408-FEAR, and bsIgG1-huCD3-FEALxCD30-T215-FEAR. (A) IC50 values ​​for cytotoxicity of Karpas-299 cells by CD3xCD30 antibodies. (B) Percentage of maximum cytotoxicity in Karpas-299 cells by the test antibody. (C-F) EC50 values ​​for induction of CD25 (C-D) or PD-1 (E-F) expression in CD4+ (C, E) or CD8+ (D, F) T cells by CD3xCD30 antibody. Data were obtained from two independent experiments using T cells from six different donors. Statistical values ​​represent the results of Wilcoxon's paired signed-rank test between the indicated clones and bsG1-huCD3-FEALxCD30-MDX060-FERR. NS: No significant difference, *: p<0.05. [Figure 14]In vitro T cell-mediated cytotoxicity of cell lines by bsG1-huCD3-FEALxCD30-MDX060-FERR. Dose-dependent T cell-mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060-FERR was tested in vitro using L-428(A), KM-H2(B), SUP-M2(C), or KI-JK(D) tumor cell lines as target cells and purified T cells as effector cells. IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, and IgG1-b12-FEAL were included as controls. The data shown represent the percentage of viable cells, and the data in each graph were obtained for one representative experiment. [Figure 15] This figure shows in vitro T cell proliferation using bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell proliferation was evaluated using a T cell-mediated cytotoxicity assay with L-428 (A,B) or KI-JK (C,D) as target cells. Diluted CD4+ (A,C) or CD8+ (B,D) T cells stained with Celltrace Violet were gated, and the proliferation index, as a measure of T cell proliferation, was calculated using a proliferation modeling tool from FlowJo. [Figure 16] In vitro expression of the T cell activation marker CD69 using bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was evaluated using a T cell-mediated cytotoxicity assay with L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker CD69 was evaluated in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 17]In vitro expression of the T cell activation marker CD25 using bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was evaluated using a T cell-mediated cytotoxicity assay with L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker CD25 was evaluated in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 18] In vitro expression of the T cell activation marker PD-1 using bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was evaluated using a T cell-mediated cytotoxicity assay with L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker PD-1 was evaluated in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 19-1] In vitro cytokine and granzyme B production induced by bsG1-huCD3-FEALxCD30-MDX060-FERR. The concentrations of 14 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, PDL-1, TNFα) and granzyme B were evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as the target cell. The concentrations of cytokines and granzyme B are shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL. [Figure 19-2]In vitro cytokine and granzyme B production induced by bsG1-huCD3-FEALxCD30-MDX060-FERR. The concentrations of 14 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, PDL-1, TNFα) and granzyme B were evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as the target cell. The concentrations of cytokines and granzyme B are shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL. [Figure 19-3] In vitro cytokine and granzyme B production induced by bsG1-huCD3-FEALxCD30-MDX060-FERR. The concentrations of 14 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, PDL-1, TNFα) and granzyme B were evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as the target cell. The concentrations of cytokines and granzyme B are shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody IgG1-b12-FEAL. [Figure 20]In vitro T cell-mediated cytotoxicity and T cell proliferation with bsG1-huCD3-FEALxCD30-MDX060-FERR at various effector-to-target ratios. (A) Dose-dependent T cell-mediated cytotoxicity with bsG1-huCD3-FEALxCD30-MDX060-FERR was tested in vitro using L-428 tumor cells as target cells and purified T cells as effector cells at E:T ratios of 1:1, 2:1, 4:1, or 8:1. bsG1-huCD3-FEALxb12-FERR was included as a control antibody. The data shown represent the percentage of viable cells obtained from one representative experiment. (B, C) The percentage of CD4+(B) or CD8+(C) T cells using diluted Celltrace Violet staining is shown as a measure of proliferating T cells. [Figure 21] Dynamics of T cell-mediated cytotoxicity and T cell proliferation in vitro with bsG1-huCD3-FEALxCD30-MDX060-FERR. (A) The dynamics of T cell-mediated cytotoxicity with bsG1-huCD3-FEALxCD30-MDX060-FERR were tested in vitro with L-428 tumor cells as target cells and purified T cells as effector cells in an E:T ratio of 4:1. Cytotoxicity was evaluated at 24, 48, and 72 hours. bsG1-huCD3-FEALxb12-FERR was included as a control antibody. The data shown are the percentage of viable cells obtained from one representative experiment. (B, C) Diluted CD4+(B) or CD8+(C) T cells with Celltrace Violet staining were gated, and the proliferation index, as a measure of T cell proliferation in cytotoxicity assays, was calculated using the FlowJo proliferation modeling tool. [Figure 22]Correlation between in vitro T cell-mediated cytotoxicity and CD30 expression levels using bsG1-huCD3-FEALxCD30-MDX060-FERR. The correlation between bsG1-huCD3-FEALxCD30-MDX060-FERR and CD30 expression levels for maximum T cell-mediated cytotoxicity (A) or IC50 concentration (B) was evaluated in eight tumor cell lines. The statistical significance of the degree of correlation between T cell-mediated cytotoxicity (maximum killing or IC50) and CD30 expression was assessed using Spearman's rank correlation test (GraphPad Prism software). [Figure 23] Fructolisides of activated T cells using bsG1-huCD3-FEALxCD30-MDX060-FERR. Isolated healthy donor T cells were stimulated for 4 days with 1 μg / mL anti-CD3 (OKT-3), 1 μg / mL anti-CD28, and 0.025 μg / mL IL-15. Once activation (CD25 upregulation) was confirmed, the T cells were incubated for 48 hours with gradually increasing concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR, bsIgG1-b12-FEALxCD30-MDX-060-FERR, bsIgG1-huCD3-FEALxb12-FERR, or IgG1-b12-FEAL. The BsG1-huCD3-FEALxCD30-MDX060-FERR-inducible T cell fructides of activated CD30+ T cells were measured as the ratio of viable T cells under each condition to the number of viable T cells under the untreated condition. (A-B) The proportion of CD25+ (A) or CD30+ (B) cells among CD4+ or CD8+ T cells 72 and 96 hours after stimulation with anti-CD3, anti-CD28, and IL-15 was determined by flow cytometry. (C) T cell fructides are shown as T cell survival rates compared to the untreated condition. Data for one representative T cell donor are shown. [Figure 24]The interference of soluble CD30 in the supernatant of CD30+ cell cultures with the antitumor activity of BsG1-huCD3-FEALxCD30-MDX060-FERR. (A) Shows soluble CD30 (sCD30) concentrations in the supernatant of different hematological malignancy cell lines as measured by ELISA. (B) In different hematological malignancy cell lines, the correlation between sCD30 concentration and the number of CD30 molecules on the cell surface, determined using quantitative flow cytometry (human IgG calibrator kit-Biocytex), was evaluated by Spearman rank correlation test (GraphPad Prism software). (C) BsG1-huCD3-FEALxCD30-MDX060-FERR was tested in an in vitro cytotoxicity assay using the CD30-positive ALCL tumor cell line DEL as the target cell and healthy donor isolated T cells as the effector cell. The following control antibodies were included: BsG1-b12-FEALxCD30-MDX060-FERR, IgG1-CD30-MDX060-FERR, BsG1-huCD3-FEALxb12-FERR, IgG1-huCD30-FEAL, and IgG1-b12-FEAL. The data shown represent the percentage of viable cells obtained from one representative experiment. [Figure 25-1] Ex vivo induction of T cell-mediated cytotoxicity and T cell proliferation by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 tumor cells. (A) bsG1-huCD3-FEALxCD30-MDX060-FERR 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) from patients with Hodgkin lymphoma (HL), acute myeloid leukemia (AML), and peripheral T-cell lymphoma (PTCL) were used as T cell sources to evaluate CD3-dependent tumor cell killing. IgG1-b12-FEAL was included as a control. The data shown represent the percentage of viable target cells. (B~D) T cell activation was evaluated by the upregulation of CD69 (B), CD25 (C), and PD-1 (D) markers on CD4+ / CD8+ T cells within the PBMC subset, and is shown as the percentage of positive cells. [Figure 25-2] Ex vivo induction of T cell-mediated cytotoxicity and T cell proliferation by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 tumor cells. (A) bsG1-huCD3-FEALxCD30-MDX060-FERR 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) from patients with Hodgkin lymphoma (HL), acute myeloid leukemia (AML), and peripheral T-cell lymphoma (PTCL) were used as T cell sources to evaluate CD3-dependent tumor cell killing. IgG1-b12-FEAL was included as a control. The data shown represent the percentage of viable target cells. (B~D) T cell activation was evaluated by the upregulation of CD69 (B), CD25 (C), and PD-1 (D) markers on CD4+ / CD8+ T cells within the PBMC subset, and is shown as the percentage of positive cells. [Figure 26] Plasma concentrations of BsG1-huCD3-FEALxCD30-MDX060-FERR after intravenous injection into SCID mice. SCID mice were injected with a single IV dose of 10 μg (0.5 mg / kg) or 100 μg (5 mg / kg) of BsG1-huCD3-FEALxCD30-MDX060-FERR. (A) Total human IgG was determined by ELISA, and the mean human IgG1 concentration was plotted over time. (B) Mean clearance rates were plotted for 0.5 or 5 mg / kg dose levels. The dotted line shows the estimated clearance rate based on the standard distribution volume of unbound, regularly distributed human IgG1 in mice. [Figure 27]C1q binding to membrane-bound bsG1-huCD3-FEALxCD30-MDX060-FERR. C1q binding to bsG1-huCD3-FEALxCD30-MDX060-FERR opsonized, activated human CD8+ T cells, or CD30+ NCEB-1 cells was evaluated by flow cytometry using FITC-labeled rabbit anti-C1q antibody. (A) Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-huCD3-FEAL, or positive control antibody IgG1-CD52-E430G to human CD8+ T cells stimulated with anti-CD3 / CD28 beads in the presence of normal human serum as a source of C1q. Data shown are geometric mean fluorescence intensity (gMFI) ± SD from replication wells of one representative experiment. (B) Binding of bsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-CD30-MDX060-FERR, or positive control antibody IgG1-7D8-E430G to CD30+NCEB-1 cells in the presence of normal human serum as a source of C1q. The data shown are gMFI from one representative experiment. [Modes for carrying out the invention]

[0020] definition As used herein, the term “antibody” is intended to mean an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, having the ability to specifically bind to an antigen under typical physiological and / or tumor-specific conditions and having a half-life of a significant period, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined period (such as a sufficient time to induce, promote, enhance and / or modulate the physiological response associated with antibody binding to the antigen and / or a sufficient time for the antibody to be internalized). Antibodies include a binding region (or binding domain as may be used herein (both are synonymous)) that can interact with the antigen, a binding region including variable regions of both the heavy and light chains of the immunoglobulin molecule, etc. The antibody may include a constant region of the antibody (Ab) that can mediate the binding of immunoglobulins 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, which is the first component in the classical pathway of complement activation.

[0021] In relation to the present invention, the term “antibody” includes monoclonal antibodies (mAbs), antibody-like polypeptides, chimeric antibodies, human antibodies, humanized antibodies, and “antibody fragments” or “fragments thereof” that possess the ability to specifically bind to an antigen (antigen-binding fragment) provided by any known technique such as enzymatic cleavage, peptide synthesis, and recombinant DNA technology. The term “antibody” also includes bispecific, tripspecific, or multispecific antibodies and / or antibodies having further modifications, such as antibody-drug conjugates and / or antibodies having modifications to the IgG Fc domain. Antibodies as defined in accordance with the present invention may have any isotype or may not have an isotype (e.g., scFv antibodies), unless otherwise disclosed herein.

[0022] It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments encompassed by the term "antibody" include: (i) a monovalent fragment consisting of Fab' or Fab fragment, a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL), and a heavy chain constant region domain 1 (CH1) domain, or a monovalent antibody described in International Publication No. 2007 / 059782; (ii) a bivalent fragment containing an F(ab')2 fragment, two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) an Fd fragment essentially consisting of a VH domain and a CH1 domain; (iv) an Fv fragment essentially consisting of a single arm of an antibody's VL domain and VH domain; and (v) a dAb fragment essentially consisting of a VH domain, also called a domain antibody (Holt et al; Trends Biotechnol-.2003 Nov;21(11):484-90) (Ward et al., Nature (341, 544546 (1989)); (vi) derived from camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers that allow the VL and VH regions to pair up to form a single protein chain that forms a monovalent molecule using recombination (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 specified or clearly indicated by the context. While such fragments generally fall within the scope of the meaning of antibody, they are unique features of the present invention, both collectively and independently, exhibiting different biological properties and uses. These and other useful antibody fragments in the context of the present invention will be discussed further herein.

[0023] Antibodies can be produced and collected either as a final product or as an intermediate for generating bispecific antibodies, for example via controlled Fab arm exchange (cFEA), from different in vitro or ex vivo expression or production systems, such as from recombinant modified host cells, from hybridomas, or from systems using cell extracts that assist in in vitro transcription and / or translation of nucleic acid sequences encoding antibodies.

[0024] As used herein, the terms “immunoglobulin heavy chain” or “immunoglobulin heavy chain” are intended to refer to one of the immunoglobulin heavy chains. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which define the immunoglobulin isotype. The heavy chain constant region of IgG typically consists of three domains: CH1, CH2, and CH3. As used herein, the term “immunoglobulin” is intended to refer to a class of structurally related glycoproteins typically consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains, and one pair of heavy (H) chains, all four of which are potentially interconnected by disulfide bonds. The structures of immunoglobulins are well characterized (e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven)). See Press, NY (1989). Within the structure of immunoglobulins, the two heavy chains are interconnected via disulfide bonds in a so-called “hinge region.” Similar to the heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of a single domain CL. Furthermore, the VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs) (or hypervariable regions that can be hypervariable in the arrangement and / or morphology of structurally defined loops). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0025] As used herein, the terms “halving,” “Fab arm,” and “arm” refer to a single heavy-light chain pair. Where a bispecific antibody is described as comprising a hapmon antibody “derived” from a first antibody and a hapmon antibody “derived” from a second antibody, the term “derived” indicates that the bispecific antibody was produced by recombining the hapmones from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, “recombining” is not intended to be limited to any particular recombination method and therefore includes all methods for producing bispecific antibodies described herein, including, for example, recombination by hapmon exchange, as well as recombination at the nucleic acid level and / or by co-expression of two hapmones in the same cell.

[0026] When the terms "first" and "second" are used herein in the context of an antibody or its domain or region, they are intended solely for the purpose of facilitating reference and are not intended to indicate a specific relative position or anything of the sort.

[0027] As used herein, the terms “antigen-binding region” or “binding region” refer to a region of an antibody that can bind to an antigen. The antigen may be any molecule, such as a polypeptide, protein, polysaccharide, or a combination thereof. The antigen may be presented, for example, on a cell, bacterium, or virion. The terms “antigen” and “target” may be used interchangeably in the context of this invention, provided that the context does not conflict with the terms. The terms “antigen-binding region” and “antigen-binding site” may be used interchangeably in the context of this invention, provided that the context does not conflict with the terms.

[0028] When used herein, "K D The term (M) refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and k d to k a It is obtained by dividing by K. D This can also be called "binding affinity."

[0029] As used herein, "k d " (sec -1 ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also called the k off value or the off-rate.

[0030] As used herein, "k a " (M -1 ×sec[[ID=1�]] -1 ) refers to the association rate constant of a particular antibody-antigen interaction. This value is also called the k on value or the on-rate.

[0031] As used herein, the term "binding" typically refers to binding of an antibody to a given antigen or target with a binding affinity corresponding to a K -6 of 1E -7 M or less, such as 5E -7 M or less, 1E -8 M or less, 5E -8 M or less, etc., 1E -9 M or less, etc., 5E -9 M or less, etc., 1E -10 M or less, etc., or 1E -11 M or less, etc., determined by biolayer interferometry using the antibody as a ligand and the antigen as an analyte, and at least ten-fold lower, at least 100-fold lower, such as at least 1,000-fold lower, at least 10,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to non-specific antigens other than the given antigen or closely related antigens (e.g., BSA, casein), and binds to the given antigen with an affinity corresponding to a K D . D [[ID=communicated]]

[0032] As used herein, the term "freeze-thaw cycle" refers to the process of freezing a pharmaceutical composition to a lower temperature, such as -75°C, and subsequently thawing it at room temperature.

[0033] As used herein, the term "k D " refers to the diffusion interaction parameter.

[0034] When used in this specification, "B 22 The term "second virial coefficient" refers to the second virial coefficient.

[0035] As used herein, the term “stable” refers to the ability of a pharmaceutical product to retain its physical and / or chemical stability and / or biological activity during storage. Therefore, if a pharmaceutical composition is stable, i.e., a stable pharmaceutical composition, it is suitable for pharmaceutical use even after a given period of storage. The quality of an antibody changes over time when exposed to factors such as temperature, agitation, and freeze-thaw cycles. A stable pharmaceutical composition retains or is negligibly affected by its physical, chemical, and biological properties throughout its shelf life. As an example, the rate of change in physical, chemical, and / or biological properties from manufacture to use is limited. In one embodiment, the % monomer, as measured by HP-SEC, does not decrease by more than 5%, such as 4%, 3%, 2%, etc., when maintained at 5°C for at least 12 months. In a further embodiment, the intact IgG% does not decrease by more than 10%, 5%, etc., when maintained at 5°C for at least 12 months. In another embodiment, the acidic form measured by icIEF does not increase by more than 20%, more than 15%, etc., or more than 10%, for example, when maintained at 5°C for at least 12 months.

[0036] As used herein, the term “buffer” refers to a pharmaceutically acceptable buffer. The term “buffer” includes, but is not limited to, agents that maintain the pH value of a solution within an acceptable range, such as acetates, histidines, TRIS® (tris(hydroxymethyl)aminomethane), citrates, succinates, glycolates, etc. Generally, as used herein, “buffer” has a pKa and buffering capacity suitable for pH ranges such as about 4 to about 8, about 5 to about 6.5, and about 5.5 to 6.

[0037] As used herein, the term “nonionic excipient” refers to a pharmaceutically acceptable nonionic excipient. For example, a nonionic excipient may be a sugar or a sugar alcohol. Sugars and polyols may also be called monosaccharides, disaccharides, and polysaccharides. Examples include, but are not limited to, sucrose, glucose, dextrose, trehalose, mannitol, and sorbitol.

[0038] As used herein, “surfactant” refers to a compound typically used in pharmaceutical formulations to prevent drug adsorption and / or aggregation to a surface. Furthermore, surfactants reduce the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, exemplary surfactants can significantly reduce surface tension when present at very low concentrations (e.g., 5% w / w or less, 3% w / w or less, 1% w / w or less, etc.). Surfactants are amphiphilic, meaning that they are typically composed of both hydrophilic and hydrophobic or lipophilic groups, and can therefore form micelles or similar self-assembling structures in aqueous solutions. Known surfactants for pharmaceutical use include glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate, and tricaprylin (anionic surfactants); benzalkonium chloride, citrimide, cetylpyridinium chloride, and phospholipids (cationic surfactants); and alpha-tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sterarates, polyoxyl hydroxystearate, polyoxylglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan esters, sucrose palmitate, sucrose stearate, tricaprylin, and TPGS (nonionic and amphoteric surfactants).

[0039] The “diluents” of interest herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing diluents of pharmaceutical compositions. Preferably, such dilution of the compositions of the present invention dilutes only the antibody concentration and not the buffer and other potential components of the composition. Therefore, in preferred embodiments, the diluent contains, for example, the same concentration of buffer present in the pharmaceutical composition of the present invention. Further exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer which is preferably acetate buffer or histidine buffer, sterile saline, Ringer's solution, or dextrose solution. In one embodiment, the diluent comprises or essentially consists of acetate buffer and sorbitol. In another embodiment, the diluent comprises or essentially consists of histidine buffer and sucrose.

[0040] As used herein, the term "CD30" refers to the human differentiated cluster 30 protein, also known as TNFRSF8 (tumor necrosis factor receptor superfamily member 8). Because CD30 is found in various species, the term "CD30" may not be limited to human CD30 unless otherwise specified in the context. The sequence of human CD30 is shown in SEQ ID NO: 39.

[0041] As used herein, the term “CD3” refers to the human differentiated cluster 3 protein, which is part of the T cell coreceptor protein complex and consists of four distinct chains. Because CD3 is found in various species, the term “CD3” may not be limited to human CD3 unless otherwise inconsistent with the context. In mammals, the complex consists of a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No Q95LI8), and two CD3ε (epsilon) chains (human CD3ε: UniProtKB / Swiss-Prot No P07766, whose sequence is incorporated herein as Sequence ID No. 42; cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No Q95LI5; or rhesus monkey CD3ε UniProtKB / Swiss-Prot No. It includes G7NCB9) and CD3 zeta chains (human CD3 zeta UniProtKB / Swiss-Prot No. P20963, cynomolgus monkey CD3 zeta UniProtKB / Swiss-Prot No. Q09TK0). These chains associate with molecules known as T cell receptors (TCRs) and generate activation signals in T lymphocytes. Both TCRs and CD3 molecules contain TCR complexes.

[0042] The term "antibody-binding region" refers to the region of an antigen containing the epitope to which the antibody binds. The antibody-binding region can be determined by epitope binning using biolayer interferometry, by alanine scanning, or by a domain shuffle assay (which uses an antigen construct in which a region of the antigen is exchanged with a region of another species to determine whether the antibody still binds to the antigen). The amino acids within the antibody-binding region involved in the interaction with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and / or crystallography of the antibody bound to that antigen.

[0043] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes typically consist of surface groups of molecules, such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural and charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents or other agents that disrupt the three-dimensional structure of the protein or its polymer, rather than the latter. Epitopes may include amino acid residues directly involved in binding, other amino acid residues not directly involved in binding, and amino acid residues that are effectively blocked or coated by the antibody when the antibody binds to the antigen.

[0044] As used herein, terms such as “monoclonal antibody,” “monoclonal antibody composition,” and “mAb” refer to preparations of antibody molecules with a single molecular composition, typically exhibiting a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can typically be produced from identical cells, which are all clones of a unique parent cell, such as hybridomas or stable cell lines. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting a single binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced from hybridomas containing B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice having a genome containing human heavy chain and light chain transgenes fused to immortalized cells. Human monoclonal antibodies may be derived from human B cells or plasma cells. Monoclonal antibodies can also be produced from recombinantly modified host cells or from systems using cell extracts that assist in in vitro transcription and / or translation of the nucleic acid sequence encoding the antibody.

[0045] As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f), encoded by a heavy chain constant region gene. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) light chain or a lambda (λ) light chain.

[0046] As used herein, the term “full-length antibody” refers to an antibody (e.g., a parent antibody or a mutant antibody) comprising one pair of heavy chains and light chains, or two pairs of heavy chains and light chains, each pair comprising constant and variable domains of the heavy and light chains as typically found in the heavy-light chain pair of the wild-type antibody of its isotype. Thus, for example, a full-length IgG1 antibody contains the VH, CH1, CH2, CH3, hinge, VL, and CL domains. In a full-length mutant antibody, the constant and variable domains of the heavy and light chains may include amino acid substitutions that modify and / or improve the functional properties of the antibody, in particular, compared to a full-length parent antibody or wild-type antibody. A full-length antibody according to the present invention may be produced by a method comprising (i) cloning a CDR sequence into one or more suitable vectors comprising complete heavy chain and light chain sequences, and (ii) expressing the resulting suitable vectors with heavy chain and light chain sequences in a suitable expression system. Producing full-length antibodies starting from either a CDR sequence or a fully variable region sequence is within the knowledge of those skilled in the art. Therefore, those skilled in the art know how to produce full-length antibodies according to the present invention.

[0047] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting a non-human antibody complementarity-determining region (CDR) that together forms an antigen-binding site onto a homologous human acceptor framework region (FR) (see, in particular, International Publication 92 / 22653 and European Patent No. 0629240). To completely reconstitute the binding affinity and binding specificity of the parent antibody, it may be necessary to replace some of the human framework residues with some of the framework residues derived from the parent antibody (i.e., non-human antibody) (reverse mutation). Structural homology modeling can help identify amino acid residues within the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody comprises a non-human CDR sequence, a human framework region which may mainly contain one or more amino acid reverse mutations to the non-human amino acid sequence, and a fully human constant domain. Further amino acid modifications, not necessarily reverse mutations, may be applied to obtain humanized antibodies with desirable properties such as specific useful affinity and biochemical characteristics, including modifications to avoid deamide and / or modifications to improve manufacturing. Furthermore, the CDR and / or framework region may be modified to improve affinity for the antigen, for example, by affinity maturation procedures.

[0048] As used herein, the term “human antibody” refers to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies may include amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. The human monoclonal antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, e.g., the standard somatic hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While somatic hybridization procedures are preferred in principle, other techniques for producing monoclonal antibodies, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of human antibody genes, may be used. Human monoclonal antibodies can be produced using transgenic or transchromosomal mice that possess a part of the human immune system, rather than mouse mice, such as HCo12 mice (see, for example, International Publication No. 03 / 059282).

[0049] As used herein, the term “Fc region” refers to the region of an antibody’s two heavy chain polypeptides, extending from the N-terminus to the C-terminus, including at least a hinge region, a CH2 region, and a CH3 region. Fc polypeptides are typically glycosylated. The Fc region of an antibody can mediate the binding of immunoglobulins to various cells of the immune system (such as effector cells) and host tissues or factors, including components of the complement system. The Fc region also typically binds to FcRn and protein A.

[0050] As used herein, "amino acids corresponding to the amino acids at the position of..." etc., refer to the positional numbers of amino acids in the human IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Unless otherwise specified or unless inconsistent with the context, amino acids in constant region sequences are numbered herein according to the EU index numbering (as described in Kabat, EA et al., 1991, Sequences of proteins of immunological interest. 5th Edition - US 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 "corresponds" an amino acid or segment in another sequence is typically aligned with the other amino acid or segment using, for example, ALIGN, ClustalW, or a similar standard sequence alignment program, with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments within a sequence, thereby determining the corresponding positions in a sequence for amino acid positions according to the present invention, are considered to be well known in the art.

[0051] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. For example, the hinge region of a human IgG1 antibody corresponds to amino acids 216–230, following the Eu numbering described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91–3242, pp. 662, 680, 689 (1991). However, the hinge region may be any of the other subtypes described herein.

[0052] As used herein, the terms “CH1 region” or “CH1 domain” refer to the CH1 region of an immunoglobulin heavy chain. For example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118–215 by Eu numbering as shown in Kabat (ibid.). However, the CH1 region may also be any of the other subtypes described herein.

[0053] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. For example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231–340 by Eu numbering as shown in Kabat (ibid.). However, the CH2 region may also be any of the other subtypes described herein.

[0054] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. For example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341–447 by Eu numbering as shown in Kabat (ibid.). However, the CH3 region may also be any of the other subtypes described herein.

[0055] As used herein, the term “Fc-mediated effector function” is intended to refer to a function resulting from the binding of a polypeptide or antibody to its target or antigen on the cell membrane, and the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor (FcgR) binding, (vi) antibody-dependent, FcγR-mediated antigen crosslinking, (vii) antibody-dependent phagocytosis (ADCP), (viii) complement-dependent cytotoxicity (CDCC), (ix) complement-enhancing cytotoxicity, (x) antibody-mediated binding of opsonized antibodies to complement receptors, (xi) opsonization, and (xii) any combination of (i) to (xi).

[0056] As used herein, the terms “inactivity,” “inactive,” or “deactivated” refer to an Fc region that cannot bind to FcγR, or has minimal binding ability, does not induce Fc-mediated crosslinking of FcγR, does not induce FcγR-mediated effector functions such as ADCC and ADCP, does not induce FcγR-mediated crosslinking of the target antigen via two Fc regions of the individual antibody, and / or cannot bind to C1q to induce complement-mediated effector functions such as CDC and CDCC. The inactivity of an antibody’s Fc region can be tested using antibodies in monospecific or bispecific format.

[0057] In the context of this invention, the term "monovalent antibody" refers to an antibody molecule capable of interacting with an antigen having only one antigen-binding domain (e.g., one Fab arm). In the context of multispecific antibodies such as bispecific antibodies, "monovalent antibody binding" refers to the binding of a multispecific antibody to a single antigen having only one antigen-binding domain (e.g., one Fab arm).

[0058] In the context of this invention, the term "monospecific antibody" refers to an antibody that has binding specificity to only one antigen or one epitope. The antibody may be a monovalent antibody of monospecificity (i.e., carrying only one antigen-binding region), a bivalent antibody of monospecificity (e.g., an antibody having two identical antigen-binding regions), or a multivalent antibody of monospecificity (e.g., an antibody having three or more identical antigen-binding regions).

[0059] The term "multispecific antibody" refers to an antibody having two or more antigen-binding domains that bind to two or more different epitopes. The term "bispecific antibody" refers to an antibody having 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 having non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity to two different epitopes, and a multispecific antibody has specificity to 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, different cells, cell type, or structure (such as extracellular matrix or vesicles and soluble proteins). Therefore, multispecific and bispecific antibodies can crosslink multiple antigens, for example, two different cells.

[0060] The term "bivalent antibody" refers to an antibody having two antigen-binding regions, which may be identical and bind to the same epitope, or they may not be identical and bind to different epitopes, which may be located on the same or different (one or more) antigens. Therefore, a bivalent antibody can be a monospecific antibody or a bispecific antibody.

[0061] 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 this invention, amino acids can be classified based on their structure and chemical properties. Therefore, the classes of amino acids may be reflected in one or both of the following tables. [Table 1] [Table 2]

[0062] Substitution of one amino acid with another can be classified as either a conservative or non-conservative substitution. In the context of this invention, a “conservative substitution” is the substitution of one amino acid with another amino acid having similar structural and / or chemical properties, and is the substitution of one such amino acid residue with 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 with isoleucine, since both are aliphatic branched hydrophobic. Similarly, an example of a conservative substitution is the substitution of aspartic acid with glutamic acid, since both are small negatively charged residues.

[0063] In the context of the present invention, substitutions in antibodies are expressed as the original amino acid - position number - substituted amino acid.

[0064] Referring to the well-known nomenclature for amino acids, a three-letter code or a one-letter code containing the code "Xaa" or "X" is used to represent any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any one 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.

[0065] Therefore, the notation "K409R" or "Lys409Arg" means that the antibody includes a substitution of lysine with arginine at amino acid position 409. A substitution of any other amino acid at a given position is referred to by the original amino acid-position, for example, "K409". In the case of a modification in which the original (one or more) amino acids and / or (one or more) substituted amino acids may contain one or more amino acids but not all (one or more) amino acids, the one or more amino acids may be separated by "," or " / ". For example, a substitution of lysine at position 409 with arginine, alanine, or phenylalanine is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409 to R,A or F". Such designations may be used interchangeably in the context of the present invention but have the same meaning and purpose.

[0066] Furthermore, the term “substitution” encompasses substitutions to any one or any other 19 natural amino acids, or to other amino acids such as non-natural amino acids. For example, substitutions of amino acid K at position 409 include each of 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 referred to as K409A, K409C, etc., or K409A, C, etc., or K409A / C / , etc. The same applies by analogy to each position referred to herein, and any one of such substitutions is specifically included herein.

[0067] As used herein, the term “host cell” is intended to refer to a cell into which a nucleic acid, such as an expression vector, has been introduced. It should be understood that such a term may include not only a specific target cell but also the offspring of such a cell. Such offspring may not be identical to the parent cell in fact, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein. Examples of recombinant host cells (i.e., host cells used for the production of recombinant proteins) include, for example, transfectomas (CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells), as well as prokaryotic cells (E. coli, etc.), and other eukaryotic hosts (plant cells and fungi, etc.).

[0068] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells expressing antibodies or target antigens, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells.

[0069] For the purposes of this invention, sequence identity between two amino acid sequences is determined over the length of the reference sequence using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented using the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later of the Needle program. The parameters used are a gap-open penalty of 10, a gap-expand penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows:

[0070] (Number of identical residues × 100) / (Alignment length - Total number of gaps in the alignment).

[0071] Retention of similar residues may also, or alternatively, be measured by a similarity score determined by using the BLAST program (e.g., BLAST 2.2.8, available from NCBI, with standard settings BLOSUM62, Open Gap=11, Extended Gap=1). Suitable variants typically exhibit similarity to the parent or reference sequence of at least about 45%, 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%). [Table 3] TIFF2026513829000004.tif188158 TIFF2026513829000005.tif222160 TIFF2026513829000006.tif180159 TIFF2026513829000007.tif221159 TIFF2026513829000008.tif176160 TIFF2026513829000009.tif221159 TIFF2026513829000010.tif226159 TIFF2026513829000011.tif136160 TIFF2026513829000012.tif206159 TIFF2026513829000013.tif193159 TIFF2026513829000014.tif204158 TIFF2026513829000015.tif203159 TIFF2026513829000016.tif228158 TIFF2026513829000017.tif115159

[0072] Further aspects and embodiments of the present invention Pharmaceutical composition In one aspect, the present invention is 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, b) Includes a buffering agent, This invention relates to a pharmaceutical composition having a pH of approximately 4.0 to approximately 8.0.

[0073] In one embodiment, the buffer is selected from the group consisting of acetates, histidine, TRIS® (tris(hydroxymethyl)aminomethane), citrates, succinates, glycolates, glutamates, and mixtures thereof. In further embodiments, the buffer is histidine, acetates, and / or mixtures thereof. In preferred embodiments, the buffer is an acetate. The acetate buffer may be selected from the group consisting of sodium acetate, potassium acetate, sodium acetate, their hydrates, and mixtures thereof. In preferred embodiments, the buffer is sodium acetate, such as sodium acetate trihydrate.

[0074] The concentration of the buffer is set so that the pH is maintained. In one embodiment, the buffer is present at concentrations such as approximately 5 to approximately 40 mM, approximately 10 to approximately 30 mM, approximately 15 to approximately 25 mM, approximately 18 to approximately 22 mM, preferably approximately 20 mM, approximately 17 mM, etc.

[0075] The pH of the pharmaceutical composition is approximately 4.0 to approximately 8.0. In one embodiment, the pH of the composition is approximately 4.5 to approximately 6.5, approximately 5.0 to approximately 6.0, approximately 5.2 to approximately 5.7, approximately 5.4 to approximately 5.6, preferably approximately 5.5. In one embodiment, the pH of the composition is approximately 5.5. In a further embodiment, the pH of the composition is approximately 6.0.

[0076] The pH of a pharmaceutical composition can be adjusted using a pH-adjusting component to obtain the desired pH of the composition. Therefore, in one embodiment, the pharmaceutical composition further comprises a pH-adjusting component. In a further embodiment, the pH-adjusting component is an acid. In a further embodiment, the acid is HCl and / or acetic acid. In a preferred embodiment, the acid is glacial acetic acid.

[0077] The concentration of the pH-adjusting component depends on several factors, including the pH-adjusting component itself. However, in one embodiment, the pH-adjusting component is present at concentrations such as about 0.1 to 30 mM, about 0.3 to 25 mM, for example, about 0.5 to 20 mM, about 0.6 to 15 mM, for example, about 1 to 10 mM, about 2 to 5 mM, for example, about 3 mM. In a preferred embodiment, the pharmaceutical composition contains 3 mM glacial acetic acid.

[0078] In one embodiment, the buffer is an acetate with a concentration of about 5-40 mM, about 10-30 mM, or about 20 mM, and the pH of the composition is about 5-6 or about 5.5. In a preferred embodiment, the buffer is an acetate with a concentration of about 10-30 mM, and the pH of the composition is about 5.5. In a more preferred embodiment, the buffer is an acetate with a concentration of about 20 mM, and the pH of the composition is about 5.5. In a further preferred embodiment, the composition contains about 17 mM sodium acetate and about 3 mM glacial acetic acid, and the pH of the composition is about 5.5.

[0079] The pharmaceutical composition may advantageously contain further components in addition to the buffer and the multispecific antibody. Therefore, in further embodiments, the pharmaceutical composition further includes a nonionic excipient.

[0080] Nonionic excipients may be known to those skilled in the art. However, in preferred embodiments, the nonionic excipient is a sugar or sugar alcohol. In further embodiments, the nonionic excipient is selected from sucrose, trehalose, mannitol, xylitol, sorbitol, and mixtures thereof. In preferred embodiments, the nonionic excipient is selected from sorbitol, sucrose, trehalose, or mixtures thereof. In more preferred embodiments, the nonionic excipient is sorbitol or trehalose. In most preferred embodiments, the nonionic excipient is sorbitol.

[0081] Nonionic excipients are added at concentrations acceptable for pharmaceutically acceptable use. In one embodiment, the nonionic excipients are present at concentrations such as about 5 to about 450 mM, about 50 to about 400 mM, for example, about 100 to about 350 mM, about 125 to about 300 mM, for example, about 150 to about 275 mM, preferably about 250 mM.

[0082] In further embodiments, the nonionic excipient is present in concentrations of approximately 350 mM, or approximately 340 mM, or approximately 330 mM, or approximately 320 mM, or approximately 310 mM, or approximately 300 mM, or approximately 290 mM, or approximately 280 mM, or approximately 270 mM, or approximately 260 mM, or approximately 250 mM, or approximately 240 mM, or approximately 230 mM, or approximately 220 mM, or approximately 210 mM, or approximately 200 mM, or approximately 1 It is present at concentrations of 90 mM, or approximately 180 mM, or approximately 170 mM, or approximately 160 mM, or approximately 150 mM, or approximately 140 mM, or approximately 130 mM, or approximately 120 mM, or approximately 110 mM, or approximately 100 mM, or approximately 90 mM, or approximately 80 mM, or approximately 70 mM, or approximately 60 mM, or approximately 50 mM, or approximately 40 mM, or approximately 30 mM, or approximately 20 mM, or approximately 10 mM.

[0083] In one embodiment, the pharmaceutical composition contains sorbitol in concentrations of approximately 100-350 mM, sorbitol in concentrations of approximately 200-300 mM, and sorbitol in concentrations of approximately 250 mM. Therefore, in further embodiments, the pharmaceutical composition contains an acetate such as sodium acetate and sorbitol, and has a pH of approximately 5.5.

[0084] In further embodiments, the ratio of the concentrations of acetate buffer to sorbitol in the pharmaceutical composition is 2:10 to 2:40, 2:15 to 2:35, 2:20 to 2:30, for example, 2:25.

[0085] The pharmaceutical composition may optionally contain further components. In one embodiment, the pharmaceutical composition further comprises a surfactant.

[0086] The surfactant may be a set 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; and alpha-tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sterarates, polyoxyl hydroxystearate, polyoxylglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan esters, close palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof. In a preferred embodiment, the surfactant is polysorbate. In a more preferred embodiment, the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80). In the most preferred embodiment, the surfactant is polysorbate 80 (PS80).

[0087] The surfactant is added at a concentration acceptable for pharmaceutically acceptable use. In one embodiment, the surfactant is present at concentrations of about 0.01 to about 0.1% w / v, about 0.01 to about 0.09% w / v, about 0.01 to about 0.06% w / v, about 0.01 to about 0.05% w / v, 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.

[0088] The overall properties of a pharmaceutical composition, such as its weight osmolality and viscosity, are influenced by the components and the concentrations of those components within the pharmaceutical composition. In one embodiment, the weight osmolality of the pharmaceutical composition is less than approximately 600 mOsm / kg, less than approximately 550 mOsm / kg, less than approximately 500 mOsm / kg, less than approximately 450 mOsm / kg, less than approximately 400 mOsm / kg, less than approximately 350 mOsm / kg, etc.

[0089] In further embodiments, the weight osmolality of the pharmaceutical composition is in the range of approximately 100 to 500 mOsm / kg, approximately 200 to 400 mOsm / kg, for example, approximately 250 to 350 mOsm / kg.

[0090] In further embodiments, the weight osmolality of the pharmaceutical composition is approximately 250 mOsm / kg, or approximately 260 mOsm / kg, or approximately 270 mOsm / kg, or approximately 280 mOsm / kg, or approximately 290 mOsm / kg, or approximately 300 mOsm / kg, or approximately 310 mOsm / kg, or approximately 320 mOsm / kg, or approximately 330 mOsm / kg, or approximately 340 mOsm / kg, or approximately 350 mOsm / kg.

[0091] In further embodiments, the viscosity of the pharmaceutical composition is less than approximately 30 cP, less than approximately 25 cP, less than approximately 20 cP, less than approximately 18 cP, less than approximately 16 cP, less than approximately 14 cP, less than approximately 12 cP, less than approximately 10 cP, less than approximately 9 cP, less than approximately 8 cP, less than approximately 7 cP, less than approximately 6 cP, less than approximately 5 cP, less than approximately 4 cP, less than approximately 3 cP, less than approximately 2 cP, etc.

[0092] In further embodiments, the viscosity of the pharmaceutical composition is in the range of about 1 to 30 cP, the same range of about 1 to 25 cP, for example, the range of about 1 to 20 cP, the range of about 1 to 18 cP, for example, the range of about 1 to 15 cP, the range of about 1 to 12 cP, for example, the range of about 1 to 10 cP, the range of about 1 to 9 cP, for example, the range of about 1 to 8 cP, the range of about 1 to 7 cP, for example, the range of about 1 to 6 cP, the range of about 1 to 5 cP, for example, the range of about 1 to 4 cP, the range of about 1 to 3 cP, etc.

[0093] In further embodiments, the viscosity of the pharmaceutical composition is approximately 2 cP, or approximately 2.5 cP, or approximately 3 cP, or approximately 3.5 cP, or approximately 4 cP, or approximately 4.5 cP, or approximately 5 cP, or approximately 5.5 cP, or approximately 6 cP, or approximately 6.5 cP, or approximately 7 cP, or approximately 7.5 cP, or approximately 8 cP, or approximately 8.5 cP, or approximately 9 cP, or approximately 9.5 cP, or approximately 10 cP, or approximately 10.5 cP, or approximately The cholecytosis is 11 cP, or approximately 11.5 cP, or approximately 12 cP, or approximately 12.5 cP, or approximately 13 cP, or approximately 13.5 cP, or approximately 14 cP, or approximately 14.5 cP, or approximately 15 cP, or approximately 15.5 cP, or approximately 16 cP, or approximately 16.5 cP, or approximately 17 cP, or approximately 17.5 cP, or approximately 18 cP, or approximately 18.5 cP, or approximately 19 cP, or approximately 19.5 cP, or approximately 20 cP.

[0094] The pharmaceutical composition contains 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, approximately 1.0 to approximately 220 mg / ml, approximately 3 to approximately 190 mg / ml, approximately 5 to approximately 160 mg / ml, approximately 10 to approximately 130 mg / ml, or approximately 20 to approximately 120 mg / ml, approximately 30 to approximately 110 mg / ml, approximately 40 to approximately 100 mg / ml, approximately 50 to approximately 90 mg / ml, approximately 60 to approximately 80 mg / ml, or approximately 65 to approximately 75 mg / ml, for example, approximately 70 mg / ml.

[0095] In further embodiments, the antibody concentration is approximately 50 to 250 mg / ml, 60 to 240 mg / ml, 70 to 220 mg / ml, 80 to 210 mg / ml, 100 to 200 mg / ml, 120 to 190 mg / ml, 130 to 180 mg / ml, 135 to 165 mg / ml, or 150 to 190 mg / ml.

[0096] In further embodiments, the antibody concentration may be approximately 10 mg / ml, or approximately 12 mg / ml, or approximately 14 mg / ml, or approximately 16 mg / ml, or approximately 18 mg / ml, or approximately 20 mg / ml, or approximately 22 mg / ml, or approximately 24 mg / ml, or approximately 26 mg / ml, or approximately 28 mg / ml, or approximately 30 mg / ml, or approximately 32 mg / ml, or approximately 34 mg / ml, or approximately 36 mg / ml, or approximately 38 mg / ml, or approximately 40 mg / ml, or approximately 42 mg / ml, or approximately 44 mg / ml, or approximately 46 mg / ml, or approximately 48 mg / ml, or approximately 50 mg / ml. or approximately 52 mg / ml, or approximately 54 mg / ml, or approximately 56 mg / ml, or approximately 58 mg / ml, or approximately 60 mg / ml, or approximately 62 mg / ml, or approximately 64 mg / ml, or approximately 66 mg / ml, or approximately 68 mg / ml, or approximately 70 mg / ml, or approximately 72 mg / ml, or approximately 74 mg / ml, or approximately 76 mg / ml, or approximately 78 mg / ml, or approximately 80 mg / ml, or approximately 82 mg / ml, or approximately 84 mg / ml, or approximately 86 mg / ml, or approximately 88 mg / ml, or approximately 90 mg / ml, or approximately 92 mg / ml, or approximately 94 mg / ml, or approximately 96 mg / ml g / ml, or approximately 98 mg / ml, or approximately 100 mg / ml, or approximately 102 mg / ml, or approximately 104 mg / ml, or approximately 106 mg / ml, or approximately 108 mg / ml, or approximately 110 mg / ml, or approximately 112 mg / ml, or approximately 114 mg / ml, or approximately 116 mg / ml, or approximately 118 mg / ml, or approximately 120 mg / ml, or approximately 122 mg / ml, or approximately 124 mg / ml, or approximately 126 mg / ml, or approximately 128 mg / ml, or approximately 130 mg / ml, or approximately 132 mg / ml, or approximately 134 mg / ml, or approximately 136 mg / ml, or approximately 1 38 mg / ml, or approximately 140 mg / ml, or approximately 142 mg / ml, or approximately 144 mg / ml, or approximately 146 mg / ml, or approximately 148 mg / ml, or approximately 150 mg / ml, or approximately 152 mg / ml, or approximately 154 mg / ml, or approximately 156 mg / ml, or approximately 158 mg / ml, or approximately 160 mg / ml, or approximately 162 mg / ml, or approximately 164 mg / ml, or approximately 166 mg / ml, or approximately 168 mg / ml, or approximately 170 mg / ml, or approximately 172 mg / ml, or approximately 174 mg / ml, or approximately 176 mg / ml, or approximately 178 mg / ml,Or approximately 180 mg / ml, or approximately 182 mg / ml, or approximately 184 mg / ml, or approximately 186 mg / ml, or approximately 188 mg / ml, or approximately 190 mg / ml, or approximately 192 mg / ml, or approximately 194 mg / ml, or approximately 196 mg / ml, or approximately 198 mg / ml, or approximately 200 mg / ml.

[0097] The pharmaceutical composition is preferably a liquid composition. Therefore, in one embodiment, the pharmaceutical composition is a liquid composition. In a further embodiment, the pharmaceutical composition is an aqueous composition.

[0098] The pharmaceutical composition is preferably a stable composition for pharmaceutical use even after storage for a given period. In one embodiment, the pharmaceutical composition is a stable pharmaceutical composition. A stable pharmaceutical composition is interpreted as being interchangeable with a stable pharmaceutical composition.

[0099] In one embodiment, the pharmaceutical composition is stable for at least two months, at least three months, at least six months, at least nine months, at least twelve months, etc.

[0100] The pharmaceutical composition is further stable over a wide range of antibody concentrations, such as approximately 10 mg / ml to approximately 250 mg / ml, approximately 20 mg / ml to approximately 70 mg / ml, or approximately 150 mg / ml, approximately 175 mg / ml, or approximately 200 mg / ml. In one embodiment, the pharmaceutical composition is stable over a wide range of antibody concentrations, such as approximately 10 mg / ml to approximately 250 mg / ml, approximately 20 mg / ml to approximately 200 mg / ml, for example, approximately 50 mg / ml to approximately 170 mg / ml.

[0101] Furthermore, the pharmaceutical composition is stable over a temperature range such as approximately 2°C to approximately 25°C. In one embodiment, the pharmaceutical composition is stable over a temperature range such as approximately 2°C to approximately 25°C.

[0102] It is remarkable that the formulation remains stable over such a wide range of antibody concentrations at temperatures fluctuating between approximately 2°C and approximately 25°C or even higher. The composition of the present invention is stable for at least 3 months, at least 6 months, and even more so for at least 9 months or at least 12 months when stored at 2°C to 8°C. Preferably, the composition is stable for pharmaceutical use for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, or at least 12 months at storage temperatures such as approximately 2 to 8°C and approximately 5°C. In one embodiment, the pharmaceutical composition is stable for pharmaceutical use for 12 months at storage temperatures such as approximately 2 to 8°C and approximately 5°C.

[0103] The pharmaceutical composition can be administered, for example, as a subcutaneous or intravenous composition. In one embodiment, the pharmaceutical composition is a subcutaneous composition and / or the composition is intended for use in subcutaneous administration. In another embodiment, the pharmaceutical composition is an intravenous composition and / or the composition is intended for use in intravenous administration.

[0104] The pharmaceutical compositions described herein can be prepared by mixing the components of the pharmaceutical composition in water. Therefore, further aspects of the present invention are a) Multispecific antibodies, antibodies such as approximately 0.5 to approximately 250 mg / ml, b) Buffering material, c) nonionic excipients, if necessary, If necessary, d) the step of mixing the surfactant in water, The present invention relates to a method for preparing a pharmaceutical composition as defined herein, comprising the step of adjusting the pH to approximately 4.0 to 8.0.

[0105] Pharmaceutical compositions and pharmaceutical preparations are used interchangeably in this specification.

[0106] Multispecific antibody format The present invention provides a pharmaceutical composition comprising a multispecific antibody capable of binding to CD30 and CD3. In one embodiment, the multispecific antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

[0107] In one embodiment, X in sequence number 9 is H. In another embodiment, X in sequence number 9 is G.

[0108] In one embodiment, the first heavy chain variable region is human or humanized. In another embodiment, the first light chain variable region is human or humanized. In one embodiment, the second heavy chain variable region is human or humanized. In another embodiment, the second light chain variable region is human or humanized. In another embodiment, the first heavy chain variable region and the first light chain variable region are human. In another embodiment, the first heavy chain variable region and the first light chain variable region are humanized. In another embodiment, the second heavy chain variable region and the second light chain variable region are human. In another embodiment, the second heavy chain variable region and the second light chain variable region are humanized. In another embodiment, the first heavy chain variable region and the first light chain variable region are human, and the second heavy chain variable region and the second light chain variable region are human. In another embodiment, the first heavy chain variable region and the first light chain variable region are human, and the second heavy chain variable region and the second light chain variable region are humanized.

[0109] As is well known to those skilled in the art, each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which may contain three CDR sequences, CDR1, CDR2, and CDR3, and four framework sequences, FR1, FR2, FR3, and FR4. This structure is preferably also found in the antibody according to the present invention. In one embodiment, one, two, three, or all of the four framework sequences are human framework sequences.

[0110] As described above, the pharmaceutical composition according to the present invention comprises a multispecific antibody containing an antigen-binding region capable of binding to human CD30, the sequence of which is shown in SEQ ID NO: 39. In particular, the antibody according to the present invention is an antibody in which the antigen-binding region capable of binding to human CD30 can bind to the extracellular domain of human CD30, such as a CD30 molecule expressed on cells, more preferably tumor cells.

[0111] As described, the pharmaceutical composition comprises an antibody comprising a CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0112] The CDR1, CDR2, and CDR3 regions can be identified from the variable heavy and light chain regions using methods known in the art. These CDR regions from the variable heavy and light chain regions are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0113] In one embodiment, the antigen-binding region that binds to CD30 is • A (first) heavy chain variable region (VH) containing the sequence of 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 of SEQ ID NO: 13; and, • Includes a (first) light chain variable region (VL) containing the sequence of 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 of SEQ ID NO: 14.

[0114] In further embodiments, the antibody used in the pharmaceutical composition according to the present invention comprises a heavy chain variable (VH) region of the antigen-binding domain that binds to CD30 as defined herein, and the sequence contains a total of up to 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 13.

[0115] In further embodiments, the antibody used in the pharmaceutical composition according to the present invention comprises a light chain variable (VL) region of the antigen-binding domain that binds to CD30 as defined herein, and the sequence contains a total of up to 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 14.

[0116] In a further embodiment, the first heavy chain variable region includes the sequence shown in Sequence ID No. 13, and the first light chain variable region includes the sequence shown in Sequence ID No. 14.

[0117] Such antigen-binding regions capable of binding to human CD30 are described, in particular, in International Publication No. 03059282 (Medarex), which is incorporated herein by reference.

[0118] The antibody used in the pharmaceutical composition according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human CD30 and the antibody. D Human CD30 can bind to it in the range of 0.1 to 20 nM, for example, in the range of 0.5 to 5 nM, 1.5 to 2 nM, etc. (monovalent binding). This binding affinity can be determined by biolayer interferometry.

[0119] In one embodiment, the antibody used in the pharmaceutical composition of the present invention can also bind to cynomolgus monkey CD30 (SEQ ID NO: 40).

[0120] As described above, the multispecific antibody used in the pharmaceutical composition according to the present invention includes an antigen-binding region capable of binding to human CD3. Furthermore, the present invention provides an antibody used in the pharmaceutical composition according to the present invention that can bind to human CD3ε (epsilon), such as human CD3ε (epsilon) identified in SEQ ID NO: 42. Such an antigen-binding region can bind to human CD3ε (epsilon) presented on T cells, such as primary human T cells.

[0121] As described, the antibody includes a CD3-binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

[0122] In one embodiment, the antigen-binding region that binds to CD3 is • A (second) heavy chain variable region (VH) containing the sequence of SEQ ID NO: 15, or a sequence having 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, • Includes a (second) light chain variable region (VL) containing the sequence of SEQ ID NO: 16, or a sequence having 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.

[0123] In further embodiments, the antibody used in the pharmaceutical composition according to the present invention comprises a heavy chain variable (VH) region of the antigen-binding domain that binds to CD3 as defined herein, and the sequence contains a total of up to 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 15.

[0124] In further embodiments, the antibody used in the pharmaceutical composition according to the present invention comprises a light chain variable (VL) region of the antigen-binding domain that binds to CD3 as defined herein, and the sequence contains a total of up to 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 16.

[0125] In further embodiments, the second heavy chain variable region includes the sequence shown in SEQ ID NO: 15, and the second light chain variable region includes the sequence shown in SEQ ID NO: 16. In one embodiment, X in SEQ ID NO: 15 is H. In another embodiment, X in SEQ ID NO: 15 is G.

[0126] Such antigen-binding regions capable of binding to human CD3 are described, in particular, in International Publication No. 2015 / 001085 (Genmab), which is incorporated herein by reference. The variants herein, such as the variant containing the VH CDR3 region as shown in Sequence ID No. 9 (wherein X is G), have a lower affinity for human CD3 than the parent antibody (wherein X is H), as described in Example 2 of International Publication No. 2017 / 009442 (Genmab), which is incorporated herein by reference.

[0127] The antibody used in the pharmaceutical composition according to the present invention has an equilibrium dissociation constant K between the antigen-binding domain that binds to human CD3. D It can bind, and in human CD3, the M nucleotide range is 5-30 nM, for example, 10-20 nM (in the case of monovalent bonding).

[0128] In one embodiment, the antibody used in the pharmaceutical composition of the present invention can also bind to cynomolgus monkey CD3 (SEQ ID NO: 43).

[0129] In further embodiments, the pharmaceutical composition is (i) A CD30 binding region comprising a first heavy chain variable region containing the sequence shown in SEQ ID NO: 13 and a first light chain variable region containing the sequence shown in SEQ ID NO: 14, and (ii) A multispecific antibody comprising a CD3 binding region comprising a second heavy chain variable region comprising the sequence shown in SEQ ID NO: 15 and a second light chain variable region comprising the sequence shown in SEQ ID NO: 16.

[0130] Antibody formats contained within pharmaceutical compositions The multispecific antibodies used in the pharmaceutical compositions of the present invention may have two or more specificities, two or three or more specificities, etc. Furthermore, the multispecific antibodies may have two or more copies of the antigen-binding region for CD3 and / or CD30. For example, in one embodiment, the antibody has two antigen-binding regions that can bind to CD3, two identical binding regions that bind to CD3, etc. For example, in another embodiment, the antibody has two antigen-binding regions that bind to CD30, two identical binding regions that bind to CD30, etc. Further antigen-binding regions may exist, for example, in the form of scFv covalently bound to the constant region.

[0131] In preferred embodiments, the pharmaceutical composition of the present invention comprises a multispecific antibody, which 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 Labrijn et al. (2019 Nat Rev Drug Discov 18(8)585-608). The bispecific antibodies of the present invention are not limited to a specific bispecific format or method of production thereof.

[0132] Examples of bispecific antibody molecules that may be included in the pharmaceutical composition according to the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody specific to two different targets via two scFv linked in tandem by an additional peptide linker, for example; and (iii) each light chain and heavy chain having short peptide bonds. (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig(trademark)) Molecule, In: Antibody Engineering, Springer Berlin) (iv) two chemically bonded bispecific (Fab') fragments; (v) a tangential bispecific antibody (DVD-Ig) containing two variable domains via Heidelberg (2010); (v) a tangab, which is a fusion of two single-stranded diabodies resulting in a tetravalent bispecific antibody having two binding sites for each target antigen; (vi) a flexibody, which is a combination of an scFv and a diabody resulting in a polyvalent molecule; (vii) a so-called "dock-and-lock" molecule based on the "dimerization and docking domain" of protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) a so-called scorpion molecule, for example, containing two scFvs fused to both ends of a human Fab arm; and (ix) a diabody.

[0133] Further examples of bispecific antibodies of different classes include, but are not limited to, (i) IgG-like molecules having a complementary CH3 domain that forces heterodimerization; (ii) recombinant IgG-like bitargeting molecules in which both sides of the molecule each contain Fab fragments or portions of Fab fragments of at least two different antibodies; (iii) IgG fusion molecules in which a full-length IgG antibody is fused to an extra Fab fragment or portion of a Fab fragment; (iv) Fc fusion molecules in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region or portion thereof; (v) Fab fusion molecules in which different Fab fragments are fused together and fused to a heavy chain constant domain, Fc region or portion thereof; and (vi) scFv systems and diabody systems and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules, different diabodies, or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, Fc region or portion thereof.

[0134] Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, International Publication No. 2011069104), so-called knob-in-to-hole molecules (Genentech, International Publication No. 9850431), CrossMAb (Roche, International Publication No. 2011117329), and electrostatically matched molecules (Amgen, European Patent No. 1870459 and International Publication No. 2009089004; Chugai, US Patent Application Publication No. 201000155133; Oncomed, International Publication No. 2010129304), and LUZ-Y molecules (Genentech, Wranik et al.). 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, International Publication No. 2010134666, International Publication No. 2014081202), Strand Exchange Engineering Domain Body (SEEDbody) molecule (EMD Examples include Serono (International Publication No. 2007110205), Biclonics molecules (Merus, International Publication No. 2013157953), FcΔAdp molecules (Regeneron, International Publication No. 201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, International Publication No. 11143545), azimetric scaffold molecules (Zymeworks / Merck, International Publication No. 2012058768), mAb-Fv molecules (Xencor, International Publication No. 2011028952), bivalent bispecific antibodies (International Publication No. 2009080254), and DuoBody® molecules (Genmab A / S, International Publication No. 2011131746).

[0135] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig molecules (International Publication No. 2009058383), two-in-one antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, International Publication No. 2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approaches (Crucell / Merus, U.S. Patent No. 7,262,028), kappa / lambda body™ molecules (NovImmune, International Publication No. 2012023053), and CovX-body (CovX / Pfizer; Doppalapudi, VR, et al. (2007. Bioorg. Med. Chem. Lett. 17, 501-506.) can be cited.

[0136] Examples of IgG fusion molecules include, but are not limited to, the bivariable domain (DVD)-Ig molecule (Abbott, U.S. Patent No. 7,612,181), bidomain bihead antibody (Unilever; Sanofi Aventis, International Publication No. 20100226923), IgG-like bispecificity molecule (ImClone / 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 molecule (Zymogenetics, International Publication No. 2010111625), HERCULES molecule (Biogen Idec, U.S. Patent No. 007951918), scFv fusion molecule (Novartis), and scFv fusion molecule (Changzhou Adam Biotech). Examples include Roche Inc. (Chinese Patent No. 102250246) and the TvAb molecule (Roche, International Publication No. 2012025525, International Publication No. 2012025530).

[0137] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), the SCORPION molecule (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, International Publication No. 2010111625), the Dual Affinity Retargeting Technology (Fc-based DART) molecule (MacroGenics, International Publication No. 2008157379, International Publication No. 2010080538), and the Dual(scFv)2-Fab molecule (National Research Center for Antibody Medicine - China).

[0138] Examples of Fab-fusion bispecific antibodies include, but are not limited to, 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.), Dock-and-Lock (DNL) molecules (ImmunoMedics, International Publication No. 2003074569, International Publication No. 2005004809), divalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15; 165(12): 7050-7.), and Fab-Fv molecules (UCB-Celltech, International Publication No. 2009040562.).

[0139] Examples of scFv-based, diabody-based, and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules (Micromet, International Publication No. 2005061547), tandem diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), DART molecules (MacroGenics, International Publication No. 2008157379, International Publication No. 2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusion (Merrimack, International Publication No. 2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al.) Examples include bi-targeted nanobody antibodies (Ablynx, Hmila et al., FASEB J. 2010) and bi-targeted heavy chain-only domain antibodies.

[0140] 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 via controlled Fab arm exchange (as described in International Publication No. 2011131746 (Genmab), etc.).

[0141] In one embodiment, the pharmaceutical composition of the present invention contains an antibody that is a bispecific DuoBody® molecule (Genmab A / S, International Publication No. 2011131746).

[0142] The multispecific antibody (bispecific antibody, etc.) used in the pharmaceutical composition of the present invention may be of any isotype. Exemplary isotypes include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably, the antibody may be selected to be the human IgG1 isotype, as shown in the examples. The human light chain constant region, either kappa or lambda, or both, such as the sequences described in SEQ ID NOs. 53 and 54, may be used. For example, in one embodiment, the light chain involved in CD30 binding includes a kappa constant region, and the light chain involved in CD3 binding includes a lambda constant region.

[0143] Accordingly, in a further embodiment, the antigen-binding region capable of binding to human CD30 is included in the heavy chain and light chain, the heavy chain comprising the VH region and the IgG1 heavy chain constant region, and the light chain comprising the VL region and the kappa light chain constant region, and the antigen-binding region capable of binding to human CD3 is included in the heavy chain and light chain, the heavy chain comprising the VH region and the IgG1 heavy chain constant region, and the light chain comprising the VL region and the lambda light chain constant region. In a further embodiment, one of the IgG1 heavy chain constant regions is as defined in SEQ ID NO: 51, the other is as defined in SEQ ID NO: 50, the kappa light chain constant region is as defined in SEQ ID NO: 53, and the lambda light chain constant region is as defined in SEQ ID NO: 54.

[0144] In one embodiment, both heavy chains of the antibody used in the pharmaceutical composition of the present invention are of the IgG1 isotype. In one embodiment, the two heavy chains of the bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. Preferably, the bispecific antibody may be selected to be of the human IgG1 isotype, as shown in the examples. The heavy chains of the selected isotype and their Fc region sequences may, preferably, be modified in the hinge, CH2 and / or CH3 regions to enable the generation and / or inactivation of the bispecific antibody.

[0145] In one embodiment, the multispecific antibody used in the pharmaceutical composition of the present invention includes an Fc region consisting of a first and a second Fc polypeptide.

[0146] In one embodiment, the first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are contained within the same polypeptide chain.

[0147] The first and second Fc polypeptides may each be of any isotype, including any human isotype such as IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotype or mixed isotype. Preferably, the Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or mixed isotype. In one embodiment, the Fc region is a human IgG1 Fc region.

[0148] In further embodiments, the multispecific antibody is a full-length antibody as defined herein.

[0149] The antibodies used in the pharmaceutical compositions according to the present invention may include modifications to the Fc region to make the antibody an inactive or deactivating antibody. Accordingly, in the antibodies disclosed herein, one or both heavy chains may be modified to induce Fc-mediated effector function to a lesser extent than an antibody that is identical except that it does not include such modifications. Fc-mediated effector function can be measured by determining Fc-mediated CD69 expression on T cells (i.e., CD69 expression as a result of CD3 antibody-mediated Fcγ receptor-dependent CD3 crosslinking, as described, for example, in International Publication No. 2015001085), by binding to the Fcγ receptor, by binding to C1q, or by inducing Fc-mediated crosslinking of FcγR. In particular, the heavy chain constant sequence may be modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type (unmodified) antibody, and such Fc-mediated CD69 expression is determined by a PBMC-based functional assay, for example, by flow cytometry as described in Example 3 of International Publication No. 2015001085. Modification of the heavy chain constant sequence and light chain constant sequence may also result in a decrease in C1q binding to the antibody. Compared to an unmodified antibody, the decrease may 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 may be modified so that the antibody mediates Fc-mediated T cell proliferation that 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 the unmodified antibody, for example in the straight portion of the curve, and such T cell proliferation is measured by a PBMC-based functional assay.

[0150] To eliminate Fc-mediated effector function, a wide range of different inactivation antibody formats have been developed by introducing amino acid substitutions and combinations thereof into the constant heavy chain region of IgG1 isotype antibodies (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. 2001 Mar 2;276(9):6591-604).

[0151] For example, examples of amino acid positions that can be modified in an IgG1 isotype antibody include positions L234 and L235. In one embodiment, the first and / or second Fc polypeptide includes amino acid substitutions corresponding to the amino acids at positions L234 and / or L235 of the human IgG1 heavy chain, the substitutions being preferably substitutions to F and E, respectively, and the amino acid positions are as defined by Eu numbering.

[0152] It is understood that, in addition to the modification of amino acid positions L234 and L235, further positions may be modified. Therefore, in further embodiments, the first and second Fc polypeptides include substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235, respectively, and the first and / or second Fc polypeptides further include substitutions of amino acids corresponding to the amino acid at position G236 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to R.

[0153] In another embodiment, the first and second Fc polypeptides each include substitutions of amino acids to F and E, corresponding to the amino acids at positions L234 and L235, respectively, and the first and second Fc polypeptides further include substitutions of amino acids corresponding to the amino acid at position G236 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to R.

[0154] In another embodiment, the first and second Fc polypeptides each include substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235, respectively, and the first and / or second Fc polypeptides further include substitutions of amino acids corresponding to the amino acid at position D265 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to A.

[0155] In another embodiment, the first and second Fc polypeptides each include substitutions of amino acids to F and E, corresponding to the amino acids at positions L234 and L235, respectively, and the first and second Fc polypeptides further include substitutions of amino acids corresponding to the amino acid at position D265 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to A.

[0156] In another embodiment, one of the first and second Fc polypeptides includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, and G236 to F, E, and R, respectively, while the other Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235E, and D265 to F, E, and A, respectively.

[0157] In further embodiments, the first Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, and G236 to F, E, and R, respectively, and the second Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235E, and D265 to F, E, and A, respectively, with the amino acid positions defined by Eu numbering.

[0158] For example, constant regions having such Fc region substitutions are provided in particular in SEQ ID NOs. 45, 46, 49, 50, 51 and 52, which can be compared with SEQ ID NO. 44, which does not have one or more such substitutions. In one embodiment, the antibody of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs. 45, 46, 49, 50, 51 and 52.

[0159] In one embodiment, the multispecific or bispecific antibody used in the pharmaceutical composition of the present invention comprises an Fc region containing different first and second CH3 regions, and as a result, the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between each of the first and second CH3 regions. Further details on these interactions and how they can be achieved are provided in International Publication Nos. 2011131746 and 2013060867 (Genmab), which are incorporated herein by reference. Stable heterodimer antibodies can be obtained in high yield, for example, by so-called Fab arm exchange, as provided in International Publication Nos. 2008 / 119353 and 2011 / 131746, based on two homodimer starting antibodies containing only minor asymmetric mutations in the CH3 region.

[0160] Accordingly, in one embodiment, in the first Fc polypeptide, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, and in the second Fc polypeptide, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, and the substitutions in the first and second Fc polypeptides are not at the same position, and the amino acid positions are as defined by Eu numbering. For example, constant regions having such Fc region substitutions are provided in particular in SEQ ID NOs: 47, 48, 49, 50, 51, and 52, which can be compared with SEQ ID NO: 44, which does not have such substitutions. In one embodiment, the antibody of the present invention contains a sequence selected from the group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, and 52.

[0161] In certain embodiments, the present invention provides a pharmaceutical composition comprising an antibody, wherein the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is R in the first Fc polypeptide, and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is L in the 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.

[0162] In further embodiments, the amino acid at the position corresponding to K409 in the first Fc polypeptide is R, and the amino acid at the position corresponding to F405 in the second Fc polypeptide is L.

[0163] Therefore, in one embodiment, one of the first and second Fc polypeptides includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, G236, and F405 to F, E, R, and L, respectively, while the other Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235E, D265, and K409 to F, E, A, and R, respectively.

[0164] In another embodiment, one of the first and second Fc polypeptides includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, G236, and K409 to F, E, R, and R, respectively, while the other Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235E, D265, and F405 to F, E, A, and L, respectively.

[0165] In further embodiments, the present invention relates to a pharmaceutical composition comprising a multispecific antibody, wherein the multispecific antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, A multispecific antibody is a bispecific antibody and contains an Fc region consisting of a first and a second Fc polypeptide. The first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain. The second Fc polypeptide and the double chain variable region are contained within the same polypeptide chain. The first Fc polypeptide includes substitutions of amino acids corresponding to positions L234, L235, and G236 to F, E, and R, respectively, and the second Fc polypeptide includes substitutions of amino acids corresponding to positions L234, L235, and D265 to F, E, and A, respectively, with the amino acid positions defined by Eu numbering. In the first Fc polypeptide, the amino acid at the position corresponding to K409 is R, and in the second Fc polypeptide, the amino acid at the position corresponding to F405 is L.

[0166] In further embodiments, the present invention relates to a pharmaceutical composition comprising a multispecific antibody, wherein the multispecific antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, A multispecific antibody is a bispecific antibody and contains an Fc region consisting of a first and a second Fc polypeptide. The first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain. The second Fc polypeptide and the double chain variable region are contained within the same polypeptide chain. The first Fc polypeptide includes substitutions of amino acids F, E, and A corresponding to the amino acids at positions L234, L235, and D265, respectively, and the second Fc polypeptide includes substitutions of amino acids F, E, and A corresponding to the amino acids at positions L234, L235, and D265, respectively, with the amino acid positions defined by Eu numbering. In the first Fc polypeptide, the amino acid at the position corresponding to K409 is R, and in the second Fc polypeptide, the amino acid at the position corresponding to F405 is L.

[0167] In one embodiment, the antibody used in the pharmaceutical composition of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19, and the light chain sequences shown in SEQ ID NOs: 18 and 20.

[0168] In further embodiments, the antibody used in the pharmaceutical composition of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19, and the light chain sequences shown in SEQ ID NOs: 18 and 20, and the antibody is a bispecific antibody.

[0169] In one embodiment, the antibody used in the pharmaceutical composition of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 35, and the light chain sequences shown in SEQ ID NOs: 18 and 20.

[0170] In one embodiment, the antibody used in the pharmaceutical composition of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs. 55 and 19 and the light chain sequences shown in SEQ ID NOs. 18 and 20.

[0171] In one embodiment, the antibody used in the pharmaceutical composition of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs. 55 and 35 and the light chain sequences shown in SEQ ID NOs. 18 and 20.

[0172] In further embodiments, the antibody used in the pharmaceutical composition of the present invention is bsG1-huCD3-FEALxCD30-MDX060-FEAR or bsG1-huCD3-FEALxCD30-MDX060-FERR. In further embodiments, the antibody used in the pharmaceutical composition of the present invention is bsG1-huCD3-FEALxCD30-MDX060-FERR. In further embodiments, the multispecific antibody is bsIgG1-huCD3-FEALxCD30-MDX0060-FERR or its biosimilar.

[0173] The constant region sequences listed in SEQ ID NOs: 44-52 and 55 do not contain C-terminal lysine (K). However, in naturally occurring human sequences from which these Fc regions originate, such C-terminal lysine may be present as part of the open reading frame. During cell culture production of recombinant antibodies, this terminal lysine can be cleaved by proteolysis by (one or more) endogenous carboxypeptidases, resulting in constant regions with the same sequence but lacking C-terminal lysine. For the purpose of antibody production, the DNA encoding this terminal lysine can be omitted from the sequence so that antibodies are produced without lysine. Deletion of C-terminal lysine from antibody-coding sequences can increase antibody uniformity with respect to the presence of C-terminal lysine. Antibodies produced from nucleic acid sequences that encode or do not encode terminal lysine are substantially identical in sequence and function, for example, because the degree of C-terminal lysine processing is typically high when using antibodies produced in CHO-based production systems (Dick, L. Wet al. Biotechnol. Bioeng. 2008;100:1132-1143). Therefore, it is understood that antibodies used in the pharmaceutical compositions according to the present invention may be produced without encoding or having C-terminal lysine as enumerated herein. Thus, antibodies can be produced without C-terminal lysine for manufacturing purposes.

[0174] In alternative embodiments, the polyspecific antibody used in the pharmaceutical composition according to the present invention is not a classical full-length antibody containing an Fc region. For example, in one embodiment, (i) The CD30 binding region and / or the CD3 binding region is Fab, (ii) The CD30 binding region and / or the CD3 binding region is scFv, (iii) The CD30 binding region is Fab and the CD3 binding region is scFv, or (iv) The CD30 binding region is scFv, and the CD3 binding region is Fab.

[0175] Binding, cytotoxicity and T cell activation A pharmaceutical composition comprising an antibody, such as the bispecific antibody 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.

[0176] As described above, preferably, the antibody used in the pharmaceutical composition according to the present invention is inactive, and furthermore, the antibody includes one or more of the following characteristics: a) When tested using flow cytometry, it is possible to bind to CD30-expressing human tumor cells (SU-DHL-1 cells, SUP-M2 cells, DL-40 cells, KARPAS-299 cells, L-82 cells, SR-786 cells, L-540 cells, KM-H2 cells, L-1236 cells, JVM-2 cells, HH cells, NCEB-1 cells and / or HDLM-2 cells, etc.) as described in the examples herein. b) When assayed as described in the examples herein, for example, when purified PBMCs, ADCC effector cells, or T cells are used as effector cells, concentration-dependent cytotoxicity of CD30-expressing human tumor cells can be mediated. c) When assayed as described in the examples herein, for example, when purified PBMCs or T cells are used as effector cells, it is possible to mediate concentration-dependent cytotoxicity of one or more human CD30-expressing tumor cell lines selected from the group consisting of SU-DHL-1 cells, L-428 cells, KM-H2 cells, SUP-M2 cells, KI-JK cells, and HDLM-2 cells. d) For example, when assayed as described in the examples herein, T cell proliferation can be induced in vitro in the presence of CD30-expressing human tumor cells. e) When assayed as described in the examples herein, T cells can be activated in vitro in the presence of one or more CD30-expressing human tumor cell lines selected from the group consisting of SU-DHL-1 cells, L-428 cells, KI-JK cells, and HDLM-2 cells. f) When assayed as described in the examples of this specification, dose-dependent production of cytokines and granzyme B by T cells can be induced in vitro. g) When assayed as described in the examples herein, CD30 expression on a subpopulation of activated T cells may not result in T cell fracturisides, and / or h) When assayed as described in the examples herein, T cell-mediated cytotoxicity can be induced even in the presence of sCD30.

[0177] In one embodiment, when the antibody described herein is assayed as described in the examples herein, using L-428 tumor cells and measured by flow cytometry, etc., the EC is less than 0.050 μg / ml, less than 0.045 μg / ml, for example less than 0.040 μg / ml, less than 0.035 μg / ml, for example less than 0.030 μg / ml, less than 0.025 μg / ml, for example less than 0.020 μg / ml. 50 It induces T cell-mediated cytotoxicity depending on the concentration.

[0178] In further embodiments, the antibodies described herein, when assayed as described in the examples herein, using L-428 tumor cells and measured by flow cytometry, etc., have maximum lysis when inducing T cell-mediated cytotoxicity of more than 80%, more than 85%, etc., for example, more than 90%.

[0179] In one embodiment, when the antibody described herein is assayed using L-428 tumor cells and measured by flow cytometry, as described in the examples herein, the CD25 expression EC is less than 0.05 μg / ml, less than 0.045 μg / ml, for example less than 0.04 μg / ml, less than 0.035 μg / ml, for example less than 0.03 μg / ml. 50 CD4 with concentration + It induces T cell activation. In further embodiments, when the antibodies described herein are assayed using L-428 tumor cells and measured by flow cytometry, etc., as described in the examples herein, the CD25 expression of EC is reduced to less than 0.005 μg / ml, less than 0.001 μg / ml, for example less than 0.0009 μg / ml, less than 0.0008 μg / ml, for example less than 0.0007 μg / ml. 50 CD8 with concentration + It induces T cell activation.

[0180] In one embodiment, when the antibody described herein is assayed using L-428 tumor cells and measured by flow cytometry, as described in the examples herein, the CD69 expression EC is less than 0.004 μg / ml, less than 0.003 μg / ml, for example less than 0.002 μg / ml, less than 0.001 μg / ml, for example less than 0.0009 μg / ml. 50 CD4 with concentration + It induces T cell activation. In further embodiments, when the antibodies described herein are assayed using L-428 tumor cells and measured by flow cytometry, etc., as described in the examples herein, the CD69 expression EC is reduced to less than 0.0035 μg / ml, less than 0.0030 μg / ml, for example less than 0.0025 μg / ml, less than 0.0020 μg / ml, for example less than 0.0015 μg / ml. 50 CD8 with concentration + It induces T cell activation.

[0181] In one embodiment, when the antibody described herein is assayed using L-428 tumor cells and measured by flow cytometry, as described in the examples herein, the EC levels of PD-1 expression are less than 0.01 μg / ml, less than 0.008 μg / ml, for example less than 0.007 μg / ml, less than 0.006 μg / ml, for example less than 0.005 μg / ml. 50 CD4 with concentration + It induces T cell activation. In further embodiments, when the antibodies described herein are assayed using L-428 tumor cells and measured by flow cytometry, etc., as described in the examples herein, the EC levels of PD-1 expression are less than 0.007 μg / ml, less than 0.006 μg / ml, for example less than 0.005 μg / ml, less than 0.004 μg / ml, for example less than 0.003 μg / ml. 50 CD8 with concentration + It induces T cell activation.

[0182] The assay may be carried out as is generally known to those skilled in the art and may optionally include an effector cell-to-target cell ratio (E:T) of 4:1 and / or an incubation period of 72 hours. The assay may be carried out by providing tumor cells, preferably labeled tumor cells, such as L-428 tumor cells; adding the antibodies described herein, preferably in serial dilutions; providing T cells, preferably labeled T cells, with an effector-to-target ratio E:T of 4:1 and incubating for 72 hours; optionally staining relevant parameters such as CD4, CD8, CD25, CD69 and / or PD-1; and finally analyzing the cells using flow cytometry such as FACS. % live cells and / or T cell proliferation / activation may be calculated from the data obtained by the analysis, as is generally known to those skilled in the art.

[0183] Production of antibodies used in the pharmaceutical composition of the present invention Conventional methods such as hybrid hybridoma and chemical conjugation (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used to prepare multispecific antibodies, such as bispecific antibodies, used in the pharmaceutical compositions of the present invention. Co-expression of two antibodies consisting of different heavy and light chains in host cells yields a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or a similar method.

[0184] As mentioned, strategies to promote the formation of functionally bispecific products during co-expression of different antibody constructs can also be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to preferential species-restricted heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which a protrusion is introduced on the first heavy chain polypeptide and on the corresponding cavity in the second heavy chain polypeptide, and as a result the protrusion can be positioned within the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation. The "protrusion" is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain. Compensatory "caves" of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains with smaller side chains (U.S. Patent No. 5,731,168). European Patent No. 1870459 (Chugai) and International Publication No. 2009089004 (Amgen) describe other strategies for promoting heterodimerization during the co-expression of different antibody domains in host cells. In these methods, one or more residues constituting the CH3-CH3 interface of both CH3 domains are replaced with charged amino acids so that homodimerization is electrostatically undesirable and heterodimerization is electrostatically favorable. International Publication No. 2007110205 (Merck) describes yet another strategy that promotes heterodimerization by utilizing the differences between IgA and IgG CH3 domains.

[0185] Another in vitro method for producing bispecific antibodies is described in WO 2008 / 119353 (Genmab), where the bispecific antibodies are formed by "Fab arm" or "half - molecule" exchange (exchange of heavy and associated light chains) between two single - specificity IgG4 or IgG4 - like antibodies upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms that may contain different sequences.

[0186] As a preferred method for preparing the bispecific CD3xCD30 antibody used in the pharmaceutical composition of the present invention, there are the methods described in WO 2011 / 131746 and WO 13 / 060867 (Genmab), as follows: a) Providing a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; b) Providing a second antibody comprising a second Fc region, wherein the Fc region comprises a second CH3 region, where the first antibody is a CD30 antibody and the second antibody is a CD3 antibody, or vice versa, providing a second antibody comprising a second Fc region, wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first and the second CH3 regions is stronger than each of the homodimeric interactions of the first CH3 region and the second CH3 region; c) Incubating the first antibody together with the second antibody under reducing conditions; d) Obtaining the bispecific CD3xCD30 antibody, and comprising.

[0187] Similarly, the multispecific antibodies (such as bispecific antibodies) used in the pharmaceutical composition according to the present invention are as follows: a) providing a first homodimeric antibody comprising a CD30 - binding region described herein and a second homodimeric antibody comprising a CD3 - binding region described herein, wherein the antibody comprises an Fc region and may comprise further features described herein; The first antibody and the second antibody have different sequences for the first and second CH3 regions, such that the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between the first and second CH3 regions. b) A step of incubating the first antibody together with the second antibody under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, c) The heterodimer multispecific antibody of the present invention as described herein, comprising the step of obtaining a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody, and a second immunoglobulin heavy chain and a second immunoglobulin light chain of a second antibody.

[0188] In one embodiment, the first antibody is incubated with the second antibody under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, where the heterodimer interaction between the first and second antibodies in the resulting heterodimer antibody is such that no Fab arm exchange occurs at 0.5 mM GSH after 24 hours at 37°C.

[0189] Although not limited to theory, in step c), the heavy chain disulfide bond in the hinge region of the parent antibody is reduced, and the resulting cysteine ​​can then form an inter-heavy chain disulfide bond with a cysteine ​​residue of another parent antibody molecule (which originally has different specificities). In one embodiment of this method, the reduction conditions in step c) include the addition of a reducing agent, for example, a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a preferred embodiment, the reducing agent is 2-mercaptoethylamine. In a further embodiment, step c) includes restoring the conditions to non-reducing or low-reducing, for example by removing the reducing agent, for example by desalting.

[0190] In a further embodiment, the multispecific antibody used in the pharmaceutical composition according to the present invention is as follows: a) A first monospecific CD30 antibody, (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A step of providing a first monospecific CD30 antibody comprising Fc regions consisting of first and second Fc polypeptides, including substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235 in the human IgG monoheavy chain, and substitutions of amino acids to A corresponding to the amino acid at position D265; b) A second monospecific CD3 antibody, (i) A CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, and (ii) A step to provide a second monospecific CD3 antibody comprising Fc regions consisting of first and second Fc polypeptides, the Fc regions comprising substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235 in the human IgG monoheavy chain, and substitutions of amino acids to A corresponding to the amino acid at position D265, A step of providing a second monospecific CD3 antibody, wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, and as a result, the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between the first and second CH3 regions, 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) A step of incubating the first antibody together with the second antibody under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, d) It may be produced by a method comprising the step of obtaining a multispecific antibody comprising a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody and a second immunoglobulin heavy chain and a second immunoglobulin light chain of a second antibody.

[0191] In a further embodiment, the multispecific antibody used in the pharmaceutical composition according to the present invention is as follows: a) A first monospecific CD30 antibody, (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A step of providing a first monospecific CD30 antibody comprising Fc regions consisting of first and second Fc polypeptides, including substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235 in the human IgG monoheavy chain, and substitutions of amino acids to R corresponding to the amino acid at position G236; b) A second monospecific CD3 antibody, (i) A CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, and (ii) A step to provide a second monospecific CD3 antibody comprising Fc regions consisting of first and second Fc polypeptides, the Fc regions comprising substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235 in the human IgG monoheavy chain, and substitutions of amino acids to A corresponding to the amino acid at position D265, The first antibody and the second antibody have different sequences in their first and second CH3 regions, resulting in a heterodimer interaction between the first and second CH3 regions being stronger than the homodimer interaction between the first and second CH3 regions, preferably with the amino acid at the position corresponding to K409 being R in the first CH3 region and the amino acid at the position corresponding to F405 being L in the second CH3 region, and the second antibody being provided as a second monospecific CD3 antibody. c) A step of incubating the first antibody together with the second antibody under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, d) It may be produced by a method comprising the step of obtaining a multispecific antibody comprising a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody and a second immunoglobulin heavy chain and a second immunoglobulin light chain of a second antibody.

[0192] The present invention further relates to a multispecific antibody used in a pharmaceutical composition according to the present invention, obtained by the method described herein.

[0193] In the above method, the step of providing a first or second homodimeric antibody that can bind to CD30 and / or CD3 is: - A step of providing cells containing the antibody or an expression vector for producing the plurality of antibodies, - A step of causing cells to produce the antibody or a plurality of antibodies, and thereafter, - The process may include the step of providing the antibody or a plurality of antibodies by obtaining the antibody or a plurality of antibodies.

[0194] In one embodiment of this method, the first and / or second homodimer antibody is a full-length antibody.

[0195] The Fc regions of the first and second homodimeric antibodies may be any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second homodimeric antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of the homodimeric antibody is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody includes the Fc regions of IgG1 and IgG4, and therefore may have interesting intermediate properties with respect to the activation of effector function.

[0196] In a further embodiment, one of the homodimer starting antibodies is engineered not to bind to protein A, and thus the heterodimer antibody is separated from the homodimer starting antibody by passing the product through a protein A column, removing the flow-through, and eluting the heterodimer antibody from the protein A column to obtain a purified heterodimer antibody composition.

[0197] As described above, the sequences of the first CH3 region and the second CH3 region of the homodimer starting antibody can be different, and as a result, the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than each of the homodimer interactions of the first CH3 region and the second CH3 region. Further details regarding these interactions and how they can be achieved are provided in International Publication No. WO 2011 / 131746 and International Publication No. WO 2013 / 060867 (Genmab), which are hereby incorporated by reference in their entirety.

[0198] In particular, stable bispecific CD3xCD30 antibodies can be obtained in high yields using the methods of the invention based on two homodimer starting antibodies that each bind to CD30 and CD3 and contain minor, fairly conservative, asymmetric mutations in the CH3 region. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions.

[0199] The multispecific antibodies (such as bispecific antibodies) used in the pharmaceutical compositions of the invention can also be obtained by co-expression of constructs encoding the first and second polypeptides in a single cell.

[0200] Thus, in a further aspect, the invention relates to a nucleic acid construct, or combination of nucleic acid constructs, encoding a multispecific antibody used in a pharmaceutical composition according to the invention, and an expression vector, or combination of expression vectors, comprising such one or more nucleic acid constructs.

[0201] Furthermore, the invention relates to a recombinant host cell capable of producing a multispecific antibody used in a pharmaceutical composition according to the invention, the host cell comprising one or more nucleic acid constructs encoding a multispecific antibody used in a pharmaceutical composition according to the invention.

[0202] Thus, the invention also provides a method for producing a multispecific antibody used in a pharmaceutical composition according to the invention, comprising (i) Culturing the recombinant host cells of the present invention under conditions in which antibodies are produced, (ii) The present invention relates to a method comprising isolating multispecific antibodies produced from a culture.

[0203] In one embodiment, the method is as follows: a) A step of providing 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, wherein the first Fc region comprises a first CH3 region, b) A step of providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region and a second antigen-binding region of a second antibody heavy chain, wherein the second Fc region comprises a second CH3 region, A step of providing a second nucleic acid construct in which the sequences of the first CH3 region and the second CH3 region are different, and as a result, the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region, and the first and second nucleic acid constructs may encode the light chain sequences of the first and second antibodies. c) A step of co-expressing the first and second nucleic acid constructs in a host cell, d) A step of obtaining the heterodimer protein from the cell culture, Preferably, the encoded amino acid at the position corresponding to F405 is L in the first CH3 region, and the encoded amino acid at the position corresponding to K409 is R in the second CH3 region, or vice versa.

[0204] Appropriate expression vectors, including promoters and enhancers, and suitable host cells for antibody production are well known in the art. Examples of host cells include yeast, bacterial and mammalian cells, Chinese hamster ovary cells (CHO), or human and human fetal kidney (HEK) cells.

[0205] The nucleic acids or one or more nucleic acids defined herein may be RNA or DNA. The nucleic acids or one or more nucleic acids defined herein may be for use in expression in mammalian cells. Accordingly, the present invention further provides one or more cells comprising the nucleic acids defined herein or one or more nucleic acids.

[0206] In the context of the present invention, nucleic acids can be expression vectors, which may be any suitable vectors, including chromosomes, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the CD30 or CD3 antibody is contained in a naked DNA or RNA vector, such as a linear expression element (e.g., described in Sykes and Johnston, Nat Biotech 17,355 59 (1997)), a compressed nucleic acid vector (e.g., described in U.S. Patent No. 6,077,835 and / or International Publication No. 00 / 70087), a plasmid vector such as pBR322, pUC 19 / 18, or pUC 118 / 119, a "midge" minimum-size nucleic acid vector (e.g., described in Schakowski et al., Mol Ther 3,793 800 (2001)), or a CaP04 precipitate construct (e.g., International Publication No. 200046147, Benvenisty and Reshef, PNAS USA 83,9551 55 (1986), Wigler et al., Cell This includes precipitated nucleic acid vector constructs such as those described in 14,725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7,603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, U.S. Patent Nos. 5,589,466 and 5,973,972).

[0207] In one embodiment, the vector is suitable for the expression of CD30 antibodies and / or CD3 antibodies in bacterial cells. Examples of such vectors include BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison, Wisconsin), etc.).

[0208] Expression vectors may also, or alternatively, be vectors suitable for expression in yeast systems. Any vector suitable for expression in yeast systems can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (discussed 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)).

[0209] Nucleic acids and / or expression vectors may also contain nucleic acid sequences encoding secretory / localization sequences that can target polypeptides, such as nascent polypeptide chains, to the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides. Nucleic acids and / or expression vectors may also contain any suitable elements that promote nucleic acid expression, i.e., transcription and / or translation, so that components of a (bispecific) antibody are expressed. Nucleic acids and / or vectors can be associated with any suitable promoters, enhancers, and other expression-promoting elements. Examples of such elements include potent expression promoters (e.g., human CMV IE promoter / enhancer, as well as RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in Escherichia coli (E. coli), antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). Nucleic acids may also contain inductive promoters, as opposed to constitutive promoters such as CMV IE.

[0210] (medical) use The pharmaceutical composition may be administered by any suitable route and manner. In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0211] The pharmaceutical composition according to the present invention is preferably intended for use as a pharmaceutical product.

[0212] The pharmaceutical composition according to the present invention is preferably intended for use in methods for treating diseases.

[0213] In particular, the pharmaceutical compositions of the present invention can be used to treat various forms of cancer. In one embodiment, the present invention relates to the use of the pharmaceutical compositions described herein for the treatment of cancer. In a further embodiment, the present invention relates to a method for treating a disease, comprising administering the pharmaceutical compositions defined herein to a subject in need thereof. In yet another embodiment, the present invention relates to a method for treating cancer in a subject, comprising administering the pharmaceutical compositions described herein to a subject in need thereof for a time sufficient to treat the cancer.

[0214] In one embodiment, the present invention provides a method for treating cancer in a subject, the method comprising the administration of 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 CD30-expressing cells in a subject, the method comprising the administration of a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0215] As described above, a suitable disease that may be targeted in the method and use of the present invention is cancer. This cancer is most preferably characterized by CD30 expression. CD30 expression in cancer can be readily determined using methods known in the art, such as PCR, immunohistochemistry, or FACS analysis, i.e., detection of CD30 transcript and / or protein expression. Antibodies described herein that can bind to human CD30 may be used, for example, in immunohistochemistry and / or FACS analysis.

[0216] In one embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of Hodgkin lymphoma or anaplastic large cell lymphoma.

[0217] In a further embodiment, the present invention relates to a pharmaceutical composition according to the present invention for use in the treatment of Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).

[0218] In one embodiment, Hodgkin lymphoma is classical Hodgkin lymphoma (cHL).

[0219] In one embodiment, non-Hodgkin lymphoma is 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).

[0220] In further embodiments, T-cell non-Hodgkin lymphoma is peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma (CTCL). In even further embodiments, T-cell non-Hodgkin lymphoma (T-NHL) is anaplastic large cell lymphoma (ALCL). In even further embodiments, peripheral T-cell lymphoma (PTCL) is anaplastic large cell lymphoma (ALCL).

[0221] In one embodiment, B-cell non-Hodgkin lymphoma (B-NHL) is mantle cell lymphoma (MCL).

[0222] In further embodiments, Hodgkin lymphoma (HL) is relapsed and refractory Hodgkin lymphoma. In further embodiments, Hodgkin lymphoma (HL) is CD30+ Hodgkin lymphoma. In further embodiments, Hodgkin lymphoma (HL) is relapsed and refractory CD30+ Hodgkin lymphoma. In further embodiments, Hodgkin lymphoma (HL) is relapsed and refractory CD30+ classical Hodgkin lymphoma.

[0223] In further embodiments, non-Hodgkin lymphoma (NHL) is relapsed and refractory non-Hodgkin lymphoma. In further embodiments, non-Hodgkin lymphoma (NHL) is CD30+ non-Hodgkin lymphoma. In further embodiments, non-Hodgkin lymphoma (NHL) is relapsed and refractory CD30+ non-Hodgkin lymphoma.

[0224] In further embodiments, the pharmaceutical compositions according to the present invention for use in the treatment of Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) are administered intravenously and / or subcutaneously, preferably subcutaneously, or have been administered intravenously and / or subcutaneously.

[0225] In a further embodiment, a patient diagnosed with cancer may undergo an evaluation of CD30 expression in cancer cells, and if CD30 is detected, which may range from low to high, such a patient may be selected for treatment with the pharmaceutical composition according to the present invention. However, including such an evaluation when selecting a patient for treatment may not always be a requirement.

[0226] The pharmaceutical compositions of the present invention, comprising multispecific antibodies, have numerous in vitro and in vivo diagnostic and therapeutic uses, including the diagnosis and treatment of disorders involving CD30-expressing cells. For example, multispecific antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to subjects, e.g., in vivo, to treat, prevent and / or diagnose various disorders. As used herein, the term “subject” is intended to include human and non-human individuals.

[0227] In one embodiment, the present invention relates to a diagnostic composition comprising a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0228] In one embodiment, the diagnostic composition is a companion diagnostic used to screen and select patients who would benefit from treatment with the pharmaceutical composition.

[0229] kit The present invention further provides a parts kit comprising a pharmaceutical composition comprising the antibody disclosed above, for example, a kit for use as a companion diagnostic / for identification within a patient population (patients who are likely to respond to treatment with an antibody as defined above herein, or patients who are likely to predict the efficacy or antitumor activity of such antibody when used in the treatment of a patient), the kit comprising the antibody as defined above and instructions for use of the kit. In one embodiment, the parts kit comprises the pharmaceutical composition described herein, a container for the pharmaceutical composition, and instructions for use of the kit. In a further embodiment, the parts kit further comprises a diluent.

[0230] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container for a pharmaceutical composition comprising a multispecific CD3xCD30 antibody and, optionally, one or more reagents for detecting crosslinking of CD30-expressing cells and CD3-expressing cells. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for an enzymatic reaction, the enzymatic reaction producing a product that can be visualized.

[0231] In a further embodiment, the present invention provides a method for detecting whether crosslinking between CD30-expressing cells and CD3-expressing cells occurs in a patient-derived sample upon administration of a pharmaceutical composition comprising any one of the embodiments disclosed herein, comprising a multispecific antibody. (i) A step of contacting a sample with a pharmaceutical composition according to any one embodiment disclosed herein, which includes a multispecific antibody, under conditions that enable the formation of a complex between the bispecific antibody and CD30-expressing cells and a plurality of CD3-expressing cells, (ii) A method comprising the step of analyzing whether a complex has been formed.

[0232] Embodiments of a pharmaceutical composition In the following embodiments, the multispecific antibody or antibody refers to a multispecific antibody that includes an antigen-binding region capable of binding to human CD30 and an antigen-binding region capable of binding to human CD3.

[0233] In one embodiment, the pharmaceutical composition described herein is a) a multispecific antibody in a concentration of approximately 0.5 to 250 mg / ml, a multispecific antibody in a concentration of approximately 20 to 200 mg / ml, b) a buffer, c) a nonionic excipient as needed, and d) a surfactant as needed, comprising or essentially consisting thereof, with a pH of approximately 5.0 to 6.5.

[0234] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 0.5 to 250 mg / ml, a multispecific antibody in a concentration of approximately 20 to 200 mg / ml, b) an acetate or histidine, c) sorbitol, trehalose, or sucrose, and d) a polysorbate, or comprising essentially these, with a pH of approximately 5.0 to 6.5.

[0235] In further embodiments, the pharmaceutical compositions described herein are a) multispecific antibodies in concentrations of approximately 0.5 to 250 mg / ml, approximately 20 to 200 mg / ml, etc., b) histidine, c) sucrose, and d) polysorbate, or comprising essentially these, and the pH of the composition is approximately 6.0.

[0236] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 0.5 to 250 mg / ml, a multispecific antibody in a concentration of approximately 20 to 200 mg / ml, b) an acetate, c) trehalose, and d) a polysorbate, or comprising essentially these, with a pH of approximately 5.5.

[0237] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody at approximately 0.5 to 250 mg / ml, a multispecific antibody at approximately 20 to 200 mg / ml, b) an acetate, c) sorbitol, and d) a polysorbate, or comprising essentially these, and the pH of the composition is approximately 5.5.

[0238] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 60-80 mg / ml, b) histidine, c) sucrose, and d) polysorbate 80, or comprising essentially these, with a pH of approximately 6.0.

[0239] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 60-80 mg / ml, b) an acetate, c) sorbitol, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0240] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 60-80 mg / ml, b) an acetate, c) trehalose, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0241] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 135-165 mg / ml, b) histidine, c) sucrose, and d) polysorbate 80, or comprising essentially these, with a pH of approximately 6.0.

[0242] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 135-165 mg / ml, b) an acetate, c) sorbitol, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0243] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 135-165 mg / ml, b) an acetate, c) trehalose, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0244] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 150-190 mg / ml, b) histidine, c) sucrose, and d) polysorbate 80, or comprising essentially these, with a pH of approximately 6.0.

[0245] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 150-190 mg / ml, b) an acetate, c) sorbitol, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0246] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 150-190 mg / ml, b) an acetate, c) trehalose, and d) polysorbate 80, or essentially consisting thereof, and the pH of the composition is approximately 5.5.

[0247] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody of approximately 0.5 to 250 mg / ml, a multispecific antibody of approximately 20 to 200 mg / ml, b) approximately 5 to 40 mM acetate or histidine, c) approximately 100 to 350 mM sorbitol, trehalose or sucrose, and d) approximately 0.01 to 0.1% w / v polysorbate, or comprising essentially these, and the pH of the composition is approximately 5.0 to approximately 6.5.

[0248] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 60-80 mg / ml of 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, with a pH of approximately 5.5-6.0.

[0249] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 135-165 mg / ml, b) a concentration of approximately 5-40 mM acetate or histidine, preferably acetate, c) a concentration of approximately 100-350 mM sorbitol, trehalose, or sucrose, preferably sorbitol, and d) a concentration of approximately 0.01-0.05% w / v polysorbate 80, with a pH of approximately 5.5-6.0.

[0250] In further embodiments, the pharmaceutical compositions described herein are a) a multispecific antibody in a concentration of approximately 150-190 mg / ml, b) a concentration of approximately 5-40 mM acetate or histidine, preferably acetate, c) a concentration of approximately 100-350 mM sorbitol, trehalose, or sucrose, preferably sorbitol, and d) a concentration of approximately 0.01-0.05% w / v polysorbate 80, with a pH of approximately 5.5-6.0.

[0251] In further embodiments, the pharmaceutical compositions described herein are a) approximately 70 mg / ml of multispecific antibody, b) approximately 20 mM of acetate, c) approximately 250 mM of sorbitol, and d) approximately 0.02% w / v of polysorbate 80, with a pH of approximately 5.5.

[0252] In further embodiments, the pharmaceutical compositions described herein are a) approximately 150 mg / ml of multispecific antibody, b) approximately 20 mM of acetate, c) approximately 250 mM of sorbitol, and d) approximately 0.02% w / v of polysorbate 80, with a pH of approximately 5.5.

[0253] In further embodiments, the pharmaceutical compositions described herein are a) approximately 170 mg / ml of multispecific antibody, b) approximately 20 mM of acetate, c) approximately 250 mM of sorbitol, and d) approximately 0.02% w / v of polysorbate 80, with a pH of approximately 5.5.

[0254] In further embodiments, the pharmaceutical compositions described herein are a) approximately 70 mg / ml of multispecific antibody, b) approximately 17 mM of acetate, sodium acetate, etc., c) approximately 250 mM sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM glacial acetic acid, and the pH of the composition is approximately 5.5.

[0255] In further embodiments, the pharmaceutical compositions described herein are The composition contains or essentially consists of a) approximately 150 mg / ml of multispecific antibody, b) approximately 20 mM of acetate, sodium acetate, etc., c) approximately 250 mM sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM glacial acetic acid, and the pH of the composition is approximately 5.5.

[0256] In further embodiments, the pharmaceutical compositions described herein are The composition contains or essentially consists of a) approximately 170 mg / ml of multispecific antibody, b) approximately 20 mM of acetate, sodium acetate, etc., c) approximately 250 mM sorbitol, d) approximately 0.02% w / v of polysorbate 80, and e) approximately 3 mM glacial acetic acid, and the pH of the composition is approximately 5.5.

[0257] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 60-80 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.05% w / v polysorbate 80, with a pH of approximately 6.0.

[0258] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 60-80 mg / ml of 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, with a pH of approximately 5.5.

[0259] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 60-80 mg / ml of 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, with a pH of approximately 5.5.

[0260] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 135-165 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.05% w / v polysorbate 80, with a pH of approximately 6.0.

[0261] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 135-165 mg / ml of 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, with a pH of approximately 5.5.

[0262] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 135-165 mg / ml of 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, with a pH of approximately 5.5.

[0263] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150-190 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.05% w / v polysorbate 80, with a pH of approximately 6.0.

[0264] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150-190 mg / ml of 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, with a pH of approximately 5.5.

[0265] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150-190 mg / ml of 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, with a pH of approximately 5.5.

[0266] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 70 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.03% w / v polysorbate 80, with a pH of approximately 6.0.

[0267] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 70 mg / ml of 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, with a pH of approximately 5.5.

[0268] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 70 mg / ml of 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, with a pH of approximately 5.5.

[0269] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.03% w / v polysorbate 80, with a pH of approximately 6.0.

[0270] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150 mg / ml of 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, with a pH of approximately 5.5.

[0271] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 150 mg / ml of 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, with a pH of approximately 5.5.

[0272] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 170 mg / ml of multispecific antibody, b) approximately 10-30 mM histidine, c) approximately 200-300 mM sucrose, and optionally d) approximately 0.01-0.03% w / v polysorbate 80, with a pH of approximately 6.0.

[0273] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 170 mg / ml of 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, with a pH of approximately 5.5.

[0274] In further embodiments, the pharmaceutical compositions described herein are The composition comprises or essentially consists of a) approximately 170 mg / ml of 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, with a pH of approximately 5.5.

[0275] In further embodiments, the pharmaceutical compositions described herein are a) Multispecific antibodies with concentrations of approximately 0.5 to 250 mg / ml, approximately 20 to 200 mg / ml, etc., wherein the antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A multispecific antibody comprising a CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs. 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein X in SEQ ID NO. 9 may be H, b) A buffering agent such as histidine or acetate, c) If necessary, a nonionic excipient such as sucrose, trehalose, or sorbitol, d) If necessary, a surfactant such as polysorbate, and a substance containing or essentially consisting thereof The pH of the composition is approximately 5.0 to 6.5.

[0276] In further embodiments, the pharmaceutical compositions described herein are a) A multispecific antibody with a concentration of approximately 0.5 to 250 mg / ml, a multispecific antibody with a concentration of approximately 20 to 200 mg / ml, etc., wherein the multispecific antibody contains or consists of the heavy chain sequence shown in SEQ ID NOs. 17 and 19, and the light chain sequence shown in SEQ ID NOs. 18 and 20, and the multispecific antibody is a bispecific antibody. b) A buffering agent such as histidine or acetate, c) If necessary, a nonionic excipient such as sucrose, trehalose, or sorbitol, d) If necessary, a surfactant such as polysorbate, and a substance containing or essentially consisting thereof The pH of the composition is approximately 5.0 to 6.5.

[0277] In further embodiments, the pharmaceutical compositions described herein are a) Multispecific antibodies with concentrations of approximately 0.5 to 250 mg / ml, approximately 20 to 200 mg / ml, etc., wherein the antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A multispecific antibody comprising a CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs. 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein X in SEQ ID NO. 9 may be H, b) Acetates such as acetates, approximately 10-30 mM, c) If necessary, add sorbitol such as sorbitol in an amount of approximately 200-300 mM, d) If necessary, containing or essentially consisting of a polysorbate such as polysorbate 80 in an amount of approximately 0.01-0.05% w / v. The pH of the composition is approximately 5.5.

[0278] In further embodiments, the pharmaceutical compositions described herein are a) A multispecific antibody with a concentration of approximately 65-75 mg / ml, a multispecific antibody with a concentration of approximately 70 mg / ml, wherein the multispecific antibody contains or consists of the heavy chain sequences shown in SEQ ID NOs. 17 and 19, and the light chain sequences shown in SEQ ID NOs. 18 and 20, and the multispecific antibody is a bispecific antibody. b) A buffering agent such as histidine or acetate, c) If necessary, a nonionic excipient such as sucrose, trehalose, or sorbitol, d) If necessary, a surfactant such as polysorbate, and a substance containing or essentially consisting thereof The pH of the composition is approximately 5.0 to 6.5.

[0279] In further embodiments, the pharmaceutical compositions described herein are a) Multispecific antibodies of approximately 65-75 mg / ml, approximately 70 mg / ml, etc., where the antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A multispecific antibody comprising a CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, wherein X in SEQ ID NO: 9 may be H, b) Acetates such as acetates, approximately 10-30 mM, c) If necessary, add sorbitol such as sorbitol in an amount of approximately 200-300 mM, d) If necessary, containing or essentially consisting of a polysorbate such as polysorbate 80 in an amount of approximately 0.01-0.05% w / v. The pH of the composition is approximately 5.5.

[0280] In further embodiments, the pharmaceutical compositions described herein are a) A multispecific antibody with a concentration of approximately 60-80 mg / ml, wherein the antibody contains or consists of the heavy chain sequences shown in SEQ ID NOs. 17 and 19, and the light chain sequences shown in SEQ ID NOs. 18 and 20, and the multispecific antibody is a bispecific antibody. b) Approximately 10-30 mM acetate, c) Approximately 200-300 mM sorbitol, d) containing or essentially consisting of approximately 0.01-0.05% w / v of polysorbate 80, The pH of the composition is approximately 5.5.

[0281] In further embodiments, the pharmaceutical compositions described herein are a) A multispecific antibody with a concentration of approximately 60-80 mg / ml, wherein the antibody is (i) A CD30 binding region comprising a first heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A multispecific antibody comprising a CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, wherein X in SEQ ID NO: 9 may be H, b) Approximately 10-30 mM acetate, c) Approximately 200-300 mM sorbitol, d) containing or essentially consisting of approximately 0.01-0.05% w / v of polysorbate 80, The pH of the composition is approximately 5.5.

[0282] The present invention will be further illustrated by the following embodiments, but these should not be construed as limiting the scope of the invention. [Examples]

[0283] Example 1-2: Preparation of CD3xCD30 bispecific antibodies by MEA-induced Fab arm exchange. The following antibodies were used in the examples: Humanized CD3 antibody IgG1-huCD3-H1L1 as described in Example 1 of International Publication No. 2015 / 001085 (Genmab). IgG1-huCD3-H1L1 is referred to herein as "IgG1-huCD3".

[0284] IgG1-huCD3-H1L1-H101G as described in Example 2 of International Publication No. 2017 / 009442 (Genmab). IgG1-huCD3-H1L1-H101G is referred to herein as "IgG1-huCD3-H101G".

[0285] CD30 antibody MDX-060, also known as HuMab 5F11, is disclosed in International Publication No. 2003 / 059282 (Medarex). hAC10 (or SGN-30) is disclosed in U.S. Patent No. 8,257,706 and U.S. Patent Application Publication No. 20100239571 (Seattle Genetics). HRS-3 is disclosed in International Publication No. 2016 / 0177846 (Affimed). HeFi-I, T405, T105, T408, and T215 are disclosed in International Publication No. 2007 / 040653 (US Government & Health).

[0286] Antibody expression The antibody sequences were cloned into a pcDNA3.3 expression vector (Invitrogen, USA) and expressed as IgG1,κ or IgG1,λ with or without Fc silencing and / or DuoBody® technology amino acid substitution in the Fc domain (see below). All antibodies were produced under serum-free conditions by co-transfecting Expi293F® cells (Thermo Fisher Scientific, USA; catalog no. A14527) with the relevant heavy and light chain expression vectors using ExpiFectamine® 293 (Thermo Fisher Scientific; catalog no. A14525), essentially as described by the manufacturer.

[0287] Production of bispecific antibodies Bispecific antibodies were produced in vitro using the DuoBody® platform technology, i.e., 2-MEA-induced controlled Fab-arm exchange (cFAE), as described in International Publication Nos. 2011147986, 2011131746, and 2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5:962-973). To enable the production of bispecific antibodies using this method, IgG1 molecules with a single mutation in the CH3 domain were produced: one parental IgG1 antibody with the F405L mutation (i.e., CD3 antibody, or control HIV-1 gp120-specific antibody), and the other parental IgG1 antibody with the K409R mutation (i.e., CD30 antibody or control antibody). In addition to these mutations, the parental IgG1 antibody contained substitutions that resulted in an Fc domain that could not interact with the IgG Fc receptor (Fc gamma receptor) and / or complement factors, such as C1q:L234F, L235E, D265A (FEA; U.S. Patent Application Publication No. 2015 / 0337049) or L234F, L235E, G236R (FER).

[0288] The combination of Fc silencing and DuoBody® technology mutation was named as follows: L234F, L235E, D265A, and F405L: FEAL L234F, L235E, D265A, and K409R: FEAR L234F, L235E, G236R, and K409R: FERR The heavy chain (HC) and light chain (LC) sequences of the parent antibody are shown in the following sequence numbers: IgG1-huCD3-FEAL: SEQ ID NO: 19(HC) and SEQ ID NO: 20(LC).

[0289] IgG1-huCD3-H101G-FEAL: SEQ ID NO: 35(HC) and SEQ ID NO: 20(LC).

[0290] IgG1-CD30-MDX060-FERR: SEQ ID NO: 17(HC) and SEQ ID NO: 18(LC).

[0291] IgG1-CD30-MDX060-FEAR: SEQ ID NO: 55(HC) and SEQ ID NO: 18(LC).

[0292] IgG1-CD30-hAC10-FEAR: SEQ ID NO: 21(HC) and SEQ ID NO: 22(LC).

[0293] IgG1-CD30-HRS-3-FEAR: SEQ ID NOs. 23(HC) and 24(LC).

[0294] IgG1-CD30-HeFi-I-FEAR: SEQ ID NOs. 25(HC) and 26(LC).

[0295] IgG1-CD30-T405-FEAR: Sequence ID 27(HC) and Sequence ID 28(LC).

[0296] IgG1-CD30-T105-FEAR: SEQ ID NOs. 29(HC) and 30(LC).

[0297] IgG1-CD30-T408-FEAR: SEQ ID NO: 31(HC) and SEQ ID NO: 32(LC).

[0298] IgG1-CD30-T215-FEAR: SEQ ID NO: 33(HC) and SEQ ID NO: 34(LC).

[0299] To produce bispecific antibodies, use PBS buffer (phosphate-buffered saline; 8.7 mM HPO4). 2- , 1.8 mM H2PO4 - , 163.9 mM Na + , 140.3 mM Cl - Two parent antibodies were mixed in equal molar ratios in 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 for 5 hours. 2-MEA was removed by dialyzing into PBS buffer using a 10 kDa molecular weight cutoff Slide-A-Lyzer cartridge (Thermo Fisher Scientific) according to the manufacturer's protocol. The samples were stored overnight at 4°C to allow for re-oxidation of the disulfide bonds and formation of intact bispecific antibodies. The efficacy of cFAE was over 95% when evaluated by electrospray ionization mass spectrometry (ESI-MS), as described by Gramer et al. (MAbs. 2013 Nov 1;5(6):962-973.).

[0300] Unbound control antibody b12 IgG1-b12 is an HIV-1 gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23) used as a negative unbound control antibody in some cases. The heavy chain and light chain sequences are included herein as SEQ ID NOs. 36 and 37 (FEAL) or SEQ ID NOs. 38 and 37 (FERR), respectively.

[0301] Example 2 - CD30 expression in human Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), and TLL cell lines 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, catalog number CP010). 4 Cells (per well) were incubated for 30 minutes at 4°C in 50 μL of staining buffer (PBS [Lonza, catalog no. BE17-517Q] supplemented with 0.1% bovine serum albumin [BSA, fraction V, Roche, catalog no. 10735086001] and 0.02% NaN3 [Sigma Aldrich, catalog no. 13412]) in a 96-well round-bottom polystyrene plate (Greiner bio-one, catalog no. 650180) containing 10 μg / mL IgG1-CD30-MDX060-FERR. In parallel, a standard curve was created using the Human IgG Calibrator Kit (Biocytex, catalog no. CP010), essentially following the manufacturer's instructions. Calibration beads containing a clearly defined number of human IgG monoclonal antibodies per bead were incubated with the same R-PE conjugate secondary antibody (Jackson ImmunoResearch, UK; catalog no. 109-116-098; 1:500 dilution), protected from light, at 4°C for 30 minutes. Cells and beads were washed with FACS buffer and analyzed by flow cytometry using a FACSCelesta flow cytometer (BD Biosciences, USA). Using a standard curve obtained with the Human IgG Calibrator Kit, the number of IgG1-CD30-MDX060-FERR antibodies bound per cell (ABC) was interpolated using GraphPad Prism software to represent an estimate of the number of CD30 molecules expressed on the cell surface.

[0302] Tables 4 and 5 show that CD30 expression exceeding the limit of quantification (LLOQ) was observed in all cell lines except SUP-T1. [Table 4] [Table 5]

[0303] Example 3 - Conjugation of CD3xCD30 bispecific antibodies against non-Hodgkin lymphomas (NHL), such as human Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) cells. The binding of CD3xCD30 bispecific antibodies to two CD30-expressing human tumor cell lines, SU-DHL-1 (ALCL;ATCC, catalog number ACC 356) and HDLM-2 (HL;ATCC, catalog number CRL-2965), was analyzed by flow cytometry.

[0304] cells (3×10 4 Cells (per well) were incubated in 50 μL of staining buffer at 4°C for 30 minutes with serial dilutions of antibody (ranging from 0.0046 to 10 μg / mL at 3-fold dilutions) in a polystyrene 96-well round-bottom plate (Greiner bio-one, catalog no. 650180). After washing twice with staining buffer, the cells were incubated with 50 μL of secondary antibody at 4°C for 30 minutes. As the secondary antibody, R-PE conjugate goat anti-human IgG (Jackson ImmunoResearch, UK; catalog no. 109-116-098) diluted 1:400 with staining buffer was used. Next, the cells were washed twice with staining buffer, resuspended in 100 μL of staining buffer supplemented with TO-PRO-3 iodide (Thermo Fisher Scientific; catalog no. T3605; 1:8000 dilution), and analyzed using a FACSCelesta flow cytometer (BD Biosciences, USA). Live cells were gated based on the absence of FSC / SSC and TOPRO-3 staining. Coupled curves were analyzed by nonlinear regression of log-transformed data (variable gradient, sigmoid dose response with four parameters) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0305] result Figure 1 shows the CD3xCD30 bispecific antibodies against SU-DHL-1 (left panel) and HDLM-2 (right panel) tumor cells: bsG1-huCD3-FEALxCD30-MDX060-FEAR (A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (B), bsG1-huCD3-FEALxCD30-HRS-3-FEAR (C), and BsG1-huCD3-FE The dose-response binding curves for ALxCD30-HeFi-I-FEAR(D), bsG1-huCD3-FEALxCD30-T405-FEAR(E), bsG1-huCD3-FEALxCD30-T105-FEAR(F), BisIgG1-huCD3-FEALxCD30-T408-FEAR(G), and bsG1-huCD3-FEALxCD30-T215-FEAR(H) are shown.

[0306] At a concentration of 1.11 μg / mL, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, and bsG1-huCD3-FEALxCD30-T105-FEAR showed similar binding to the single-specific bivalent CD30 parent antibodies IgG1-CD30-MDX060-FEAR, IgG1-CD30-hAC10-FEAR, IgG1-CD30-HRS-3-FEAR, and IgG1-CD30-T105-FEAR (Figure 1I).

[0307] In contrast, at a concentration of 1.11 μg / mL, the binding of bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR to SU-DHL-1 and HDLM-2 cells was lower than that of the single-specific bivalent CD30 parental antibodies IgG1-CD30-T405-FEAR, IgG1-CD30-T408-FEAR, and IgG1-CD30-T215-FEAR (Figure 1I).

[0308] Overall, the binding of bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, BsG1-huCD3-FEALxCD30-HeFi-I-FEAR, and bsG1-huCD3-FEALxCD30-T105-FEAR at a concentration of 1.11 μg / mL was higher than that of bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR at the same concentration (Figure 1I).

[0309] The negative control antibody BsG1-huCD3-FEALxb12-FEAR included in these experiments did not bind to SU-DHL-1 cells or HDLM-2 cells, indicating that neither SU-DHL-1 cells nor HDLM-2 cells express CD3.

[0310] In conclusion, CD30 antibody clones T405, T408, and T215 showed reduced binding in monovalent format compared to bivalent format, while clones MDX060, hAC10, HRS-3, and T105 showed efficient binding to CD30-expressing tumor cells in both monovalent and bivalent formats.

[0311] Figure 2 shows the dose-response binding curves of bsG1-huCD3-FEALxCD30-MDX060-FERR for (A) HDLM-2(HL) cells, (B) L-428(HL) cells, (C) DEL(ALCL) cells, and (D) KI-JK(ALCL) cells. BsG1-huCD3-FEALxCD30-MDX060-FERR showed similar maximum binding compared to the monospecific bivalent CD30 parent antibody IgG1-CD30-MDX060-FERR. The negative control antibodies bsG1-huCD3-FEALxb12-FEAR, IgG1-huCD3-FEAL, and IgG1-b12-FEAL did not bind 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-FEER to HL and ALCL cell lines.

[0312] Table 6 shows the EC results for the binding of bsG1-huCD3-FEALxCD30-MDX060-FEAR to HDLM-2, L-428, DEL, and KI-JK cells, as evaluated in two independent experiments. 50 Shows the value. EC 50 The values ​​ranged from 0.05 to 0.30 μg / mL. [Table 6]

[0313] Figures 8, 9, and 10 show the dose-response binding curves of bsG1-huCD3-FEALxCD30-MDX060-FERR for CD30-expressing HL cell lines (Figure 8), ALCL cell lines (Figure 9), and NHL cell lines (Figure 10). In all cell lines, BsG1-huCD3-FEALxCD30-MDX060-FERR showed similar maximum binding compared to the monovalent control antibody BsG1-b12-FEALxCD30-MDX060-FERR. The monospecific bivalent CD30 parent antibody IgG1-CD30-MDX060-FERR showed dose-dependent binding to all cell lines, but exhibited lower maximum binding compared to BsG1-huCD3-FEALxCD30-MDX060-FERR. The negative control antibodies bsG1-huCD3-FEALxb12-FEAR, IgG1-huCD3-FEAL, and IgG1-b12-FEAL did not bind to any of these cell lines, indicating that these cells do not express CD3 on their cell surface. These data confirm the efficient binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HL cell lines, T-NHL (ALCL and CTCL cell lines, etc.), and B-NHL (MCL cell lines, etc.).

[0314] Table 7 shows the EC binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to HL, T-NHL, and B-NHL cell lines as evaluated in two or three independent experiments. 50 Shows the value. EC 50 The values ​​ranged from 0.12 to 0.35 μg / mL. [Table 7]

[0315] Example 4 - In vitro induction of T cell-mediated cytotoxicity and T cell proliferation using CD3xCD30 bispecific antibodies. 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. CD3-dependent tumor cell killing was evaluated using CD3-positive ADCC effector cells type IV (Clean Cells, Montaigu, France) or purified T cells (described in Example 5) as the T cell source.

[0316] SU-DHL-1 (ALCL), HuT78 (ALCL), HDLM-2 (HL), NCEB-1 (MCL), or L540 (HL) cells were seeded at a density of 10,000 cells / well in 96-well round-bottom polystyrene plates (Greiner bio-one, catalog number 650180). Effector cells were labeled with 0.5 μM CFSE (carboxyfluorescein succinimimidyl ester; Cell Signalling Technology, Danvers, Massachusetts; catalog number C34554) 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). Cells were incubated at 37°C for 72 hours with serial dilutions of bispecific CD3xCD30, b12xCD30, or CD3xb12 antibodies, or monospecific bivalent CD30 antibodies (final concentrations ranging from 10 to 0.041 μg / mL; 3-fold dilution). In some experiments, the bsG1-huCD3-FEALxCD30-MDX060-FEAR mutant was used with a CD3-binding arm containing the H101G mutation, which reduces affinity for CD3 (International Publication No. 2017 / 009442, Genmab). After washing twice with 100 μL of staining buffer, cells were resuspended in staining buffer containing TO-PRO-3 iodide (Thermo Fisher Scientific; catalog no. T3605; 1:4000 dilution) and analyzed using a FACSCelesta flow cytometer (BD Biosciences, USA).

[0317] The viability of tumor cell samples treated with 5 μM staurosporine (Sigma-Aldrich, USA, catalog number S6942) was set to 0%, and the viability of untreated tumor cell samples was set to 100%.

[0318] The "percentage of living cells" was calculated as follows: Surviving cells % = ([Cell count of sample - Cell count of staurosporin-treated target cells] / [Cell count of untreated target cells - Cell count of staurosporin-treated target cells]) × 100.

[0319] CFSE-positive cells were counted as a measure of the absolute number of T cells, and T cell proliferation was evaluated.

[0320] The dose-response curves were analyzed using nonlinear regression (sigmoid dose-response with variable gradient) with GraphPad Prism V8 software (GraphPad Software, San Diego, California, USA).

[0321] result CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30-positive tumor cell lines SU-DHL-1 or HDLM-2 cells as target cells and ADCC effector cell type IV cells (Clean Cells, Montaigu, France) as effector cells.

[0322] Figure 3 shows bsG1-huCD3-FEALxCD30-MDX060-FEAR (A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (B), bsG1-huCD3-FEAL xCD30-HRS-3-FEAR(C), BsG1-huCD3-FEALxCD30-HeFi-I-FEAR(D), bsG1-huCD3-FEALxCD30-T405-FEAR(E), The images show that bsG1-huCD3-FEALxCD30-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 viable cell percentage) in SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel).

[0323] The single-specific bivalent CD30 antibodies IgG1-MDX060-FEAR(A), IgG1-CD30-hAC10-FEAR(B), IgG1-CD30-HRS-3-FEAR(C), IgG1-CD30-HeFi-I-FEAR(D), IgG1-CD30-T405-FEAR(E), IgG1-CD30-T105-FEAR(F), IgG1-CD30-T408-FEAR(G), and IgG1-CD30-T215-FEAR(H) did not induce T cell-mediated cytotoxicity. The control antibody bsG1-huCD3-FEALxb12-FEAR also did not induce T cell-mediated cytotoxicity in SU-DHL-1 or HDLM-2 cells.

[0324] Furthermore, the CD3xCD30 bispecific antibody was tested in an in vitro cytotoxicity assay using different MCL, ALCL, and HL cell lines as target cells and purified T cells or ADCC effector cell type IV cells as effector cells. Figures 4A-B show that T cell-mediated cytotoxicity of SU-DHL-1, HuT78, or NCEB-1 cells induced by bsG1-huCD3-FEALxCD30-MDX060-FEAR is more potent compared to a variant of this antibody (bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR) with a reduced affinity CD3 binding arm. Similar maximum T cell-mediated cytotoxicity of HDLM-2 cells was induced by both bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR (Figure 4B). Incubation with the bivalent monospecific antibodies IgG1-huCD3-FEAL or IgG1-b12-FEAR, included as controls, did not induce cytotoxicity in these cell lines. Potent and similar T cell-mediated cytotoxicity in L540 cells was induced at the lowest concentration tested (0.014 μg / mL; Figure 4C) by bsG1-huCD3-FEALxCD30-MDX060-FEAR and bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR. The control antibodies bsG1-b12-FEALxCD30-MDX060-FEAR or IgG1-MDX060-FEAR did not induce T cell-mediated cytotoxicity in L540 cells.

[0325] In conclusion, bsG1-huCD3-FEALxCD30-MDX060-FEAR induced potent killing of various CD30-expressing MCL, ALCL, and HL tumor cell lines. BsG1-huCD3-FEALxCD30-MDX060-FEAR, which contains H at position 101 of the VH CDR3 in the CD3 arm, is more potent in killing CD30-expressing MCL and ALCL tumor cells compared to mutants with a low-affinity CD3 arm containing G at position 101.

[0326] 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 evaluated as a measure of absolute T cell count. Figure 4D shows that T cell-mediated cytotoxicity of HDLM-2 and NCEB-1 cells induced by bsG1-huCD3-FEALxCD30-MDX060-FEAR or bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR (see Figure 4B) was associated with a dose-dependent increase in T cell count. Generally, in this assay, similar T cell counts were counted after incubation with bsG1-huCD3-FEALxCD30-MDX060-FEAR or bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR. In co-cultures of NCEB-1 cells incubated with bsG1-huCD3-FEALxCD30-MDX060-FEAR at concentrations higher than 1 μg / mL, a decrease in T cell count was observed. The control antibody IgG1-b12-FEAL did not affect T cell count in these experiments.

[0327] In conclusion, BsG1-huCD3-FEALxCD30-MDX060-FEAR induced T cell proliferation in the presence of various CD30-expressing ALCL, HL, and MCL tumor cell lines.

[0328] Example 5 - Conjugation of CD3xCD30 bispecific antibodies to human CD30, cynomolgus monkey CD30, or rhesus monkey CD30 expressed in Expi293F cells. The binding of bispecific CD3xCD30 antibodies and monospecific bivalent CD30 antibodies to the plasma membrane of Expi293 cells transiently transfected with human CD30 or cynomolgus monkey CD30 was analyzed by flow cytometry.

[0329] Transient expression of human, cynomolgus monkey, or rhesus monkey CD30 in HEK-293F or HEK-293 cells The following codon-optimized constructs were generated for the expression of various full-length CD30 mutants: human (Homo sapiens) CD30 (huCD30; Uniprot accession number P28908), cynomolgus monkey (Macaca fascicularis) CD30 (mfCD30; Uniprot accession number A0A2K5VW07) (SEQ ID NO: 40), and rhesus monkey (Macaca mulatta) CD30 (mmCD30; Uniprot accession number A0A1D5RK03) (SEQ ID NO: 41). The constructs contained restriction sites suitable for cloning and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999; 234(2): 187-208). Full-length human CD30, cynomolgus monkey, and rhesus monkey CD30 codon-optimized constructs were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen) and expressed using the Expi293F expression platform (Thermo Fisher Scientific, Waltham, Massachusetts, USA, catalog number A14527) essentially as described by the manufacturer. In a separate series of experiments, full-length human CD30 or cynomolgus monkey CD30 constructs were expressed in HEK-293 cells.

[0330] Conjugation of CD3xCD30 bispecific antibodies to human, cynomolgus monkey, or rhesus monkey CD30 expressed in Expi293 cells. cells (3×10 4Cells (per well) were incubated for 30 minutes at 4°C in 100 μL of staining buffer containing serially diluted antibody (ranging from 0.005 to 10 μg / mL in a 3-fold dilution) in a 96-well polystyrene round-bottom plate (Greiner bio-one, catalog no. 650180). The experiment was performed in technical double-dose. After washing twice with staining buffer, cells were incubated for 30 minutes at 4°C in 50 μL of secondary antibody. As the secondary antibody, R-PE conjugated goat anti-human IgG (Jackson ImmunoResearch, UK; catalog no. 109-116-098) diluted 1:400 with staining buffer was used. Cells were washed twice with staining buffer, resuspended in 30 μL of staining buffer containing 0.4% EDTA, and analyzed using an iQue Screener (Intellicyt Corporation, USA). We analyzed the coupled curves using nonlinear regression (sigmoid dose-response with variable gradient) with GraphPad Prism V 9.0.0 software (GraphPad Software, San Diego, California, USA).

[0331] Conjugation of CD3xCD30 bispecific antibodies to human, cynomolgus monkey, or rhesus monkey CD30 expressed in HEK293 cells. cells (3×10 4Cells (per well) were incubated in 50 μL of staining buffer containing serially diluted antibody (ranging from 0.0002 to 50 μg / mL at 4-fold dilution) in a 96-well polystyrene round-bottom plate (Thermo Scientific, catalog no. 163320) for 30 minutes at 4°C. The experiment was performed in technical double-row. After washing twice with staining buffer, cells were incubated in 50 μL of secondary antibody at 4°C for 30 minutes. As the secondary antibody, R-PE conjugated goat anti-human IgG (Jackson ImmunoResearch, UK; catalog no. 109-116-098) diluted 1:200 with staining buffer was used. Cells were washed twice with staining buffer and resuspended in 30 μL of staining buffer containing 0.4% EDTA and a 1:10,000 diluted ToPro-3 survival marker (Invitrogen, catalog no. T3605). Cells were analyzed using iQue Screener (Intellicyt Corporation, USA). Coupled curves were analyzed using nonlinear regression (sigmoid dose-response with variable gradient) with GraphPad Prism V 9.0.0 software (GraphPad Software, San Diego, California, USA).

[0332] Binding of CD3xCD30 bispecific antibodies to human T cells or cynomolgus monkey PBMCs. Cynomolgus monkey PBMCs (Tebu-Bio; Netherlands, catalog number PBMCMFA-10) or purified human T cells were seeded in 96-well round-bottom polystyrene plates. The T cells were derived from human donor buffy coats (Sanquin, Amsterdam, Netherlands) and isolated using RosetteSep human T cell enrichment cocktail (Stemcell Technologies, France, catalog number 15061) according to the manufacturer's instructions. (3 × 10⁶ cells) 4Cells (per well) were incubated in 50 μL of staining buffer at 4°C for 30 minutes with serial dilutions of the antibodies IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR (ranging from 0.0001 to 10 μg / mL at 3-fold dilution). After washing twice with staining buffer, the cells were incubated in 50 μL of secondary R-PE conjugate-conjugated goat anti-human IgG antibody at 4°C for 30 minutes (1:400 dilution). After washing twice with staining buffer, T cells were stained for the T cell markers CD3 (1:100; Miltenyi biotec, clone 10D12, conjugated to APC), CD4 (1:50; eBioscience, clone OKT4, conjugated to APC-Cy7), CD8 (1:100; Biolegend, clone RPA-T8, conjugated to AF700), and the 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-stained samples using Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42) were used for flow cytometer calibration. After incubation at 4°C for 30 minutes, cells were washed twice with staining buffer, resuspended in 100 μL of staining buffer, and analyzed using FACS Fortessa (BD Biosciences). Data were processed using FlowJo (BD Biosciences).

[0333] result The CD30-targeted bispecific antibodies bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR, and bsG1-b12-FEALxCD30-MDX060-FEAR did not bind to wild-type Expi293F cells (Figure 5A), but showed dose-dependent binding to Expi293F cells transfected with huCD30 (Figure 5B) or mfCD30 (Figure 5C). The binding of these bispecific antibodies was comparable to that of the monospecific bivalent CD30 antibody IgG1-CD30-MDX060-FEAR. As expected, the negative control antibody bsG1-huCD3-FEALxb12-FEAR did not bind to wild-type or huCD30 or mfCD30 transfected Expi293F cells. Similarly, bsG1-huCD3-FEALxCD30-MDX060-FERR showed dose-dependent binding to HEK293 cells transfected with huCD30 (Figure 11A) or HEK293 cells transfected with mfCD30 (Figure 11B), while the negative control antibody bsG1-huCD3-FEALxb12-FERR did not show binding to HEK293 cells transfected with huCD30 or mfCD30. This indicates that the CD30-targeted antibody MDX060 efficiently binds to both human CD30 and cynomolgus monkey CD30 expressed in HEK cells in both bivalent and monovalent forms.

[0334] Figure 5D shows that the CD3xCD30 bispecific antibody bsG1-huCD3-FEALxCD30-MDX060-FEAR and the control bispecific antibody bsG1-huCD3-FEALxb12-FEAR efficiently bound to primary human and cynomolgus monkey T cells. This indicates that these CD3-targeted bispecific antibodies efficiently bind to both endogenously expressed human and cynomolgus monkey CD3 on T cells. The bivalent parent antibody IgG1-CD30-MDX060 did not bind to human or cynomolgus monkey T cells, indicating that CD30 was not expressed on these cells.

[0335] The binding of a panel of CD3xCD30 bispecific antibodies and monospecific parental CD30 antibodies to Expi293F cells transfected with huCD30 or mmCD30 was evaluated by flow cytometry. The CD3xCD30 bispecific antibodies bsG1-huCD3-FEALxCD30-MDX060-FEAR (Figure 6A), bsG1-huCD3-FEALxCD30-hAC10-FEAR (Figure 6B), bsG1-huCD3-FEALxCD30-HRS-3-FEAR (Figure 6C), bsG1-huCD3-FEALxCD30-T405-FEAR (Figure 6E), bsG1-huCD3-FEALxCD30-T105-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, parental monospecific CD30 antibody clones showed equal binding to cells expressing either huCD30 or mmCD30. In contrast, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR and parental monospecific CD30 antibody IgG1-CD30-HeFi-I_FEAR showed binding to huCD30 but not to mmCD30 (Figure 6D).

[0336] Example 6 - Evaluation of conformational stability of single-specificity and bispecificity inactivated antibody variants by DSF analysis The protein stability characteristics of bivalent monospecific CD30, CD3, and bispecific CD3xCD30 IgG1 antibody mutants with deactivating mutations in the constant heavy chain region were evaluated using differential scanning fluorescence (DSF).

[0337] Samples of IgG1-CD30-MDX060-FEAR, IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and BsG1-huCD3-FEALxCD30-MDX060-FERR were formulated in PBS pH 7.4 at a concentration of approximately 1 mg / mL.

[0338] To evaluate conformational stability, DSF was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad), which can detect changes in fluorescence intensity caused by the binding of the exogenous dye Sypro-Orange (5000-fold concentrate in DMSO, catalog no. S5692, Sigma-Aldrich) to the hydrophobic regions exposed during IgG unfolding. Sypro-Orange was diluted 320-fold in PBS pH 7.4 (Hyclone GE Healthcare). The thermal fusion curve can be derived by measuring the increase in fluorescence during controlled, stepwise thermal denaturation of the analyzed IgG. Therefore, 5 μL of replicated antibody solution (1 mg / mL in PBS) was added to 20 μL of diluted Sypro-Orange in PBS pH 7.4 in an iQ 96-well PCR plate. Fluorescence was recorded in stepwise increments of 0.5°C / increment and with a duration of 15 seconds + the time required to record fluorescence in all wells, with increasing temperatures ranging from 25°C to 95°C. The data was analyzed using Bio-Rad CFX Manager Software 3.0, and the melting point was determined from the fluorescence-to-temperature graph by the software.

[0339] result Figure 7 and Table 8 show the melting temperature (T) of IgG1-CD30-MDX060-FERR. m The temperature was 69.0°C, which corresponds to the T of IgG1-CD30-MDX060-FEAR at pH 7.4 (64.5°C). m This indicates that it is higher than IgG1-CD30-MDX060-FERR has higher conformational stability than IgG1-CD30-MDX060-FEAR, suggesting that IgG1-CD30-MDX060 containing the FER skeleton has higher conformational stability than IgG1-CD30-MDX060 containing the FEA skeleton. The melting temperature of BsG1-huCD3-FEALxCD30-MDX060-FERR was determined to be 64.5°C, which is higher than the T values ​​determined for the two parent antibodies IgG1-huCD3-FEAL (62.5°C) and IgG1-CD30-MDX060-FERR (69.0°C). mIt is between these two points. [Table 8]

[0340] Example 7 - Simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to T cells and tumor cells We studied the simultaneous binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to tumor cells and naive T cells.

[0341] Frozen T cells isolated from healthy donors were thawed and labeled with 0.25 mM Celltrace Violet (Pacific Blue; Invitrogen, catalog number C34557A) at 37°C for 15 minutes. L-428 tumor cells were labeled with Celltrace FarRed (APC; Invitrogen catalog number C34564A) at 37°C for 15 minutes and added to the T cells in a 1:1 E:T ratio. Serial dilutions of bsG1-huCD3-FEALxCD30-MDX060-FERR or the control antibody bsG1-huCD3-FEALxb12-FEAR, bsG1b12FEALxCD30MDX060-FERR, or IgG1-b12-FEAL were added (6 × 10⁻¹⁰ -5 Cells were incubated at 4°C for 2 hours with a final concentration in the range of ~10 μg / mL (3-fold dilution). After incubation, the survival marker 7-AAD (BD Bioscience, catalog no. 559925) was added (100-fold final dilution), and the cells were analyzed using a FACS Celesta flow cytometer (BD Biosciences).

[0342] result Figure 12 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR is a measure of bsG1-huCD3-FEALxCD30-MDX060-FERR mediated cross-linking (co-binding) of tumor cells to T cells. + CD30 +This demonstrates the induction of double-positive events (cells expressing both CellTrace Far Red and CellTrace Violet on flow cytometry staining). The increase in double-positive events was antibody concentration-dependent and showed a bell-shaped curve. Increased crosslinking of tumor cells and T cells was not observed in samples incubated with the control antibodies bsG1-huCD3-FEALxb12-FEAR, bsG1b12FEALxCD30MDX060-FERR, or IgG1-b12-FEAL, or in samples incubated without the antibody (A). Figure 12B shows the co-binding of bsG1-huCD3-FEALxCD30-MDX060-FERR to tumor cells and naive T cells, detected by the percentage of cells expressing both CellTrace Far Red and CellTrace Violet (B).

[0343] These data are bsG1huCD3FEALxCD30-MDX060-FERR CD30 + Tumor cells and CD3 + This demonstrates that it can simultaneously bind to and crosslink T cells.

[0344] Example 8 - In vitro induction of T cell-mediated cytotoxicity and T cell activation by CD3xCD30 bispecific antibodies We evaluated T cell-mediated cytotoxicity in Karpas-299 tumor cells and associated T cell activation using a panel of CD3xCD30 bispecific antibodies. The following antibodies were evaluated: bsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR.

[0345] T cells were obtained from healthy human donors (Buffycoat, Sanquin, Amsterdam, Netherlands) and isolated using the RosetteSep (Stemcell Technologies, France, catalog number 15061) human T cell enrichment cocktail according to the manufacturer's instructions. The T cells were labeled with Celltrace Violet (Invitrogen, catalog number C34557A; final concentration 5 μM) at 37°C for 15 minutes. In parallel, Karpas-299 tumor cells were labeled with Celltrace FarRed (Invitrogen, catalog number C34564A; final concentration 2 μM) at 37°C for 15 minutes. After labeling, 5 × volume of ice-cold DBSI was added and incubated at room temperature for 5 minutes. The cells were pelleted, resuspended in culture medium, and tumor cells were seeded at a density of 50,000 cells / well in 96-well plates (Greiner-bio-one, Netherlands, catalog number 655180). Serial dilutions of bispecific CD3xCD30 antibody were added (final concentration ranging from 1,000 to 0.051 ng / mL; 3-fold dilution), and the plates were incubated at room temperature for 15 minutes. T cells were added to tumor cells in an effector-to-target (E:T) ratio of 4:1, and the plates were incubated at 37°C for 72 hours. After washing twice with PBS / 0.1% BSA / 0.02% azide (staining buffer), the cells were stained with the T cell markers CD4 (1:50; Biolegend, catalog number 300521, conjugated to Pacific Blue), CD8 (1:100; BD Biosciences, conjugated to FITC), and the T cell activation markers CD69 (1:50; Biolegend, catalog number 310934, conjugated to BV650), CD25 (1:100; Invitrogen, catalog number 25-0259-42, conjugated to PE-Cy7), and CD279 / PD-1 (1:50; Biolegend, catalog number 329930, conjugated to BV605). The flow cytometer was calibrated using a single-stained sample containing Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42).After incubation at 4°C for 30 minutes, the plates were washed twice with staining buffer, and the cells were stained with 7-AAD (diluted 1:100 with staining buffer) at 4°C for 10 minutes. The cells were analyzed using FACS Celesta (BD Biosciences). The data were processed using FlowJo (BD Biosciences).

[0346] Dose-response curves were created using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0347] result Figures 13A and 13B show that all CD3xCD30 antibodies induce T cell-mediated cytotoxicity in Karpas-299 cells. CD3xCD30 bispecific antibodies produced using CD30 clone MDX060 (bsG1-huCD3-FEALxCD30-MDX060-FERR and bsG1-huCD3-FEALxCD30-MDX060-FEAR) were more effective in killing Karpas-299 cells compared to all other clones tested. Indeed, CD3xCD30 bispecific antibodies based on MDX060 showed significantly lower IC50 compared to CD3xCD30 bispecific antibodies produced using CD30 clones HRS-3, HeFi-I, T105, T405, T408, or T215. 50The values ​​are shown (bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T105-FEAR, bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR; Figure 13A). Furthermore, bsG1-huCD3-FEALxCD30-MDX060-FERR and bsG1-huCD3-FEALxCD30-MDX060-FEAR induced higher maximal killing compared to CD3xCD30 bispecific antibodies prepared using CD30 clones hAC10, HeFi-I, T405, T408, or T215 (bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR; Figure 13B). Figures 13C and D show CD4 + T cell (Figure 13C) or CD8 + As a measure of T cell activation in T cells (Figure 13D), bsG1-huCD3-FEALxCD30-MDX060-FERR and bsG1-huCD3-FEALxCD30-MDX060-FEAR were more effective than any other CD3xCD30 bispecific antibody in inducing CD25 expression (EC 50 This indicates that the values ​​were low. Similar results were observed for the expression of PD-1 (Figure 13E and F) and CD69 (data not shown). No difference in T cell-mediated killing or T cell activation was observed between the two MDX060-based CD3xCD30 bispecific antibodies containing FEAR or FERR mutations.

[0348] The bispecific antibody bsG1-huCD3xCD30-MX060 was also more effective in inducing T cell-mediated cytotoxicity in L-428 cells compared to other CD3xCD30 bispecific antibodies tested in the panel (data not shown).

[0349] Mean IC of T cell-mediated cytotoxicity induced by the panel of CD3xCD30 bispecific antibodies used in these experiments 50 Concentration, maximum solubility, and EC of T cell activation 50 The concentrations (CD25 expression) are summarized in Table 9.

[0350] In conclusion, these data demonstrate that bsG1huCD3xCD30-MDX060 is more effective than any other CD3xCD30 bispecific antibodies evaluated in inducing T cell-mediated cytotoxicity. [Table 9]

[0351] Example 9: In vitro induction of T cell-mediated cytotoxicity, T cell proliferation, and T cell activation by -bsG1-huCD3-FEALxCD30-MDX060-FERR T cell-mediated cytotoxicity of tumor cells, as well as related T cell proliferation and activation by bsG1-huCD3-FEALxCD30-MDX060-FERR, were evaluated in HL and ALCL cell lines.

[0352] T cells were obtained from healthy human donor Buffycoat (Sanquin, Amsterdam, Netherlands) and isolated using the RosetteSep (Stemcell Technologies, France, catalog number 15061) human T cell enrichment cocktail according to the manufacturer's instructions. The T cells were labeled with Celltrace Violet (Invitrogen, catalog number C34557A; final concentration 5 μM) at 37°C for 15 minutes. In parallel, tumor cells L-428, KI-JK, KM-H2, or SUP-M2 were labeled with Celltrace FarRed (Invitrogen, catalog number C34564A; final concentration 2 μM) at 37°C for 15 minutes. After labeling, 5 × volume of ice-cold DBSI was added and incubated at room temperature for 5 minutes. Cells were pelleted, resuspended in culture medium, and seeded at a density of 50,000 cells / well in 96-well plates (Greiner-bio-one, Netherlands, catalog number 655180). Serial dilutions of bsG1-huCD3-FEALxCD30-MDX060-FERR or control antibodies IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, and IgG1-b12-FEAL were added (final concentrations ranging from 1,000 to 0.051 ng / mL; 3-fold dilution), and the plates were incubated at room temperature for 15 minutes. T cells were added to the tumor cells in an effector-to-target (E:T) ratio of 4:1, and the plates were incubated at 37°C for 72 hours.After washing twice with PBS / 0.1% BSA / 0.02% azide (staining buffer), the cells were stained with the T cell markers CD4 (1:50; Biolegend, catalog number 300521, conjugated to Pacific Blue), CD8 (1:100; BD Biosciences, catalog number 345772, conjugated to FITC), and the T cell activation markers CD69 (1:50; Biolegend, catalog number 310934, conjugated to BV650), CD25 (1:100; Invitrogen, catalog number 25-0259-42, conjugated to PE-Cy7), and CD279 / PD-1 (1:50; Biolegend, catalog number 329930, conjugated to BV605). A single-stained sample containing Ultracomp beads (5 μL; Invitrogen, catalog no. 01-2222-42) was used for calibrating the flow cytometer. After incubation at 4°C for 30 minutes, the plate was washed twice with staining buffer, and the cells were stained with 7-AAD (diluted 1:100 with staining buffer) at 4°C for 10 minutes. The cells were analyzed using FACS Celesta (BD Biosciences), and the data were processed using FlowJo (BD Biosciences).

[0353] The percentage of live target cells was calculated using the following formula: % Live target cells = (Absolute number of live single Celltrace FarRed-labeled cells under each condition / Absolute number of live single Celltrace FarRed-labeled cells under the condition containing only target cells and T cells without antibody addition) × 100.

[0354] T cell proliferation, CD4 + or CD8 + T cells were evaluated by gating with diluted Celltrace Violet staining. The proliferation index was calculated using FlowJo's proliferation modeling tool. The generated peaks were automatically fitted, and the proliferation index value was calculated according to the following formula: Proliferation index = total cell volume / cell volume at the start of culture = (G0+G1+G2+G3+G4+G5+G6) / (G0+G1:2+G2:4+G3:8+G4:16+G5:32+G6:64).

[0355] Gn = number of cells at generation n peak (n = 0 to 6).

[0356] Dose-response curves were created using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0357] result Figure 14 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR induced dose-dependent T cell-mediated cytotoxicity in vitro in L-428(HL), KM-H2(HL), SUP-M2(ALCL), and KI-JK(ALCL) cell lines. Table 10 summarizes the mean IC50 concentrations of T cell-mediated cytotoxicity induced by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cells. No cytotoxicity was observed in cells incubated with the control antibodies IgG1-huCD3-FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, or IgG1-b12-FEAL, or in samples incubated without the antibodies.

[0358] Figures 15, 16, 17, and 18 show that T cell-mediated cytotoxicity of L-428 and KI-JK cells induced by bsG1-huCD3-FEALxCD30-MDX060-FERR is mediated by CD4 + and CD8 +This demonstrates association with T cell proliferation (Figure 15) and the expression of T cell activation markers CD69 (Figure 16), CD25 (Figure 17), and PD-1 (Figure 18). The mean EC50 concentrations of T cell proliferation and activation induced by bsG1-huCD3-FEALxCD30-MDX060-FERR in these experiments are summarized in Table 10.

[0359] Therefore, bsG1-huCD3-FEALxCD30-MDX060-FERR induced dose-dependent T cell-mediated cytotoxicity in HL and ALCL cell lines in vitro, which was associated with T cell proliferation and activation. [Table 10]

[0360] Example 10: In vitro induction of cytokine production using bsG1-huCD3-FEALxCD30-MDX060-FERR Cytokine and granzyme B production induced by bsG1-huCD3-FEALxCD30-MDX060-FERR was evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments 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 concentrations of 14 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, PDL-1, TNFα) and granzyme B were measured using a bead-based multiplex immunoassay (Luminex) custom-made by the R&D System.

[0361] result In the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR, increases in the concentrations of granzyme B and cytokines IFNγ, IL-13, and TNFα (>2000 pg / mL) were mainly observed in the supernatant from co-cultures of L-428 cells and T cells. Compared to the control antibody IgG1-b12-FEAL, moderate increases were observed in the concentrations of CD40, IL-10, IL-12, IL-1β, IL-2, IL-4, IL-6, and IP-10 cytokines. Levels of IL-8, MCP-1, and PDL1 were not regulated compared to the control antibody IgG1-b12-FEAL (Figure 19).

[0362] Therefore, T cell-mediated cytotoxicity and T cell activation mediated by bsG1-huCD3-FEALxCD30-MDX060-FERR were associated with dose-dependent production of cytokines and granzyme B.

[0363] Example 11 - In vitro induction of T cell-mediated cytotoxicity using purified T cells as effector cells at various effector-to-target ratios with bsG1-huCD3-FEALxCD30-MDX060-FERR To determine the optimal effector-to-target cell ratio for T cell-mediated tumor cell killing in the presence of the bispecific antibody bsG1-huCD3-FEALxCD30-MDX060-FERR, in vitro cytotoxicity assays were performed at various effector-to-target cell (E:T) ratios using the CD30-positive tumor cell line L-428 as the target cell and purified T cells as the effector cells.

[0364] T cell-mediated cytotoxicity was evaluated essentially as described in Example 9, except that T cells were added to tumor cells in various effector-to-target (E:T) cell ratios of 1:1, 2:1, 4:1, or 8:1.

[0365] result Figure 20A shows that dose-dependent T cell-mediated cytotoxicity was induced by bsG1-huCD3-FEALxCD30-MDX060-FERR at all E:T ratios, with the greatest tumor cell killing (less than 20% surviving tumor cells) observed at E:T ratios of 4:1 and 8:1. In consistency with this, CD4 + and CD8 + T cell proliferation was observed at all E:T cell ratios, most notably at 4:1 and 8:1 E:T ratios (Figure 20B-C). Specific T cell-mediated cytotoxicity or T cell proliferation was not induced by the control antibody bsG1-huCD3-FEALxb12-FERR at any of the E:T ratios tested.

[0366] In summary, these data indicate that bsG1-huCD3-FEALxCD30-MDX060-FERR-induced T cell-mediated cytotoxicity of L-428 tumor cells in vitro was most effective with E:T ratios of 4:1 and 8:1.

[0367] Example 12: Dynamics of T cell-mediated cytotoxicity and T cell proliferation in vitro using bsG1-huCD3-FEALxCD30-MDX060-FERR To evaluate the dynamics of T cell-mediated tumor cell killing in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR, in vitro cytotoxicity assays were performed at various incubation periods using the CD30-positive tumor cell line L-428 as the target cell and purified T cells as the effector cells.

[0368] Except for evaluating tumor cell injury and T cell proliferation at 24, 48, and 72 hours, T cell-mediated cytotoxicity was evaluated essentially as described in Example 9.

[0369] result Figure 21 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR induced dose-dependent T cell-mediated cytotoxicity at 48 and 72 hours, but no significant T cell-mediated cytotoxicity was observed at 24 hours. Dose-dependent CD4+ and CD8 + T cell proliferation was induced by bsG1-huCD3-FEALxCD30-MDX060-FERR after 72 hours, but no T cell proliferation was observed after 24 or 48 hours (Figures 21B and C).

[0370] In summary, these data indicate that bsG1-huCD3-FEALxCD30-MDX060-FERR induced T cell-mediated cytotoxicity and T cell proliferation in tumor cells in a time-dependent manner.

[0371] Example 13: In vitro correlation between CD30 expression levels and bsG1-huCD3-FEALxCD30-MDX060-FERR-induced T cell-mediated cytotoxicity. T cell-mediated killing of eight CD30-expressing tumor cell lines by bsG1-huCD3-FEALxCD30-MDX060-FERR was determined using an in vitro cytotoxicity assay described in Example 9, with 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. CD30 expression levels for these tumor cell lines were evaluated by quantitative flow cytometry, as detailed in Example 2.

[0372] result Figure 22 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR induced T cell-mediated cytotoxicity in all cell lines in vitro, with a maximum target cell killing rate of 68% to 98%. Maximum T cell-mediated tumor cell killing by bsG1-huCD3-FEALxCD30-MDX060-FERR was significantly correlated with CD30 expression levels (Figure 22A) (Spearman r = 0.8571; P = 0.0107).

[0373] Figure 22B shows the EC of T cell-mediated killing in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR for each cell line. 50The data is plotted against CD30 expression levels, showing a negative but not statistically significant trend (Spearman r = -0.6190; P = 0.1150).

[0374] Therefore, these data demonstrate a positive correlation between CD30 expression levels and in vitro bsG1-huCD3-FEALxCD30-MDX060-FERR-induced maximal T cell-mediated cytotoxicity.

[0375] Example 14: Activated T cell fractides induced by -BsG1-huCD3-FEALxCD30-MDX060-FERR activated CD30 + BsG1-huCD3-FEALxCD30-MDX060-FERR-inducible T cell flutorides were evaluated in vitro.

[0376] A 96-well plate (Greiner-bio-one, Netherlands, catalog number 655180) was coated with 100 μL of a solution of 1 μg / mL anti-human CD3 (clone OKT3, Invitrogen, catalog number 16-0037-85) in PBS. The plate was incubated at 37°C for 4 hours. After removing the antibody solution, the wells were washed with 100 μL of PBS. T cells were obtained from healthy human donor buffy coats (Sanquin, Amsterdam, Netherlands) and isolated using RosetteSep® human T cell enrichment cocktail (Stemcell Technologies, France, catalog number 15061) according to the manufacturer's instructions. Purified T cells were placed in T cell medium (Roswell Park Memorial Institute [RPMI]-1640 medium (Lonza, catalog no. BE12-115F) containing 25 mM HEPES and L-glutamine) supplemented with 10% thermo-inactivated donor bovine serum containing iron (DBSI; Gibco, catalog no. 20731-030) and penicillin / streptomycin (pen / strep; Lonza, catalog no. DE17-603E), supplemented with 25 mM HEPES and L-glutamine, in a mixture of 2 × 10⁶ cells. 6The T cells were resuspended at a concentration of cells / mL, and 100 μL of T cell suspension (containing 200,000 T cells) was added to each well of an anti-CD3 coated plate. Furthermore, 100 μL of T cell culture medium supplemented with 2 μg / mL of anti-CD28 (clone CD28.2, Invitrogen, catalog no. 16-0289-85) and 0.05 μg / mL of IL-15 (ThermoFisher, catalog no. PHC9151) was added to each well. The T cells were then incubated at 37°C for 96 hours.

[0377] After 96 hours, T cells were collected and 2 × 10⁶ 6The cells were resuspended in T cell medium at a concentration of cells / mL. Flow cytometry was performed to measure the expression of CD30 and T cell activation markers. Briefly, aliquots of cells were washed with PBS / 0.1% BSA / 0.02% azide (staining buffer), stained with 50 μl of 1000-fold dilution of FVS510 viability dye (BD Biosciences, catalog no. 564406), and incubated at room temperature for 15 minutes. Next, the cells were washed with staining buffer and stained for CD30 (1:50; Biolegend, catalog number 333906, conjugated to PE), T cell marker CD4 (1:50; Biolegend, catalog number 300506, conjugated to FITC), CD8 (1:100; Biolegend, catalog number 301028, conjugated to AF700), and T cell activation marker CD69 (1:50; Biolegend, catalog number 310910, conjugated to APC), CD25 (1:100; Invitrogen, catalog number 25-0259-42, conjugated to PE-Cy7), and CD279 / PD1 (1:50; Biolegend, catalog number 329924, conjugated to BV605). A single-stained sample containing Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42) was used for calibrating the flow cytometer. After incubation at 4°C for 30 minutes, the plate was washed twice with staining buffer. Cells were analyzed using FACS Celesta (BD Biosciences), and the data were processed using FlowJo (BD Biosciences).

[0378] To evaluate whether BsG1-huCD3-FEALxCD30-MDX060-FERR can induce fractolides in activated T cells, stimulated T cells were seeded in 96-well plates at a density of 200,000 cells / well. Subsequently, 50 μL of BsG1-huCD3-FEALxCD30-MDX060-FERR or a control antibody, i.e., bsG1-b12-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxb12-MDX060-FERR, or IgG1-b12, was added to each well (final concentrations ranging from 0.003 to 3.3 μg / mL at a 3-fold dilution in T cell medium). The plates were incubated at 37°C for 48 hours.

[0379] After washing twice with staining buffer, cells were stained with FVS510 viability dye, resulting in staining for T cell markers CD4 and CD8, as well as T cell activation markers CD69, CD25, and CD279 / PD1. Flow cytometry analysis was performed using FACS Celesta (BD Biosciences), and the data were processed using FlowJo (BD Biosciences).

[0380] Dose-response curves were created using GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0381] result Figure 23 shows that the cell markers CD25 (T cell activation) (A) and CD30 (B) were expressed in 54–63% and 21–27% of T cells, respectively, after 72 hours of incubation. CD25 and CD30 expression was further induced after 96 hours of incubation (CD25: 80–83% and CD30: 27–33%). Figure 23C shows that increasing the dose of BsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxb12-MDX060-FERR, or IgG1-b12 was not associated with a decrease in activated T cell viability.

[0382] Therefore, CD30 expression on a subpopulation of activated T cells did not result in T cell fluctuators during incubation with BsG1-huCD3-FEALxCD30-MDX060-FERR.

[0383] Interference of sCD30 with the antitumor activity of Example 15-BsG1-huCD3-FEALxCD30-MDX060-FERR Cell surface CD30 shedding and soluble CD30 (sCD30) generation in 17 different hematological CD30 + Evaluation was performed using tumor cell lines.

[0384] Three days after seeding cells in fresh medium, the concentration of sCD30 in 25 μL of undiluted supernatant collected from the cell culture was measured by an ELISA assay for the quantitative detection of human CD30 using the "Human sCD30 ELISA Kit" (Invitrogen, catalog number BMS240) according to the manufacturer's instructions.

[0385] result Figure 24A shows the concentration of sCD30 in the cell culture supernatant. As shown, various concentrations of sCD30 were detected in the cell culture supernatants from different cell lines. Figure 24B shows that the concentration of sCD30 in the cell culture supernatant was significantly correlated with the CD30 membrane expression level (Spearman r = 0.5912; P = 0.0260) when measured by quantitative flow cytometry as described above in Example 2 (Human IgG Calibrator kit, Biocytex, catalog number CP010).

[0386] To evaluate whether sCD30 can block potent BsG1-huCD3-FEALxCD30-MDX060-FERR-induced T cell-mediated cytotoxicity, T cell-mediated cytotoxicity by BsG1-huCD3-FEALxCD30-MDX060-FERR was evaluated in DEL tumor cells (ALCL) showing high levels of sCD30 (129 ng / mL) in the supernatant. The T cell-mediated cytotoxicity assay was performed as previously described (Example 8). Figure 24C shows that BsG1-huCD3-FEALxCD30-MDX060-FERR induced potent T cell-mediated cytotoxicity in this cell line with a maximum tumor cell killing rate of 86%, demonstrating that BsG1-huCD3-FEALxCD30-MDX060-FERR was still able to induce potent T cell-mediated cytotoxicity in vitro in the presence of sCD30.

[0387] Therefore, sCD30 concentrations varied among cell types and correlated with the level of CD30 expression on the cell surface. Furthermore, BsG1-huCD3-FEALxCD30-MDX060-FERR was still able to induce potent T cell-mediated cytotoxicity in vitro in the presence of sCD30.

[0388] Example 16 - Ex vivo cytotoxicity of bsG1-huCD3-FEALxCD30-MDX060-FERR using patient-derived peripheral blood mononuclear T cells as effector cells. CD3xCD30 bispecific antibodies were tested in an ex vivo cytotoxicity assay using CD30-positive tumor cell lines as target cells and primary patient-derived T cells as effector cells. Peripheral blood mononuclear cells (PBMCs, Discovery Life Sciences, Table 11) from patients with Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and acute myeloid leukemia (AML) were used as T cell sources to evaluate CD3-dependent tumor cell killing.

[0389] L-428 tumor cells were labeled with Celltrace FarRed (Invitrogen, catalog number C34564A; final concentration 2 μM) at 37°C for 15 minutes. After labeling, 5 × volume of ice-cold DBSI was incubated at room temperature for 5 minutes. The cells were pelleted, resuspended in culture medium, and seeded at a density of 50,000 cells / well in 96-well plates (Greiner-bio-one, Netherlands, catalog number 655180). Serial dilutions of bsG1-huCD3-FEALxCD30-MDX060-FERR and the control antibody IgG1-b12-FEAL were added (final concentrations ranging from 1,000 to 0.051 ng / mL; 3-fold dilution), and the plates were incubated at room temperature for 15 minutes. The PBMCs were thawed, counted, and resuspended in culture medium (RPMI1640 / 10% FBS / 1% penicillin-streptomycin / 1% glutamate). They were then added to tumor cells at an effector-to-target (E:T) ratio of 8:1, and the plates were incubated at 37°C for 72 hours. After washing twice with PBS / 0.1% BSA / 0.02% azide (staining buffer), the cells were stained, and different cell populations were distinguished using CD3 (T cells; Invitrogen, catalog no. 48-0037), CD14 (monocytes / macrophages; Biolegend; catalog no. 301834), CD19 (B cells; Biolegend; catalog no. 302246), CD16 (monocytes / macrophages; BD Biosciences; catalog no. 556618), CD56 (NK cells; BD Biosciences; catalog no. 564849), and CD66b (granulocytes; Biolegend; catalog no. 305116).Cells were further stained with the T cell markers CD4 (1:50; Biolegend, catalog number 300521, conjugated in Pacific Blue), CD8 (1:100; BD Biosciences, catalog number 345772, conjugated in FITC), and the T cell activation markers CD69 (1:50; Biolegend, catalog number 310934, conjugated in BV650), CD25 (1:100; Invitrogen, catalog number 25-0259-42, conjugated in PE-Cy7), and CD279 / PD-1 (1:50; Biolegend, catalog number 329930, conjugated in BV605). Single-stained samples, including those containing Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42), were used for flow cytometer calibration. After incubation at 4°C for 30 minutes, the plates were washed twice with staining buffer, and the cells were stained with 7-AAD (diluted 1:100 with staining buffer) at 4°C for 10 minutes. The cells were analyzed using FACS Celesta (BD Biosciences), and the data were processed using FlowJo (BD Biosciences). Dose-response curves were generated using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0390] The percentage of live target cells was calculated using the following formula: % Live target cells = (Absolute number of live single Celltrace FarRed-labeled cells under each condition / Absolute number of live single Celltrace FarRed-labeled cells under the condition containing only target cells and T cells without antibody addition) × 100.

[0391] result Figure 25A shows that bsG1-huCD3-FEALxCD30-MDX060-FERR induced dose-dependent cytotoxicity of L-428 tumor cells after 72 hours, mediated by T cells derived from both healthy control donors and different HL and NHL patient donors. The cytotoxicity was associated with T cell activation and proliferation, as exemplified by the upregulation of CD69, CD25, and PD-1 (Figures 25B-D). No T cell-mediated cytotoxicity was observed for the control antibody IgG1-b12-FEAL. No T cell-mediated cytotoxicity was observed for donor E (AML), which may be due to the low frequency of T cells in the PBMC sample (Table 11).

[0392] In summary, these data indicate that peripheral blood T cells derived from HL and NHL patients can induce T cell-mediated cytotoxicity in tumor cell lines in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR. [Table 11]

[0393] Example 17 - Evaluation of the pharmacokinetic properties of BsG1-huCD3-FEALxCD30-MDX060-FERR in SCID mice Female tumor-free SCID mice (CB-17 / IcrHan®Hsd-Prkdcscid mice, Envigo) aged 11-12 weeks (3 mice per group) were intravenously (IV) injected with a single dose of BsG1-huCD3-FEALxCD30-MDX060-FERR at a dose of 1 μg (0.05 mg / kg), 10 μg (0.5 mg / kg), or 100 μg (5 mg / kg). Since BsG1-huCD3-FEALxCD30-MDX060-FERR does not cross-react with mouse proteins, the experiment was set up to test antibody clearance in the absence of target-mediated clearance.

[0394] Blood samples of 40 μL were collected by buccal vein puncture or saphenous vein puncture 10 minutes, 4-6 hours, 24 hours, 2 days, 7 days, 14 days, and 21 days after antibody administration. The blood was collected in a K2-EDTA-containing vial (Sarstedt, Microvette CB300, catalog number 16.444.100) and centrifuged at 10,000 g for 10 minutes. The plasma supernatant was transferred to a labeled Eppendorf vial and stored at -80°C until plasma IgG concentration measurement.

[0395] Human IgG concentrations were determined using whole-human IgG enzyme-linked immunosorbent assay (ELISA). Mouse anti-human IgG-κ clone MH16 (CLB Sanquin, Netherlands; catalog number M1268), coated at a concentration of 2 μg / mL on a 96-well Microlon ELISA plate (Greiner, Germany) and incubated overnight at 4°C in 100 μL of PBS (BioTrading, catalog number K654F500PP), was used as the capture antibody. After blocking the plate with PBSA (PBS containing 0.2% bovine serum albumin [BSA]) at room temperature (RT) for 1 hour, the sample was added, serially diluted with PBSA, and incubated on a plate shaker at RT for 1 hour. The plates were washed three times with 300 μL of PBST (PBS supplemented with 0.05% Tween 20), followed by incubation at room temperature for 1 hour with goat anti-human IgG immunoglobulin (Jackson, West Grace, Pennsylvania; catalog no. 109-035-098; 1:10.000 in PBST supplemented with 0.2% BSA). After washing the plates three times with 300 μL of PBST, they were incubated with light-protected 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, catalog nos. 11112422001 and 11112597001). The reaction was stopped by adding 100 μL of 2% oxalic acid (Sigma-Aldrich, catalog no. 33506), and the mixture was incubated at room temperature for 10 minutes. Absorbance was measured at 405 nm using an ELx808 absorbance microplate reader (Biotek, Winuskie, Vermont).

[0396] A standard curve was created from the reference antibody human IgG1λ (pure protein 30C, catalog number BP078) for further dilution by 3-fold dilution in PBSTA (concentration range: 1 mg / mL [3 μL]). A second standard curve was created using the injected substance, and a concentration of 1 mg / mL (3.6 μL of antibody) was prepared from a 5 mg / kg dose for further dilution by 3-fold dilution in PBSTA.

[0397] result Calibration curves were calculated from reference standards by interpolating unknowns using a 4-parameter logistic fit curve in Microsoft Excel. Human IgG1 concentrations in plasma samples were calculated from the equation of the plotted calibration curve (Figure 26A), and the area under the curve (AUC) was calculated using GraphPad Prism software. IgG clearance up to the final day of blood collection (day 21) was determined by the equation D*1.000 / AUC (where D is the injection dose (1 mg / kg)) (Figure 26B).

[0398] BsG1-huCD3-FEALxCD30-MDX060-FERR does not cross-react with mouse proteins, and therefore its pharmacokinetic properties are expected to be comparable to other unbound wild-type human IgG1 molecules. Expected human IgG plasma concentrations for all dose groups were calculated to be approximately 100 μg / mL (approximately 5 mg / kg), 10 μg / mL (approximately 0.5 mg / kg), or 1 μg / mL (approximately 0.05 mg / kg). Samples from animals treated with 0.05 mg / kg were not available for any of the time points.

[0399] The plasma clearance rate of BsG1-huCD3-FEALxCD30-MDX060-FERR was comparable to the predicted plasma clearance rate of normal human IgG1. The mean maximum human IgG plasma concentration (Cmax) was comparable to the predicted Cmax of normal human IgG1.

[0400] Therefore, the pharmacokinetic profile of BsG1-huCD3-FEALxCD30-MDX060-FERR is comparable to that predicted for normal human IgG1 in non-tumor-carrying SCID mice in the absence of target binding.

[0401] Example 18: Evaluation of C1q binding to -BsG1-huCD3-FEALxCD30-MDX060-FERR The binding of complement protein C1q to the membrane-bound BsG1-huCD3-FEALxCD30-MDX060-FERR, or the parental antibodies that generated BsG1-huCD3-FEALxCD30-MDX060-FERR, i.e., IgG1-huCD3-FEAL and IgG1-CD30-MDX060-FERR, was evaluated using either CD3-expressing cells or CD30-expressing cells.

[0402] A. C1q binding to BsG1-huCD3-FEALxCD30-MDX060-FERR bound to CD3-expressing cells The binding of complement protein C1q to BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1-huCD3-FEAL bound to CD3, and stimulated human CD8 + The tests were performed using T cells. IgG1-CD52-E430G was included as a positive control, which has VH and VL domains based on the CD52 antibody CAMPATH-1H and an Fc-enhancing skeleton known to efficiently bind to C1q when bound to the cell surface. IgG1-b12-FERR and IgG1-b12 were included as unbound negative control antibodies.

[0403] Human CD8 + T cells, RosetteSep(trademark) human CD8 +T cells were purified (concentrated) from buffy coats obtained from healthy volunteers (Sanquin) by negative selection using a T cell enrichment cocktail (Stemcell Technologies, catalog no. 15023C.2) according to the manufacturer's instructions. The purified T cells were resuspended in T cell medium supplemented with 10% thermo-inactivated donor bovine serum containing iron (DBSI; Gibco, catalog no. 20731-030) and penicillin / streptomycin (pen / strep; Lonza, catalog no. DE17-603E) (Roswell Park Memorial Institute [RPMI]-1640 medium (Lonza, catalog no. BE12-115F) containing 25 mM HEPES and L-glutamine).

[0404] Anti-CD3 / CD28 beads (Dynabeads® Human T-activator CD3 / CD28; ThermoFisher Scientific, catalog no. 11132D) were washed with PBS and resuspended in T-cell medium. The beads were then enriched with human CD8. + The beads were added to T cells in a 1:1 ratio and incubated at 37°C and 5% CO2 for 48 hours. Next, the beads were removed using a magnet, the cells were washed twice with PBS, and then counted again.

[0405] Activated CD8 of BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1-huCD3-FEAL + Binding to T cells was confirmed by flow cytometry using BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1-huCD3-FEAL (30 μg / mL), as well as R-phycoerythrin (PE) conjugated goat anti-human IgG F(ab')2 (diluted 1:200 in GMB FACS buffer; Jackson ImmunoResearch, catalog no. 109-116-098).

[0406] activated CD8 +T cells were seeded in a round-bottom 96-well plate (30,000 cells / well), pelletized, and resuspended in 30 μL of assay medium (RPMI-1640 containing 25 mM HEPES and L-glutamine, supplemented with 0.1% (BSA; Roche, catalog no. 10735086001) bovine serum albumin fraction V and penicillin / streptomycin). Subsequently, 50 μL of BsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-huCD3-FEAL, IgG1-b12-FERR, IgG1-CD52-E430G, or IgG1-b12 (final concentration 1.7 × 10⁶ at a 3-fold dilution in assay medium) was used. -4 (~30 μg / mL) was added to each well and incubated at 37°C for 15 minutes to bind the antibody to the cells.

[0407] Human serum (20 μL / well; Sanquin, Lot 20L15-02) was added as a source of C1q to a final concentration of 20%. After incubating the cells on ice for 45 minutes, they were washed twice with cold GMB FACS buffer and incubated with 50 μL of fluorescein isothiocyanate (FITC) conjugated rabbit anti-human C1q (final concentration 20 μg / mL (DAKO, Catalog No. F0254); diluted 1:75 in GMB FACS buffer) in or without allophycocyanin-conjugated mouse anti-CD8 (BD Biosciences, catalog no. 555369; diluted 1:50 in GMB FACS buffer) for 30 minutes in the dark at 4°C. Cells were washed twice with cold GMB FACS buffer and resuspended in 20 μL of GMB FACS buffer supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, catalog no. 03690) and 4',6-diamidino-2-phenylindole (DAPI) viability dye (1:5,000; BD Pharmingen, catalog no. 564907). C1q binding to viable cells (identified by DAPI exclusion) was analyzed by flow cytometry using iQue3 Screener (Intellicyt Corporation). Data were analyzed using iQue software (Intellicyt Corporation, ForeCyt® Enterprise Client Edition 6.2 [R3], version 6.2.652). Binding curves were analyzed using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism software.

[0408] result Figure 27A shows that dose-dependent C1q binding was observed with membrane-bound IgG1-CD52-E430G, but no C1q binding was observed with membrane-bound BsG1-huCD3-FEALxCD30-MDX060-FERR or IgG1-huCD3-FEAL, or with the unbound control antibody.

[0409] C1q binding to BsG1-huCD3-FEALxCD30-MDX060-FERR bound to B.CD30-expressing cells. The binding of complement protein C1q to CD30-bound BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1-huCD30-MDX060-FERR was tested using NCEB-1 mantle cell lymphoma cells. IgG1-7D8-E430G (anti-CD20), which does not contain inactive mutations and retains the ability to bind to C1q, was included as a positive control. IgG1-b12-FERR was included as a non-binding negative control antibody.

[0410] NCEB-1 cells were suspended at a concentration of 2 × 10⁶ cells / mL in assay medium (RPMI-1640 (Lonza, Switzerland, catalog number BE12-115F)) containing 0.1% bovine serum albumin (BSA, fraction V, Roche, catalog no. 10735086001) and 1% penicillin / streptomycin (Gibco, catalog no. 15140-122). Tumor cells (100,000 cells in 50 μL) were placed in a 96-well round-bottom plate (Greiner). The antibody was added to Bio (catalog number 650180). Next, 30 μL of BsG1-huCD3-FEALxCD30-MDX060-FERR, IgG1-CD30-MDX060-FERR, or a control antibody (final concentration of 5.6 × 10⁻⁵ to 10 μg / mL at a 3-fold dilution in assay medium) was added to each well, and incubated at 37°C for 15 minutes to bind the antibody to the cells.

[0411] To confirm antibody binding, half of the cells (40 μL of cell suspension containing the antibody) were seeded on different plates. Binding of BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1-CD30-MDX060-FERR to NCEB-1 cells was confirmed by flow cytometry using R-phycoerythrin (PE) conjugated goat anti-human IgG F(ab')2 (diluted 1:500 in FACS buffer; Jackson ImmunoResearch, catalog no. 109-116-098).

[0412] Human serum (10 μL / well; Sanquin, Lot 21K04-01) was added to the remaining 40 μL of cell suspension (20% of the final concentration), incubated on ice for 45 minutes, washed with FACS buffer, and incubated with 25 μL of FITC conjugate rabbit anti-C1q antibody (final concentration 20 μg / mL (DAKO, catalog no. F0254); diluted with FACS buffer) for 30 minutes in the dark at 4°C. Cells were washed with cold FACS buffer, resuspended in 30 μL of FACS buffer supplemented with TO-PRO®-3 viability stain (1:5000; ThermoFisher, catalog no. T3605), and measured using iQue3 Screener (Intellicyt Corporation). Data were analyzed using iQue software (Intellicyt Corporation, ForeCyt® Enterprise Client Edition 6.2[R3], version 6.2.652). The combined curves were analyzed using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism software.

[0413] result Figure 27B shows that dose-dependent C1q binding was observed with membrane-bound IgG1-CD20-E430G, but no C1q binding was observed with membrane-bound BsG1-huCD3-FEALxCD30-MDX060-FERR or IgG1-CD30-MDX060-FERR or the unbound control antibody.

[0414] Therefore, these results demonstrate that bsG1-huCD3-FEALxCD30-MDX060-FERR does not bind to C1q, confirming a functionally inactive skeleton.

[0415] Example 19 - Three Formulations; Basic Properties and Stability Materials and methods Preparation of test formulations Table 12 shows the compositions of the three formulations tested. [Table 12]

[0416] Preparation 1 was prepared by dissolving L-histidine monohydrochloride monohydrate and sucrose in water. The pH was adjusted to 6.0 using NaOH or HCl. The solution was then filtered through a 0.22 μm bottle-top filter in a flow cabinet. For preparations containing polysorbate 80, 10% polysorbate 80 was added and gently mixed until complete dissolution was achieved.

[0417] Formulations 2 and 3 were prepared by dissolving sodium acetate trihydrate in water. Glacial acetic acid was added stepwise in water, and then stepwise into the sodium acetate trihydrate solution. D-sorbitol (formulation 2) or trehalose (formulation 3) was added, and the pH was adjusted to 5.5 with NaOH or HCl. The solution was then filtered through a 0.22 μm bottle-top filter in a flow cabinet. For formulations containing polysorbate 80, 10% polysorbate 80 was added and gently mixed until complete dissolution was achieved.

[0418] Sample preparation The antibody used in the sample was bispecific huCD3xCD30-MDX060 (bsIgG1-huCD3-FEALxCD30-MDX060-FERR). The preparation described herein is shown in Example 1.

[0419] Stability tests were conducted on formulations 1, 2, and 3 (including polysorbate 80) at antibody concentrations of 20 mg / mL and 100 mg / mL.

[0420] Freeze-thaw and stirring studies were conducted at an antibody concentration of 20 mg / mL in formulations 1, 2, and 3, with and without polysorbate 80.

[0421] High-concentration studies were conducted on antibody concentrations ranging from 20 to 200 mg / mL in formulations 1, 2, and 3 (including polysorbate 80).

[0422] B22 / k DMeasurements were performed for antibody concentrations in the range of 2.5 to 20 mg / mL in formulations 1, 2, and 3 (including polysorbate 80).

[0423] The samples were received frozen and thawed at 5°C. The time required to completely thaw the sample bulk ranged from 24 to 72 hours.

[0424] Vials containing formulation buffer (including polysorbate 80) were used as blanks for stability testing, appearance, and osmolality by weight. Freeze-thaw cycles, agitation, and vials specifically for high-concentration studies were maintained at 5°C before testing.

[0425] Preparation of samples containing 20 mg / mL antibody (for each formulation) The antibody is placed in a Thermo Scientific® Slide-A-Lyzer® G2 dialysis cassette, as shown below. Dialysis was performed using (3.5K MWCO, 70 mL). All subsequent steps were performed. The procedure was performed using specific formulations that did not contain polysorbate 80, i.e., formulations 1, 2, or 3, respectively. First, the cassette membrane was hydrated in the dialysis buffer, i.e., the specific formulation that did not contain polysorbate 80, for at least 2 minutes. The dialysis cassette was removed, and any excess buffer was gently removed. The sample was transferred to the dialysis cassette. The dialysis cassette was placed in 1 L of the desired buffer and incubated for 2 hours with gentle mixing. The dialysis cassette was removed, the buffer was discarded, 1 L of fresh buffer was added to the container, and incubated overnight. All incubations were performed at 5°C with gentle agitation. The following day, the sample was removed, and the protein concentration was quantified using A280. The concentration was adjusted to 20 ± 2 mg / mL by volumetric correction as needed by adding the required amount of buffer, or by concentrating the sample using Pierce® Protein Concentrator (PES, 30K MWCO, 20-100 mL). The remaining volume was subjected to sterile filtration, and the protein concentration was quantified using A280, revealing an actual concentration (measured concentration) of approximately 20 mg / mL.

[0426] For samples containing polysorbate 80, 10% polysorbate 80 was added after dialysis and concentration to reach a final concentration of 0.02% (w / v).

[0427] Aliquots were prepared in 2 mL glass cryovials (fluid X vials) with a final volume of 1.5 mL under sterile conditions.

[0428] Preparation of samples containing 100 mg / mL antibody (for each formulation) A 20 mg / mL antibody sample was concentrated to a final concentration of 100 mg / mL using Pierce® Protein Concentrators (PES, 30K MWCO, 20-100). Prior to concentration, the protein concentrator was rinsed with a specific formulation that did not contain polysorbate 80. The protein concentration was quantified using A280 and volumetrically corrected to 100 ± 10 mg / mL. Next, the sample was dialyzed using Thermo Scientific® Slide-A-Lyzer® G2 Dialysis Cassettes (3.5K MWCO, 70 mL). All subsequent steps were performed using a specific formulation that did not contain polysorbate 80. First, the cassette membrane was hydrated in dialysis buffer, i.e., a specific formulation that did not contain polysorbate 80, for at least 2 minutes. The dialysis cassette was removed, and excess buffer was gently removed. The sample was transferred to the dialysis cassette. The dialysis cassette was placed in 1 L of the desired buffer and incubated for 2 hours with gentle mixing. The dialysis cassette was removed, the buffer was discarded, 1 L of fresh buffer was added to the container, and it was incubated overnight. All incubations were performed at 5°C with gentle agitation. The following day, the samples were removed and quantified using A280. The samples were concentrated to a target concentration of 100 ± 2 mg / mL by adding the required buffer volume as needed, or by concentrating the samples using Pierce® Protein Concentrators (PES, 30K MWCO, 20-100 mL). The remaining volume was sterile filtered and quantified using A280, showing an actual concentration (measured concentration) of approximately 70 mg / mL (66 mg / mL, 74 mg / mL, and 79 mg / mL for formulations 1, 2, and 3, respectively). The initial target concentration was 100 mg / mL for all three formulations. However, due to protein loss during the process caused by gel formation, it was decided to concentrate to approximately 70 mg / mL to minimize sample loss.

[0429] For samples containing polysorbate 80, 10% polysorbate 80 was added after dialysis and concentration to reach a final concentration of 0.02% (w / v).

[0430] Aliquots were prepared in 2 mL glass cryovials (fluid X vials) with a final volume of 1.5 mL under sterile conditions.

[0431] Handling of samples Vials were removed from the incubator at the specified time and number. Samples were removed from the incubator within 3 days of the intended pull date. Additional vials were maintained under the specified storage conditions and used when additional sample volume was needed. The remaining volume of the removed sample was kept at 5°C.

[0432] Freeze-thaw Antibodies at a concentration of 20 mg / mL were subjected to five freeze-thaw cycles. All three formulations, including those with and without polysorbate 80, were prepared. Vials were kept at 5°C before use.

[0433] Three aliquots were prepared for each formulation. One aliquot was used as a control, tested under control conditions without freeze-thaw stress, where the sample was maintained at 5°C (5°C control). One aliquot was subjected to five simulated cycles (5°C to room temperature), and another aliquot was subjected to five freeze-thaw cycles (-75°C to room temperature). After sample preparation, the aliquots were stored at 5°C and then analyzed together.

[0434] Each freeze-thaw cycle consisted of freezing the aliquots overnight at -75°C and then thawing them at room temperature (2-4 hours). The control sample was removed from storage at 5°C and left at room temperature for the same amount of time as the -75°C sample.

[0435] stirring A 20 mg / mL antibody concentration was subjected to a single agitation cycle. All three formulations, including those with and without polysorbate 80, were prepared. The vials were kept at 5°C before use.

[0436] Two aliquots were used for each formulation. One aliquot was tested as a control without agitation stress. The other aliquot was agitated at 500 rpm for 24 hours at room temperature. The vial was placed upright. After sample preparation, the aliquots were stored at 5°C and analyzed together. The control sample was removed from storage at 5°C and left at room temperature for 24 hours (simultaneously with agitation).

[0437] high concentration High-concentration studies were conducted. Samples were concentrated to a range of antibody concentrations from 20 to 200 mg / mL. All three formulations, including polysorbate 80, were tested.

[0438] The sample was concentrated using an Amicon Ultra-4 Centrifugal Filter Unit (10kDa) and quantified using an A280.

[0439] Thermal stability due to fluorescence and static light scattering The conformational and colloidal stability were determined by combined fluorescence / static light scattering (SLS) measurements at UNcle Instruments (Unchained Labs, LLC) by increasing thermal stress to induce protein unfolding and aggregation.

[0440] Conformational stability is assessed by determining the unfolding state transition caused by increased thermal stress, detected by changes in the intrinsic fluorescence of Trp (and Tyr) residues in the protein due to changes in the local environment during protein unfolding. When buried tryptophan residues are exposed, the maximum emission wavelength shifts to longer wavelengths. The fluorescence ratio at wavelengths 350 / 330 nm is plotted to show the conformational changes of the protein with respect to temperature. Fluorescence analysis is performed at the temperature at which the protein begins to unfold (unfolding onset temperature - T). onset ) and the midpoint of the protein transition from the folded state to the unfolded state (melting temperature - T) m ) provides.

[0441] Colloid stability is also provided by the UNcle instrument by SLS measurement when the temperature is gradually increased. The sample was irradiated with laser light scattered by the molecules in the solution. The intensity of static light scattering is proportional to the average molecular weight of the species in the solution. Therefore, this analysis is sensitive to protein aggregation across a temperature gradient. Static light scattering was measured at 266 nm to detect smaller aggregates and at 473 nm to detect larger aggregated species. Using the results at 266 nm, the onset of the aggregation temperature (T agg ) was determined from these data. These data are best analyzed by a large change in the count intensity, with higher counts indicating that more light was scattered due to the formation of protein aggregates. T agg is determined based on a 10% increase in count.nm from the baseline.

[0442] Based on intrinsic antibody fluorescence and static light scattering, the midpoint of unfolding (melting temperature, Tm) and the onset of aggregation temperature (Tagg) were determined simultaneously.

[0443] The antibody batch was diluted to 1 mg / mL in PBS pH 7.4 and centrifuged before analysis. Samples were analyzed in triplicate. Fluorescence and SLS were measured by increasing the temperature from 25 to 95 °C at a temperature gradient of 0.5 °C / min. T m was determined by the UNcle software based on the fluorescence ratio (350 / 330 nm) method. T agg was determined by the UNcle software based on the SLS 266 nm data.

[0444] k D and B 22 Determination k D and B 22 Both are parameters for studying colloid stability and aggregation tendency, which are useful for selecting conditions to prevent aggregation. The second virial coefficient B 22k is a thermodynamic measure of protein-protein interactions in solution, determined by the slope of a Debye plot calculated based on scattering intensity. Also known as the diffusion interaction parameter. D This is measured using the diffusion coefficient determined by DLS at various protein concentrations. Both parameters are simultaneously determined by DLS measurements at 25°C using a concentration range of 0–20 mg / mL. Data analysis was performed using UNcle Analysis software. Negative k D and B 22 The value indicates enhancement of self-association, and a positive k D and B 22 The values ​​indicate repulsive interactions between proteins.

[0445] exterior The external appearance was determined by visual inspection.

[0446] viscosity Viscosity was measured using the Viscosizer TD System (Malvern Panalytical Ltd) according to the manufacturer's instructions.

[0447] Osmolality The gravimetric osmolality was measured using an A20 Advanced Automator (Advanced Instruments, LLC) according to the manufacturer's instructions.

[0448] particle size Particle size was measured using an MFI 5200 imaging-based particle counter (Protein Simple, Inc.) according to the manufacturer's instructions.

[0449] Absorbance A 280 Protein concentration "A280" is a spectrophotometric method that measures ultraviolet absorbance at 280 nm. Determining protein concentration by ultraviolet absorption (280 nm) depends on the presence of aromatic amino acids in the protein. The concentration of the solution can be calculated using the Beer-Lambert law: (c = A / ε·l); where c is the molar concentration, l is the cuvette path length (in cm), and ε is the molar extinction coefficient (in M). -1 cm -1 ) and A is the absorbance at 280 nm.

[0450] Protein concentration was determined by UV / Vis spectroscopy (absorbance measurement at 280 nm (A280)) using a NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific).

[0451] Size exclusion chromatography (SEC) Monomer purity was measured using HPSEC. SEC is a chromatographic method in which molecules in solution are separated by their size and / or molecular weight. SEC chromatograms of unstressed samples typically showed one major peak corresponding to the monomer antibody, accounting for over 90% of the total peak area. This single major SEC peak reflected the uniformity of antibody size in each formulation. For comparison of SEC data between different formulations, the peak area values ​​of species eluted before the major peak were combined and reported as a percentage of HMW (high molecular weight) species, usually representing the aggregated form. Similarly, the peak area values ​​of species eluted after the major peak were combined and reported as a percentage of LMW (low molecular weight) species, usually representing the degraded form.

[0452] Size exclusion chromatography was performed on Agilent 1100 and 1200 HPLC systems using a TOSOH, TSK-gel G3000SWxL (7.8 × 300 mm) column (Sigma, catalog number 08541).

[0453] Imaging capillary isoelectric focusing (icIEF) and cation exchange chromatography (CIEX) Acidity and basicity were measured using cIEF and CIEX.

[0454] The imaged capillary isoelectric focusing electrophoresis was performed using an iCE 3 Analyzer equipped with a PrinCE Autosampler. Specifically, iCE IEF separates antibody isomers based on the difference in their isoelectric points (pI), which arise from the total charge on the molecules as a function of the surrounding pH.

[0455] Cation exchange chromatography was performed using a Waters Alliance HPLC system equipped with a CIEX column and a UV detector. A Proteomix® WCX-NP5 weak cation exchange column (4.6 × 250 mm) was used. pH and salt gradients were used to separate species in solution.

[0456] Reducing and non-reducing microchip capillary electrophoresis - sodium dodecyl sulfate Intact IgG purity (%) and (HC+LC) purity (%) were measured using CE-SDS. Capillary electrophoresis (both reducing and non-reducing) was performed using a PA800 Plus (Sciex) instrument according to the manufacturer's instructions.

[0457] result 1. Osmolality and Viscosity Three formulations were tested with antibody concentrations of 0 mg / mL, 20 mg / mL, and 100 mg / mL. The gravimetric osmolality and viscosity of the formulations at 0 months (t=0M) are shown in Tables 13 and 14. [Table 13] [Table 14]

[0458] The gravimetric osmolality is within the acceptable range of 200–600 mOsm / kg and within the target gravimetric osmolality of 300 mOsm / kg. Furthermore, all formulations exhibit low viscosity (<10 cP). Viscosity is comparable across different formulations.

[0459] 2. Stirring and freeze-thaw cycle Antibodies at a concentration of 20 mg / mL were subjected to one agitation cycle or five freeze-thaw cycles at month 0 (t=0M) in three different formulations (Table 15) with or without PS80. No difference in antibody quality was observed between formulations with and without PS80 after freeze-thaw cycles (-75°C to room temperature) and control (5°C to room temperature). [Table 15] TIFF2026513829000030.tif22971

[0460] 3.T m , T agg and k D The stability of bispecific antibodies in various formulations at 0 months (t=0M) is determined by T m , T agg This was measured by examining B shown in Tables 16-18. 22 and k D .

[0461] T m The fluorescence ratio was measured at 0 months (t=0M) using the 350 / 330 nm method. The data showed that for all three buffer formulations, equivalent T values ​​were obtained for BisG1-huCD3-FEAL / CD30-MDX60-FERR. m This was shown. [Table 16]

[0462] A 10% increase in counts.nm from the threshold at 25℃ and 0 months (t=0M) indicates that T aggThe following was determined. The data showed that for bispecific antibodies, the acetate / sorbitol and acetate / trehalose buffers increased the T-initiation of agglutination compared to histidine buffer. [Table 17]

[0463] Protein aggregation, B 22 and k D Further research was conducted using B at 0 months (t=0M). 22 and k D The antibody concentration ranges used to determine this were 2.5, 5, 10, 15, and 20 mg / mL. [Table 18]

[0464] Positive B for acetate / sorbitol buffer and acetate / trehalose buffer 22 / k D The values ​​suggest increased antibody solubility in acetate-buffered formulations compared to histidine-buffered formulations.

[0465] Example 20 - Long-term stability of bispecific antibodies in three formulations Three formulations of bsIgG1-huCD3-FEALxCD30-FERR were prepared at two concentrations (20 mg / mL and 100 mg / mL) and maintained at different temperatures (5°C, 25°C, and 40°C) for 12 months. Samples were analyzed at 0 months (t=0M), 1 month (t=1M), 2 months (t=2M), 3 months (t=3M), 6 months (t=6M), 9 months (t=9M), and 12 months (t=12M).

[0466] The materials and methods were carried out as described in Example 19.

[0467] The compositions of the three formulations tested are detailed in Table 12 of Example 19.

[0468] 1. Visual inspection and particle inspection [Table 19] TIFF2026513829000035.tif148161

[0469] Overall, Table 19 shows that the samples were essentially free of visible particles (EFVP) at all time points. Slight variations in color are due to different interpreters. [Table 20] [Table 21]

[0470] For all formulations, the sample concentrations remain stable for 12 months at 5, 25, and 40°C.

[0471] 2. Monomer purity [Table 22] [Table 23] [Table 24]

[0472] Tables 22-24 show the measured monomer purity values, expressed as polymer%, monomer%, and decomposition%. Monomer purity is comparable for all formulations. Monomer purity % is stable at 5°C and 25°C for 12 months for samples at concentrations of 20 mg / mL and 100 mg / mL, where monomer purity % exceeds 95%. A slight decrease is observed at 25°C, but it is still considered stable. However, at 40°C, monomer purity % is less than 95% after 2 months, indicating increased aggregation and fragmentation.

[0473] 3. Intact IgG% and LC+HC% [Table 25]

[0474] The intact IgG purity is equivalent for all formulations. Intact IgG purity remains stable at 5°C for 12 months for samples at concentrations of 20 mg / mL and 100 mg / mL. A slight decrease in IgG purity is observed at 25°C.

[0475] After 3 months, at 40°C, the intact IgG purity % decreased to approximately 90%, and as shown in Table 25, the low molecular weight species (LMWS) and high molecular weight species (HMWS) increased. [Table 26]

[0476] Table 26 shows that the (HC+LC) purity percentage is equivalent for all formulations. The (HC+LC) purity percentage is stable at 5°C for at least 9 months for samples at concentrations of 20 mg / mL and 100 mg / mL. A slight decrease in (HC+LC) purity percentage is observed at 25°C and 5°C at 12 months.

[0477] After 3 months at 40°C, the (HC+LC) purity was less than 95%, and LMWS and HMWS increased.

[0478] 4. Acidic form % and basic form % [Table 27] [Table 28] [Table 29] [Table 30]

[0479] As shown in Tables 27-30, the acidity percentage was comparable for all formulations. The acidity percentage remained stable at 5°C for 12 months for samples at concentrations of 20 mg / mL and 100 mg / mL, with a slight increase observed at 25°C. After 3 months at 40°C, the acidity percentage increased compared to 0 months (t=0M), while the basicity percentage remained relatively constant compared to the starting level (i.e., 0 months at 40°C (t=0M)). This was demonstrated by both electrophoretic and chromatographic overlays.

[0480] Furthermore, the pH was measured for formulations 1+PS80, 2+PS80, and 3+PS80 at antibody concentrations of 20 mg / mL and 100 mg / mL in formulations maintained at 5°C, 25°C, and 40°C for 12 months (12M), 6 months (6M), and 3 months (3M), respectively. The results showed that the pH of the formulations was stably maintained over time at different temperatures for both antibody concentrations (data not shown).

[0481] Example 21 - High-Concentration Formulation Three formulations of bsIgG1-huCD3-FEALxCD30-FERR were prepared at five concentrations (20 mg / mL, 50 mg / mL, 100 mg / mL, 150 mg / mL, and 200 mg / mL). The samples were analyzed after preparation, i.e., at t=0M.

[0482] The materials and methods were carried out as described in Example 19.

[0483] The three formulations tested are described in detail in Table 12 of Example 19.

[0484] 1. Visual inspection [Table 31]

[0485] Table 31 shows that higher viscosity was observed at higher antibody concentrations. However, no visible particles were detected.

[0486] 2. Actual concentration Processing a formulation can alter the concentration of antibodies within it. Therefore, the intended target concentration may differ from the actual concentration (measured concentration) measured in the processed formulation. This can depend on the antibody concentration and the formulation's composition. [Table 32] Table 32 shows that the maximum target concentrations (i.e., the target concentrations) for formulations 2 and 3 were met with final recovery rates of 64% and 69%, respectively (200 mg / mL ± 10%). Compared with acetate buffer, formulation 1 (histidine buffer) yielded lower maximum protein concentrations (142.1 mg / mL) and recovery rates (49%).

[0487] 3.Purity [Table 33] [Table 34] [Table 35]

[0488] Tables 33-35 show that monomer purity was considered equivalent across all formulations, despite a moderate increase in polymers being observed at higher concentrations. Similar IgG and (HC+LC) purities were observed across all formulations, and the acidic, neutral, and basic percentages were comparable among them.

[0489] In conclusion, it appears possible to achieve higher concentrations with the three different buffer formulations. However, the data showed better recovery rates for acetate buffer than for histidine buffer.

Claims

1. A pharmaceutical composition, 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, b) Including a buffering agent, A pharmaceutical composition having a pH of 4.0 to 8.

0.

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

3. The pharmaceutical composition according to claim 2, wherein the buffering agent is histidine, an acetate, and / or a mixture thereof.

4. The pharmaceutical composition according to claim 3, wherein the buffering agent is an acetate.

5. The pharmaceutical composition according to claim 4, wherein the buffering agent is selected from the group consisting of sodium acetate, potassium acetate, sodium acetate, hydrates thereof, and mixtures thereof.

6. The pharmaceutical composition according to claim 5, wherein the buffering agent is sodium acetate such as sodium acetate trihydrate.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the buffering agent is present at a concentration of about 5 to about 40 mM, about 10 to about 30 mM, about 15 to about 25 mM, about 18 to about 22 mM, preferably about 20 mM, about 17 mM, etc.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pH of the composition is approximately 4.5 to approximately 6.5, approximately 5.0 to approximately 6.0, approximately 5.2 to approximately 5.7, approximately 5.4 to approximately 5.6, preferably approximately 5.

5.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pH of the composition is about 5.

5.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pH of the composition is about 6.

0.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition further comprises a pH adjusting component.

12. The pharmaceutical composition according to claim 11, wherein the pH adjusting component is an acid.

13. The pharmaceutical composition according to claim 12, wherein the acid is HCl and / or acetic acid such as glacial acetic acid.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition further comprises a nonionic excipient.

15. The pharmaceutical composition according to claim 14, wherein the nonionic excipient is a sugar or a sugar alcohol.

16. The pharmaceutical composition according to claim 15, wherein the nonionic excipient is selected from sucrose, trehalose, mannitol, xylitol, sorbitol, and mixtures thereof.

17. The pharmaceutical composition according to claim 16, wherein the nonionic excipient is selected from sorbitol, sucrose, trehalose, or a mixture thereof.

18. The pharmaceutical composition according to claim 17, wherein the nonionic excipient is sorbitol or trehalose.

19. The pharmaceutical composition according to claim 18, wherein the nonionic excipient is sorbitol.

20. The pharmaceutical composition according to any one of claims 14 to 19, wherein the nonionic excipient is present at a concentration of about 5 to about 450 mM, about 50 to about 400 mM, for example, about 100 to about 350 mM, about 125 to about 300 mM, for example, about 150 to about 250 mM, preferably about 250 mM.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the pharmaceutical composition comprises an acetate such as sodium acetate and sorbitol, and has a pH of about 5.

5.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the weight osmolality of the pharmaceutical composition is less than about 600 mOsm / kg, less than about 550 mOsm / kg, less than about 500 mOsm / kg, less than about 450 mOsm / kg, less than about 400 mOsm / kg, less than about 350 mOsm / kg, etc.

23. The pharmaceutical composition according to any one of claims 1 to 21, wherein the weight osmolality of the pharmaceutical composition is in the range of about 100 to 500 mOsm / kg, about 200 to 400 mOsm / kg, for example, in the range of about 250 to 350 mOsm / kg.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the viscosity of the pharmaceutical composition is less than about 30 cP, 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, etc.

25. The pharmaceutical composition according to any one of claims 1 to 23, wherein the viscosity of the pharmaceutical composition is in the range of about 1 to 30 cP, the same range of about 1 to 25 cP, for example, the range of about 1 to 20 cP, the range of about 1 to 18 cP, for example, the range of about 1 to 15 cP, the range of about 1 to 12 cP, for example, the range of about 1 to 10 cP, the range of about 1 to 9 cP, for example, the range of about 1 to 8 cP, the range of about 1 to 7 cP, for example, the range of about 1 to 6 cP, the range of about 1 to 5 cP, for example, the range of about 1 to 4 cP, the range of about 1 to 3 cP, etc.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the pharmaceutical composition further comprises a surfactant.

27. The pharmaceutical composition according to claim 26, wherein 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; and alpha-tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxyl hydroxystearate, polyoxylglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan esters, close palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof.

28. The pharmaceutical composition according to claim 27, wherein the surfactant is a polysorbate.

29. The pharmaceutical composition according to claim 28, wherein the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80), preferably polysorbate 80 (PS80).

30. The pharmaceutical composition according to any one of claims 26 to 29, wherein the surfactant is present at a concentration of about 0.01 to about 0.1% w / v, about 0.01 to about 0.09% w / v, about 0.01 to about 0.06% w / v, about 0.01 to about 0.05% w / v, 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. The pharmaceutical composition according to any one of claims 1 to 30, wherein the concentration of the antibody is approximately 0.5 to approximately 250 mg / ml, approximately 1.0 to approximately 220 mg / ml, approximately 3 to approximately 190 mg / ml, approximately 5 to approximately 160 mg / ml, approximately 10 to approximately 130 mg / ml, approximately 20 to approximately 120 mg / ml, approximately 30 to approximately 110 mg / ml, approximately 40 to approximately 100 mg / ml, approximately 50 to approximately 90 mg / ml, approximately 60 to approximately 80 mg / ml, or approximately 65 to approximately 75 mg / ml, or approximately 20 to approximately 200 mg / ml.

32. The pharmaceutical composition according to any one of claims 1 to 31, wherein the concentration of the antibody is approximately 50 to approximately 250 mg / ml, approximately 60 to approximately 240 mg / ml, etc., or approximately 70 to approximately 220 mg / ml, etc., approximately 80 to approximately 210 mg / ml, etc., approximately 100 to approximately 200 mg / ml, etc., approximately 120 to approximately 190 mg / ml, etc., approximately 140 to approximately 180 mg / ml, etc., or approximately 150 to approximately 170 mg / ml, etc.

33. The pharmaceutical composition according to any one of claims 1 to 32, wherein the pharmaceutical composition is a liquid composition.

34. The pharmaceutical composition according to any one of claims 1 to 33, wherein the pharmaceutical composition is an aqueous composition.

35. The pharmaceutical composition according to any one of claims 1 to 34, wherein the pharmaceutical composition is a stable pharmaceutical composition.

36. The pharmaceutical composition according to any one of claims 1 to 35, wherein the pharmaceutical composition is stable for at least two months, at least three months, at least six months, at least nine months, at least twelve months, etc.

37. The pharmaceutical composition according to any one of claims 1 to 36, wherein the pharmaceutical composition is stable over a wide range of antibody concentrations, such as about 50 mg / ml to about 170 mg / ml, including about 10 mg / ml to about 250 mg / ml and about 20 mg / ml to about 200 mg / ml.

38. The pharmaceutical composition according to any one of claims 1 to 37, wherein the pharmaceutical composition is stable over a temperature range such as about 2°C to about 25°C.

39. The pharmaceutical composition according to any one of claims 1 to 38, wherein the composition is stable for at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, or at least twelve months at a storage temperature such as 2 to 8°C or 5°C.

40. The pharmaceutical composition according to any one of claims 1 to 39, wherein the pharmaceutical composition is stable for at least 12 months at a storage temperature of about 5°C.

41. a) Multispecific antibodies of approximately 0.5 to 250 mg / ml, multispecific antibodies of approximately 20 to 200 mg / ml, b) Buffering agent, c) If necessary, a nonionic excipient, d) If necessary, a surfactant and This includes, or is essentially derived from, The pharmaceutical composition according to any one of claims 1 to 40, wherein the pH of the composition is about 5.0 to about 6.

5.

42. The pharmaceutical composition according to any one of claims 1 to 41, wherein the composition is a subcutaneous composition and / or the composition is for use in subcutaneous administration.

43. The pharmaceutical composition according to any one of claims 1 to 42, wherein the composition is an intravenous composition and / or the composition is for use in intravenous administration.

44. The multispecific antibody described above (i) A CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) CD3 binding region comprising a second heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. A pharmaceutical composition according to any one of claims 1 to 43, including the above.

45. The pharmaceutical composition according to claim 44, wherein the first heavy chain variable region and / or the first light chain variable region is human.

46. The pharmaceutical composition according to any one of claims 44 to 45, wherein the second heavy chain variable region and / or the second light chain variable region is humanized.

47. A pharmaceutical composition according to any one of claims 44 to 46, wherein X in Sequence ID No. 9 is H.

48. The pharmaceutical composition according to any one of claims 44 to 47, wherein the first heavy chain variable region includes the sequence shown in Sequence ID No. 13, and the first light chain variable region includes the sequence shown in Sequence ID No.

14.

49. The pharmaceutical composition according to any one of claims 44 to 48, wherein the second heavy chain variable region includes the sequence shown in Sequence ID No. 15, and the second light chain variable region includes the sequence shown in Sequence ID No.

16.

50. The pharmaceutical composition according to any one of claims 49, wherein X in Sequence ID No. 15 is H.

51. The pharmaceutical composition according to any one of claims 44 to 50, wherein the multispecific antibody is a bispecific antibody.

52. The pharmaceutical composition according to any one of claims 44 to 51, wherein the multispecific antibody comprises an Fc region consisting of a first and a second Fc polypeptide.

53. The pharmaceutical composition according to any one of claims 44 to 52, wherein the Fc region is an IgG1 Fc region, preferably a human IgG1 Fc region.

54. The pharmaceutical composition according to any one of claims 44 to 53, wherein the multispecific antibody is a full-length antibody.

55. The pharmaceutical composition according to any one of claims 44 to 54, wherein the multispecific antibody includes an inactive Fc region.

56. The pharmaceutical composition according to any one of claims 44 to 55, wherein the first and / or second Fc polypeptide comprises amino acid substitutions corresponding to the amino acids at positions L234 and / or L235 of the human IgG1 heavy chain, wherein the substitutions are preferably substitutions to F and E, respectively, and the amino acid positions are as defined by Eu numbering.

57. The pharmaceutical composition according to any one of claims 55 to 56, wherein the first and second Fc polypeptides each comprise substitutions of amino acids to F and E corresponding to amino acids at positions L234 and L235, respectively, and the first and / or second Fc polypeptide further comprises a substitution of amino acid corresponding to an amino acid at position G236 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to R, and the amino acid position is as defined by Eu numbering.

58. The pharmaceutical composition according to claim 57, wherein the first and second Fc polypeptides each comprise substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235, and the first and second Fc polypeptides further comprise substitutions of amino acids corresponding to the amino acid at position G236 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to R.

59. The pharmaceutical composition according to any one of claims 55 to 56, wherein the first and second Fc polypeptides each comprise substitutions of amino acids to F and E corresponding to amino acids at positions L234 and L235, respectively, and the first and / or second Fc polypeptide further comprises a substitution of amino acid corresponding to an amino acid at position D265 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to A, and the amino acid positions are as defined by Eu numbering.

60. The pharmaceutical composition according to claim 59, wherein the first and second Fc polypeptides each comprise substitutions of amino acids to F and E corresponding to the amino acids at positions L234 and L235, and the first and second Fc polypeptides further comprise substitutions of amino acids corresponding to the amino acid at position D265 in the human IgG1 heavy chain, wherein the substitution is preferably a substitution to A.

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

62. The pharmaceutical composition according to claim 61, wherein the first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are contained within the same polypeptide chain.

63. The pharmaceutical composition according to claim 62, wherein the first Fc polypeptide comprises substitutions of amino acids corresponding to the amino acids at positions L234, L235, and G236 to F, E, and R, respectively, and the second Fc polypeptide comprises substitutions of amino acids corresponding to the amino acids at positions L234, L235E, and D265 to F, E, and A, respectively, wherein the amino acid positions are as defined by Eu numbering.

64. The pharmaceutical composition according to any one of claims 44 to 63, wherein in the first Fc polypeptide, at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, and in the second Fc polypeptide, at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, wherein the substitutions in the first and second Fc polypeptides are not at the same position, and the amino acid positions are as defined by Eu numbering.

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

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

67. The multispecific antibody described above (i) A CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. Includes, The multispecific antibody is a bispecific antibody and includes an Fc region composed of a first and a second Fc polypeptide. The first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain. The second Fc polypeptide and the double chain variable region are contained within the same polypeptide chain. The first Fc polypeptide comprises substitutions of amino acids corresponding to the amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and the second Fc polypeptide comprises substitutions of amino acids corresponding to the amino acids at positions L234, L235, and D265 with F, E, and A, respectively. The aforementioned amino acid positions are as defined by Eu numbering, The pharmaceutical composition according to any one of claims 44 to 66, wherein the amino acid at the position corresponding to K409 in the first Fc polypeptide is R, and the amino acid at the position corresponding to F405 in the second Fc polypeptide is L.

68. The pharmaceutical composition according to any one of claims 44 to 67, wherein the multispecific antibody comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19, and the light chain sequences shown in SEQ ID NOs: 18 and 20.

69. The pharmaceutical composition according to any one of claims 44 to 68, wherein the multispecific antibody comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19 and the light chain sequences shown in SEQ ID NOs: 18 and 20, and the antibody is a bispecific antibody.

70. The pharmaceutical composition according to any one of claims 1 to 69, wherein the multispecific antibody comprises a lambda (λ) light chain and a kappa (κ) light chain (for example, an antibody having a heavy chain and a lambda light chain including a binding region that can bind to CD3, and a heavy chain and a kappa light chain including a binding region that can bind to CD30).

71. The pharmaceutical composition according to any one of claims 1 to 70, wherein the antigen-binding region capable of binding to human CD30 is included in the heavy chain and the light chain, the heavy chain includes the VH region and the IgG1 heavy chain constant region, the light chain includes the VL region and the kappa light chain constant region, and the antigen-binding region capable of binding to human CD3 is included in the heavy chain and the light chain, the heavy chain includes the VH region and the IgG1 heavy chain constant region, and the light chain includes the VL region and the lambda light chain constant region.

72. The pharmaceutical composition according to claim 71, wherein one IgG1 heavy chain constant region is as defined in SEQ ID NO: 51, the other is as defined in SEQ ID NO: 50, the kappa light chain constant region is as defined in SEQ ID NO: 53, and the lambda light chain constant region is as defined in SEQ ID NO:

54.

73. The aforementioned antibody (i) A CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. It includes, and in sequence number 9, X is H, The multispecific antibody is a bispecific antibody and includes an Fc region composed of a first and a second Fc polypeptide. The first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are contained within the same polypeptide chain. The first Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, and G236 to F, E, and R, respectively, and the second Fc polypeptide includes substitutions of amino acids corresponding to the amino acids at positions L234, L235, and D265 to F, E, and A, respectively, wherein the amino acid positions are as defined by Eu numbering. The pharmaceutical composition according to any one of claims 1 to 72, wherein the amino acid at the position corresponding to K409 in the first Fc polypeptide is R, and the amino acid at the position corresponding to F405 in the second Fc polypeptide is L.

74. The pharmaceutical composition according to any one of claims 1 to 73, wherein the antibody is bsIgG1-huCD3-FEALxCD30-FERR or a biosimilar thereof.

75. a) The antibody in a concentration of approximately 0.5 to 250 mg / ml, the antibody in a concentration of approximately 20 to 200 mg / ml, etc., wherein the antibody is (i) A CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and (ii) A CD3 binding region comprising a second heavy chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region including the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, wherein X in SEQ ID NO: 9 may be H, b) A buffering agent such as histidine or acetate, c) If necessary, a nonionic excipient such as sucrose, trehalose, or sorbitol, d) If necessary, a surfactant such as polysorbate, and a substance containing or essentially consisting thereof A pharmaceutical composition according to any one of claims 1 to 74, wherein the pH of the composition is about 5.0 to about 6.

5.

76. A pharmaceutical composition according to any one of claims 1 to 75, for use as a pharmaceutical product.

77. A pharmaceutical composition for use as a pharmaceutical according to claim 76 for use in a method of treating a disease.

78. A pharmaceutical composition according to any one of claims 1 to 75, for use in the treatment of cancer.

79. The pharmaceutical composition according to claim 78, for use in the treatment of Hodgkin lymphoma, such as classical Hodgkin lymphoma, or non-Hodgkin lymphoma (NHL).

80. The pharmaceutical composition for use according to claim 79, wherein the non-Hodgkin lymphoma is T-cell non-Hodgkin lymphoma (T-NHL).

81. The pharmaceutical composition for use according to claim 80, wherein T-NHL is cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL).

82. A pharmaceutical composition for use according to any one of claims 79 to 81, wherein T-cell non-Hodgkin lymphoma (T-NHL) is anaplastic large cell lymphoma (ALCL).

83. The pharmaceutical composition for use according to claim 78, wherein the non-Hodgkin lymphoma is B-cell non-Hodgkin lymphoma (B-NHL).

84. A pharmaceutical composition for use according to any one of claims 78 to 83, administered intravenously and / or subcutaneously, preferably subcutaneously.

85. Use of the pharmaceutical composition according to any one of claims 1 to 75 for the treatment of cancer.

86. It is a parts kit, a. A pharmaceutical composition according to any one of claims 1 to 75, b. A container for the pharmaceutical composition, c. Instructions for use of the aforementioned kit, A parts kit that includes this.

87. The parts kit according to claim 86, wherein the parts kit further comprises a diluent.

88. A method for preparing a pharmaceutical composition according to any one of claims 1 to 75, a) Multispecific antibodies, antibodies such as approximately 0.5 to approximately 250 mg / ml, b) Buffering agent, c) nonionic excipients, if necessary, If necessary, d) surfactant and The process of mixing in water, A method comprising the step of adjusting the pH to approximately 4.0 to 8.0.

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