Antibodies binding to CD30 and CD3

Abstract

To provide: CD30 targeting compounds that are efficacious and safe, have good manufacturability and / or have a long shelf-life; and improved CD30-targeting cancer therapy.SOLUTION: The invention provides multispecific antibodies binding to CD3 and CD30. The invention further provides pharmaceutical compositions comprising antibodies, nucleic acids encoding the antibodies, host cells that produce the antibodies, methods of producing the antibodies, and uses of the antibodies, in particular for cancer therapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a multispecific antibody that binds to CD30 and CD3. A pharmaceutical composition comprising the antibody, a nucleic acid encoding the antibody, a host cell producing the antibody, and a method for producing the antibody. The present invention further provides methods and uses of the antibodies, particularly for cancer therapy. [Background technology]

[0002] CD30, also known as Ki-1 or TNFRSF8, is a 120 kD transmembrane glycoprotein. Protein receptor and member of the tumor necrosis factor receptor (TNFR) superfamily (Smith et al. (1994) Cell 76:959-962). D30 is a single-pass type I protein with six cysteine-rich repeats in its extracellular domain. It is a membrane protein (Durkop et al. (1992) Cell 68:421 In addition, a soluble form of CD30 (sCD30) has been shown to be involved in the pathogenesis of ulcerative colitis (UC) ( Giacomelli et al.Clin Exp Immunol.1998;1 11:532-5) and inflammatory diseases, and CD30-positive hematological malignancies (Josimo vic-Alasevic et al. (1989) Eur J Immunol) sCD30 has been detected in the serum of patients with AD. Cleavage of the extracellular portion of CD30 by plasma membrane-anchored metalloproteinases such as AM10 Representing objects (Nagata et al., PNAS 2005, Hansen et al. l., FASEB, 2004).

[0003] In normal tissues, CD30 expression is primarily restricted to subsets of activated T and B lymphocytes. (Bowen et al. (1996) J Immunol. 156:442-9, Shanebeck et al. (1995)Eur J Immunol.25:2 147-53). CD30 expression has been detected in a variety of lymphoid tumors. Hodgkin's lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) have high CD30 expression. In particular, the Reed-Sternberg (R The majority of S) cells are positive for CD30 (Frizzera et al. 1992) Semin. Diagn. Pathol. 9:291-296).

[0004] For example, the CD30-targeting antibody-drug conjugate brentuximab vedotin (BV, SGN- 35) is used to treat cancers such as cHL, ALCL, and CTCL? It is recommended to use it (Younes et al. J Clin Onc ol.2012 Jun 20;30(18):2183-9, Pro et al.B lood.2017 Dec 21;130(25):2709-2717, Shea et al.Curr Hematol Malig Rep.2020 Feb;15 (1):9-19). The tetravalent bispecific CD30×CD16A antibody AFM13 was used to treat cHL and It is being developed as an NK cell-mediated immunotherapy for CD30-positive lymphomas (Rot he et al.Blood.2015 Jun 25;125(26):4024- 31).

[0005] In addition, CD30-targeted CAR-T cell therapy has been shown to be effective for cHL and CD30-positive lymphomas. Other CD30 antibodies have been developed in WO2003059282 (Medarex). , US8257706(Seattle genetics), US2010 / 0239 571 (Seattle Genetics), WO2007 / 040653 (U.S. Government and Health), and WO2016 / 0177846 (Affimed).

[0006] Pohl et al.(1993 Int.J.Cancer,54:820-82 7) The hybridoma cells producing the CD30 monoclonal antibody HRS-3 and the CD3 monoclonal Fusion with OKT-3-producing hybridoma cells (hybrid hybridoma We report the CD3 × CD30 bispecific antibody OKT-3 / HRS-3 generated by the ELISA technique. Posted.

[0007] WO2008 / 119567 discloses a CD30 and CD3 interspecies-specific bispecific single chain molecule The generation and characterization of

[0008] US2020 / 0095330 identifies two anti-CD30 clones (8D10 and 10C2 This is due to the chemical heteroconjugation of anti-CD3 (orthoclone OKT-3), which was named They describe a CD3xCD30 bispecific antibody that inhibits CD3xCD30 expression.

[0009] However, none of these CD3×CD30 bispecific antibodies have been tested in a clinical setting. It hasn't been done yet.

[0010] Thus, there is a need for improved CD30-targeted cancer therapies that are effective and safe. and a method for producing a compound targeted to CD30, which has good manufacturability and / or a long shelf life. There is a need. Summary of the Invention

[0011] The present invention relates to T cell inducing antibodies that bind to human and cynomolgus monkey CD30 and CD3. The present inventors have discovered that some CD30-binding antibodies are available in a bivalent (monoclonal) format. Although the antibody binds well to CD30-expressing cells, it also binds to CD30-expressing cells with monovalent CD30 binding. We found that the mat showed strongly reduced binding and cytotoxicity. Bispecific antibodies with superior binding and tumor cell killing were identified. A functionally inactive Fc backbone that is suitable for development into a pharmaceutical product due to its low molecular weight and solubility. A bispecific antibody having the same function as the IL-11 antibody has been identified.

[0012] In one aspect, the present invention provides a method for producing (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively. A first heavy chain variable region comprising the C3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. a first light chain variable region comprising DR1, CDR2, and CDR3 sequences; A region, (ii) CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 8, and 9, respectively. A second heavy chain variable region comprising the R3 sequence, and the R4 sequence is set forth in SEQ ID NOs: 10, 11, and 12, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences as set forth above. 3. A binding region; The present invention relates to a multispecific antibody comprising the

[0013] In a further aspect, the present invention provides a nucleic acid construct encoding a multispecific antibody according to the invention. The present invention relates to a combination of nucleic acid constructs.

[0014] In a further aspect, the present invention relates to an expression vector or a delivery vector comprising a nucleic acid construct according to the present invention. Regarding the vehicle.

[0015] In a further aspect, the present invention makes it possible to produce multispecific antibodies according to the present invention. With respect to recombinant host cells, the host cell may contain one or more vectors encoding the multispecific antibodies according to the invention. The nucleic acid construct includes:

[0016] In a further aspect, the present invention relates to a method for the preparation of a multispecific antibody according to the present invention and a pharma- ceutically acceptable and a carrier.

[0017] In a further aspect, the present invention relates to a compound according to the invention for use as a medicament, for example in the treatment of cancer. The present invention relates to a multispecific antibody, a nucleic acid construct, a delivery vehicle, or a pharmaceutical composition for do.

[0018] In a still further aspect, the present invention relates to a method for producing a multispecific antibody according to the invention. Regarding the law. [Brief description of the drawings]

[0019] [Figure 1]Binding of bispecific CD3xCD30 antibodies and their monospecific, bivalent CD3 and CD30 counterparts to SU-DHL-1 or HDML-2 cells. (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 IgG1-CD30 -hAC10-FEAR, (C)bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-HRS-3-FEA R, (D)BsIgG1-huCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-HeFi-I-FEAR, (E)bs IgG1-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- Dose-dependent binding of T408-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-T408-FEAR, and (H) bsIgG1-huCD3-FEALxCD30-T215-FEAR, bsG1-huCD3-FEALxb12-FEAR, and IgG1-CD30-HRS-3-FEAR to SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel). (I) Binding of CD3xCD30 bispecific and CD30 monospecific antibodies to SU-DHL-1 (left panel) or HDLM-2 cells (right panel) at a concentration of 1.11 μg / mL. The antibody clones used for the CD30 arm are indicated on the x-axis. Data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Diagram 2] Binding of bsG1-huCD3xCD30-MDX060 to HL and ALCL cell lines. Binding of bsG1-huCD3xCD30-MDX060 to (A) HDLM-2 (HL), (B) L-428 (HL), (C) DEL (ALCL), or (D) KI-JK (ALCL) cells was assessed by flow cytometry. Bispecific antibodies bsG1-huCD3xb12 and bsG1-b12xCD30-MDX060 and 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 indicated. Data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Diagram 3]Induction of in vitro cytotoxicity by CD3xCD30 bispecific antibodies in SU-DHL-1 or HDLM-2 cells. CD3xCD30 bispecific antibodies were tested in an in vitro cytotoxicity assay using CD30 positive tumor cell line SU-DHL-1 cells (left panel) or HDLM-2 cells (right panel) as target cells and T cells (CD3 positive ADCC effector cells type IV cells, Clean Cells, Montaigu, France) as effector cells. 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 (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-HRS-3-FEAR. Antibody bsG1-huCD3-FEALxb12-FEAR was included as a control in all experiments. Data shown are the percentage of viable cells and data for each graph are from one representative experiment. [Figure 4]In vitro T cell-mediated cytotoxicity and induction of T cell proliferation by CD3xCD30 bispecific antibodies in several ALCL and HL cell lines. (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 type IV cells (C) as effector cells. CD3xCD30 bispecific antibodies contained the huCD3-FEAL Fab arm or the huCD3-H101G-FEAL variant, which has a lower affinity for CD3, and the CD30-specific MDX060-FEAR Fab arm. IgG1-huCD3 and IgG1-b12 (A, B) or bsG1-b12-FEALxCD30-MDX060-FEAR and IgG1-CD30-MDX060-FEAR (C) were included as controls. Data shown are the percentage of viable cells, and data for each graph were obtained from one representative experiment. (D) The number of CFSE-positive cells was assessed as a measure of absolute T cell numbers in cytotoxicity assays using HDLM-2 cells (left panel) or NCEB-1 cells (right panel) as target cells. [Diagram 5]Binding of CD3xCD30 bispecific antibodies to full length human and cynomolgus CD30 transfected into Expi293F cells. (AC) Binding of monovalent and bivalent CD30 antibodies to wild type Expi293F cells (A) or Expi293F cells transiently transfected with full length human CD30 (B) or cynomolgus CD30 (C). Cells were incubated with increasing concentrations of the following antibodies: 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 technical replicates. (D) Binding of antibodies IgG1-CD30-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX060-FEAR, and bsG1-huCD3-FEALxb12-FEAR to human or cynomolgus T cells. Data are presented as mean fluorescence intensity (MFI) values ​​determined by flow cytometry of one representative experiment. [Figure 6]Binding of CD3xCD30 bispecific antibodies to full-length human and rhesus 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 rhesus CD30 (right panel) was assessed by flow cytometry. The following antibodies were evaluated: (A) 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-FEALxC (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-HRS-3-FEAR. The antibody bsG1-huCD3-FEALxb12-FEAR was included in all experiments as a negative control. Data shown are mean fluorescence intensity (MFI) values ​​determined by flow cytometry for one representative experiment. [Figure 7] Thermal stability of antibodies with different non-activating mutations as determined by Differential Scanning Fluorometry (DSF). Conformational protein stability at increasing temperatures was assessed in duplicate by DSF. Melting curves of 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 assessed by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. Data shown are 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 assessed by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. Data shown are 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. 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 assessed by flow cytometry. Bispecific antibodies bsG1-huCD3-FEALxb12-FERR and bsG1-b12-FEALxCD30-MDX060-FERR and monospecific antibodies IgG1-CD30-MDX060-FERR, IgG1-huCD3-FEAL, and IgG1-12-FEAL were included as controls. Data shown are 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 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 CD30 (B) was assessed by flow cytometry. bsG1-huCD3-FEALxb12-FERR was included as a negative control. 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 total viable cells in the presence of 6x10-5 to 10 μg / mL of bsG1huCD3FEALxCD30-MDX060-FERR or control antibodies bsG1-huCD3-FEALxb12-FEAR, bsG1b12FEALxCD30MDX060-FERR, or IgG1-b12-FEAL. The percentage of double-positive events in samples incubated without antibody is shown by the dotted line. (B) Example of the gating strategy for double positive cells in samples incubated with 0.12 μg / mL of bsG1huCD3FEALxCD30-MDX060-FERR. [Figure 13]Induction of T cell-mediated cytotoxicity and T cell activation in vitro by CD3xCD30 bispecific antibodies. A panel of CD3xCD30 bispecific antibodies was tested in in vitro cytotoxicity assays using the CD30 positive tumor cell line Karpas-299 as target cells and T cells purified from healthy human donor buffy coats as effector cells. In these assays, CD25 expression was assessed 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 of Karpas-299 cells by the tested antibodies. (CF) EC50 values ​​for induction of CD25 (CD) or PD-1 (EF) expression in CD4+ (C, E) or CD8+ (D, F) T cells by CD3xCD30 antibodies. Data are from two independent experiments performed on T cells obtained from six different donors. Statistical values ​​represent the results of Wilcoxon signed rank test between the indicated clones and bsG1-huCD3-FEALxCD30-MDX060-FERR. NS: not significant, *: p<0.05. [Figure 14]T cell-mediated cytotoxicity of cell lines in vitro 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. Data shown are percentage of viable cells and data for each graph were obtained for one representative experiment. [Figure 15] In vitro T cell proliferation by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell proliferation was assessed in a T cell-mediated cytotoxicity assay using L-428 (A, B) or KI-JK (C, D) as target cells. CD4+ (A, C) or CD8+ (B, D) T cells with diluted Celltrace Violet staining were gated and the proliferation index was calculated using the proliferation modeling tool from FlowJo as a measure for T cell proliferation. [Figure 16] Expression of the T cell activation marker CD69 in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was assessed in a T cell-mediated cytotoxicity assay using L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker CD69 was assessed in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 17]Expression of the T cell activation marker CD25 in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was assessed in a T cell-mediated cytotoxicity assay using L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker CD25 was assessed in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 18] Expression of the T cell activation marker PD-1 in vitro by bsG1-huCD3-FEALxCD30-MDX060-FERR in L-428 and KI-JK cell lines. T cell activation was assessed in a T cell-mediated cytotoxicity assay using L-428 (A, B) or KI-JK (C, D) as target cells. Expression of the T cell activation marker PD-1 was assessed in CD4+ (A, C) or CD8+ (B, D) T cells. [Figure 19] In vitro cytokine and granzyme B production 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, INFα) and granzyme B were evaluated in supernatants collected during in vitro T cell-mediated cytotoxicity experiments using L-428 as target cells. Cytokine and granzyme B concentrations are shown for samples treated with different concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR or control antibody IgG1-b12-FEAL. [Figure 20]In vitro T cell-mediated cytotoxicity and T cell proliferation by bsG1-huCD3-FEALxCD30-MDX060-FERR at varying effector to target ratios. (A) Dose-dependent T cell-mediated cytotoxicity by 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. Data shown are the percentage of viable cells from one representative experiment. (B, C) The percentage of CD4+ (B) or CD8+ (C) T cells with diluted Celltrace Violet staining is shown as a measure of proliferating T cells. [Figure 21] Kinetics of T cell-mediated cytotoxicity and T cell proliferation in vitro with bsG1-huCD3-FEALxCD30-MDX060-FERR. (A) Kinetics of 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 an E:T ratio of 4:1. Cytotoxicity was assessed after 24, 48, and 72 hours. bsG1-huCD3-FEALxb12-FERR was included as a control antibody. Data shown are the percentage of viable cells from one representative experiment. (B, C) CD4+ (B) or CD8+ (C) T cells with diluted Celltrace Violet staining were gated and the proliferation index was calculated using the proliferation modeling tool from FlowJo as a measure for T cell proliferation in the cytotoxicity assay. [Figure 22]Correlation between in vitro T cell-mediated cytotoxicity by bsG1-huCD3-FEALxCD30-MDX060-FERR and CD30 expression level. The correlation between maximum T cell-mediated killing by bsG1-huCD3-FEALxCD30-MDX060-FERR (A) or IC50 concentration of T cell-mediated cytotoxicity (B) versus CD30 expression level 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 evaluated using the Spearman rank correlation test (GraphPad Prism software). [Figure 23] Fratricide of activated T cells with bsG1-huCD3-FEALxCD30-MDX060-FERR. Isolated healthy donor T cells were stimulated with 1 μg / mL anti-CD3 (OKT-3), 1 μg / mL anti-CD28, and 0.025 μg / mL IL-15 for 4 days. After confirmation of activation (CD25 upregulation), T cells were incubated with increasing concentrations of bsG1-huCD3-FEALxCD30-MDX060-FERR, bsIgG1-ctrlxCD30-MDX-060-FERR, bsIgG1-CD3xb12, or IgG1-b12-FEAL for 48 hours. BsG1-huCD3-FEALxCD30-MDX060-FERR-induced T cell fratricide of activated CD30+ T cells was measured as the percentage of viable T cells in each condition relative to the number of viable T cells in the condition without any antibody added. (AB) The percentage of CD25+ (A) or CD30+ (B) cells among CD4+ or CD8+ T cells was determined by flow cytometry 72 and 96 hours after stimulation with anti-CD3, anti-CD28, and IL-15. (C) T cell fratricide is shown as the percent T cell survival relative to the condition without any antibody added. Data for one representative T cell donor is shown. [Figure 24]Soluble CD30 in the supernatant of CD30+ cell cultures and its interference with the antitumor activity of BsG1-huCD3-FEALxCD30-MDX060-FERR. (A) Soluble CD30 (sCD30) concentrations in the supernatants of different hematological tumor cell lines measured by ELISA. (B) The correlation between sCD30 concentrations and the number of CD30 molecules on the cell surface, determined using quantitative flow cytometry (human IgG calibrator kit-Biocytex), in different hematological tumor cell lines 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 target cells and healthy donor isolated T cells as effector cells. 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. Data shown are the percentage of viable cells from one representative experiment. [Diagram 25]Induction of T cell-mediated cytotoxicity and T cell proliferation ex vivo 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 Hodgkin's lymphoma (HL), acute myeloid leukemia (AML), and peripheral T cell lymphoma (PTCL) patients were used as a source of T cells to evaluate CD3-dependent tumor cell killing. IgG1-b12-FEAL was included as a control. Data shown are the percentage of viable target cells. (B-D) T cell activation was assessed by upregulation of CD69 (B), CD25 (C), and PD-1 (D) markers on CD4+ / CD8+ T cells within PBMC subsets, shown as the percentage of positive cells. [Figure 26] Plasma concentration of BsG1-huCD3-FEALxCD30-MDX060-FERR after intravenous injection in 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 mean human IgG1 concentration plotted over time. (B) Mean clearance rate plotted for 0.5 or 5 mg / kg dose levels. Dotted line indicates estimated clearance rate based on standard volume of distribution of unbound normal human IgG1 in mice. [Figure 27]C1q binding to membrane-bound bsG1-huCD3-FEALxCD30-MDX060-FERR. Binding of C1q to bsG1-huCD3-FEALxCD30-MDX060-FERR-opsonized, activated human CD8+ T cells, or CD30+ NCEB-1 cells was assessed by flow cytometry using a 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 replicate wells from 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. Data shown are gMFI from one representative experiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition As used herein, the term "antibody" refers to an antibody that is capable of binding to a cell for at least about 30 minutes, at least about 45 minutes, minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours for at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, A significant period of time, such as at least about 3, 4, 5, 6, 7 days or more, or any other relevant feature. Induce, promote, enhance, or increase the physiological response associated with antibody binding to an antigen for a period of time that is specifically defined. and / or for a period of time sufficient to modulate and / or for the antibody to be internalized. Specific to the antigen under typical physiological and / or tumor-specific conditions, with a half-life of 10-20 minutes or less. Immunoglobulin molecules and fragments of immunoglobulin molecules that have the ability to bind heterologously The term "antibody" is intended to refer to a molecule that interacts with an antigen. A binding region that can be used in the present specification (or both have the same meaning) a binding region that contains the variable regions of both the heavy and light chains of an immunoglobulin molecule, Antibodies are involved in the functioning of various cells of the immune system (e.g., effector cells) and components of the complement system, e.g. For example, C1q, the first component in the classical pathway of complement activation, is involved in the activation of host tissues or factors. It may include a constant region of an antibody (Ab) capable of mediating immunoglobulin binding.

[0021] In the context of the present invention, the term "antibody" refers to a monoclonal antibody (mAb), -like polypeptides, chimeric antibodies, human antibodies, humanized antibodies, as well as enzymatic cleavage and peptide synthesis and antigens (antigen-binding fragments) provided by any known technique, such as recombinant DNA technology. "Antibody fragments" or "antibody fragments thereof" that retain the ability to specifically bind to the antibody The term "antibody" includes bispecific, trispecific, or multispecific antibodies, and / or or antibodies with further modifications, such as antibody-drug conjugates and / or IgG Fc domains. Unless otherwise limited by the disclosure herein, the present invention includes antibodies having modifications in the An antibody defined according to the above may have any isotype or may have no isotype. It is possible that the antibody may not be specifically designed for use with the scFv antibody.

[0022] It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include: (i) Fab' or a Fab fragment, a light chain variable domain (VL), a heavy chain variable domain (VH), A monovalent fragment consisting of a light chain constant region (CL) and a heavy chain constant region domain 1 (CH1) domain. (ii) F(ab ') 2 The fragment consists of two Fab fragments linked by a disulfide bridge at the hinge region. (iii) a bivalent fragment comprising a VH and CH1 domain; Fd fragment, (iv) consisting essentially of the VL and VH domains of a single arm of an antibody Fv fragment, (v) H consisting essentially of a VH domain, also called a domain antibody olt et al;Trends Biotechnol-.2003 Nov;21 (11):484-90, dAb fragments Ward et al., Nature 341, 544546 (1989), (vi) Camelidae or Nanobody Revets et al;Expert Opin Biol Ther.2005 Jan;5( 1):111-24, and (vii) isolated complementarity determining regions (CDRs). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes. However, they can be encoded using recombinant methods such that the VL and VH regions are monovalent. These antibodies combine to form a molecule known as a single-chain antibody or single-chain Fv (scFv). The proteins are linked by a synthetic linker that allows them to be made into a single protein chain. See, for example, Revets et al., Expert Opin Biol Ther. .2005 Jan;5(1):111-24 and Bird et al.,Scien ce 242, 423-426 (1988). Such single chain antibodies can be Unless otherwise stated in the paragraph or clearly indicated by the context, the term antibody includes Although such fragments are generally included within the meaning of antibody, they are Collectively, and each independently, they are unique features of the present invention and have distinct biological properties and useful These and other useful antibody fragments in the context of the present invention are described herein. This is discussed further in.

[0023] As a final product, or for example, bispecific antibodies can be synthesized by controlled Fab-arm exchange (cF Antibodies, either as intermediates for production through different in vitro or can be produced in ex vivo expression or production systems and from there, e.g., recombinantly modified From the host cell, cells that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody are Cell extracts may be harvested from hybridomas or systems using the method.

[0024] As used herein, the term "immunoglobulin heavy chain" or "immunoglobulin heavy chain" refers to The term is intended to refer to one of the heavy chains of an immunoglobulin. The heavy chain variable region (abbreviated herein as VH) and the immunoglobulin isotype The heavy chain constant region of IgG consists of a heavy chain constant region (abbreviated as CH herein) that defines the The region typically consists of three domains, CH1, CH2, and CH3. The term "immunoglobulin" as used herein typically refers to an immunoglobulin that is made up of two pairs of polypeptide chains, Structurally related glycoproteins consisting of a pair of light (L) low molecular weight chains and a pair of heavy (H) chains The term is intended to refer to a class of molecules, all four of which are potentially interconnected by disulfide bonds. The structures of immunoglobulins are well characterized (e.g., Fundam et al., J. Immunol. 1999, 144:1311-1323). ental Immunology Ch.7(Paul,W.,ed.,2nd ed. (See Raven Press, NY (1989)). In the structure, the two heavy chains are held together by disulfide bonds in the so-called "hinge region". Similar to the heavy chains, each light chain typically comprises several regions, the light chain variable region (LVVL region). The light chain constant region is composed of a light chain constant region (abbreviated herein as VL) and a light chain constant region. Typically, it is composed of one domain, CL. Furthermore, the VH and VL regions are in frame. The complementarity determining regions (CDRs) are interspersed with more conserved regions called FRs. Hypervariable regions (or sequence and / or structurally defined regions) are also called CDRs. Each VH and VH fragment can be further divided into two subdomains, each of which can be hypervariable in the form of a loop. VL typically consists of three amino acid residues arranged from amino terminus to carboxy terminus in the following order: and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0025] As used herein, the terms "half molecule," "Fab-arm," and "arm" refer to The term refers to one heavy-light chain pair. A bispecific antibody is one half "derived from" a first antibody. When a molecule is described as including a molecular antibody, a half-molecule antibody "derived from" a second antibody, the term "derived from" is used interchangeably with "a molecular antibody." The term "bispecific antibody" refers to an antibody that binds to the first and second antibodies by any known method. and recombining the half molecules from each of the antibodies into the resulting bispecific antibody. In this context, "recombining" refers to any particular recombination method. Thus, for example, the invention may be combined with half-molecule exchange. and at the nucleic acid level and / or by co-expression of the two half molecules in the same cell. Methods for producing the bispecific antibodies described herein, comprising recombining Includes all of the above.

[0026] The terms "first" and "second" are used herein in the context of an antibody or a domain or region thereof. When used in the specification, it is intended merely for ease of reference and does not imply a specific relative position. It is not intended to indicate that

[0027] As used herein, the term "antigen-binding region" or "binding region" refers to a region that binds to an antigen. An antigen refers to a region of an antibody that can bind to a polypeptide, protein, polysaccharide, or other The antigen can be any molecule, such as a cell, a bacterium, or a combination of the above. The terms "antigen" and "target" are used interchangeably unless otherwise specified by context. and may be used interchangeably in the context of the present invention. The terms "position" and "position" are used interchangeably in the context of the present invention, unless otherwise contradicted by the context. It can be done.

[0028] As used herein, "K D The term "(M)" refers to the equilibrium state of a particular antibody-antigen interaction. Dissociation constant, k d k a It is obtained by dividing by K D Also, "binding affinity" It is called.

[0029] As used herein, "kd " (sec -1 The term ) refers to a specific antibody-antigen interaction. This value is the dissociation rate constant for k off This is also referred to as the off-value or off-rate.

[0030] As used herein, "k a " " -1 xsec -1 The term ) refers to a specific antibody-antibody This value is the association rate constant of the original interaction, k on It is also called the value or on-rate.

[0031] As used herein, the term "binding" refers to the binding of an antibody to a given antigen or target, typically Specifically, biolayer interferometry uses antibodies as ligands and antigens as analytes. When it is determined that -6 M or less, for example, 5E -7 M or less, 1E -7 M or less, e.g. 5E -8 M or less, for example, 1E -8 M or less, for example, 5E -9 M or less, for example, 1E -9 M or less, for example, 1E -10 M or less, or, for example, 1E -11 K below M D Corresponding to The term refers to binding with a binding affinity that is greater than or equal to the binding affinity of a given antigen, and refers to binding with a non-specific antigen other than the given antigen (e.g., less than its affinity for binding to BSA, casein) or closely related antigens at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1,000 times lower e.g., at least 10,000 times lower, e.g., at least 100,000 times lower K D with an affinity corresponding to

[0032] As used herein, the term "CD30" refers to TNFRSF8 (tumor necrosis factor receptor 8). Human Cluster of Differentiation 30 protein, also known as a member of the human mitochondrial cell line superfamily 8) CD30 is found in a variety of species, and therefore the term "CD30" is used interchangeably with Unless otherwise contradicted by the context, the sequence of human CD30 may not be limited to human CD30. The columns are set forth in SEQ ID NO:39.

[0033] As used herein, the term "CD3" refers to a member of the T cell coreceptor protein complex. CD3 is a human cluster of differentiation 3 protein that is composed of four distinct chains. It is found in a variety of species, and therefore the term "CD3" is not contradictory depending on the context. In mammals, the complex may be CD3γ( gamma) chain (human CD3 gamma chain UniProtKB / Swiss-Prot No P09 693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No Q95LI7), CD3δ (delta) chain (human CD3δ UniProtKB / Swi ss-Prot No. P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No Q95LI8), two CD3ε (epsilon) chains ( CD3ε: UniProtKB / Swiss-Prot No. P07766, The sequence herein is incorporated as SEQ ID NO: 42, and is incorporated herein by reference. iProtKB / Swiss-Prot No Q95LI5, or rhesus CD3ε UniProtKB / Swiss-Prot No G7NCB9), and CD3 ζ chain (Zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No P2 0963, Macaca fascicularis CD3ζ UniProtKB / Swiss-Prot No These chains associate with a molecule known as the T cell receptor (TCR). Together, the TCR and CD3 molecules generate an activation signal in T lymphocytes. , including the TCR complex.

[0034] The term "antibody binding region" refers to the region of an antigen that contains the epitope to which an antibody binds. Antibody binding regions were identified by epitope binning using biolayer interferometry. by Ninscan or domain shuffling assays (where a region of an antigen is identified as a region of another species) (using the exchanged antigen construct and determining whether the antibody still binds to the antigen) The amino acids in the antibody binding region involved in the interaction with the antibody can be determined by hydrogen / Determined by deuterium exchange mass spectrometry and / or by crystallography of antibody binding to that antigen It can be done.

[0035] The term "epitope" means an antigenic determinant specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof. They usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational epitopes are epitopes whose binding disrupts the three-dimensional structure of a protein or its multimers. in that the binding to the latter is not lost in the presence of denaturing solvents or other agents that inhibit the binding of the former. An epitope is a set of amino acid residues that are directly involved in binding and those that are not directly involved in binding. Other amino acid residues that are not effective in blocking or blocking the antibody when it is bound to the antigen, for example The amino acid residues may be barred.

[0036] "Monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb" and like terms as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody is a polypeptide that typically exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are typically produced from cultures, such as hybridomas, stable cell lines, etc. They can be produced by the same cell that is a clone of every unique parent cell. The term "human monoclonal antibody" refers to an antibody derived from human germline immunoglobulin sequences. Human monoclonal antibody refers to an antibody that exhibits a single binding specificity and has variable and constant regions that correspond to the same antibody. has a genome containing human heavy and light chain transgenes fused to an immortalized cell. Transgenic or transchromosomal non-human animals, e.g., transgenic mice It can be produced by a hybridoma containing B cells obtained from a mouse. Monoclonal antibodies can be derived from human B cells or plasma cells. Monoclonal antibodies can also be derived from recombinantly modified 2. In vitro transcription and / or translation of nucleic acid sequences encoding antibodies The antibody can be produced from a system that uses cell extracts that support the antibody.

[0037] As used herein, the term "isotype" refers to an immunoglobulin class (e.g., , IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM ), or IgG1m(za) and IgG1m( f) and any of their allotypes. In addition, each heavy chain isotype can be divided into kappa ( It can be combined with either kappa (κ) or lambda (λ) light chains.

[0038] The term "full antibody" as used herein refers to a pair of heavy and light chains, or two The antibody comprises paired heavy and light chains, each pair being identical to the heavy-light chain pair typically found in a wild-type antibody of that isotype. The antibody (e.g., parent antibody or antibody fragment) may comprise heavy and light chain constant and variable domains as found in Thus, for example, a full-length IgG1 antibody has VH, CH1, CH2, It contains the CH3, hinge, VL, and CL domains. The chain and light chain constant and variable domains are particularly similar to the antibody when compared to the full-length parent or wild-type antibody. The full length polypeptides of the present invention may contain amino acid substitutions that modify and / or improve the functional properties of the polypeptides. The antibody may be prepared by (i) incorporating the CDR sequences into one or more suitable vectors containing the complete heavy and light chain sequences. and (ii) cloning the heavy and light chain sequences into a suitable expression system. and expressing the resulting suitable vector. It is possible to produce full length antibodies when starting with either the DR sequence or the complete variable region sequence. It is within the knowledge of a person skilled in the art to know how to generate a full-length antibody according to the present invention. I know the law.

[0039] As used herein, the term "humanized antibody" refers to an antibody that has a high level of human variable domains. A human antibody constant domain and a non-human variable domain modified to contain sequence homology with each other. This refers to a genetically engineered non-human antibody that contains multiple amino acids that together form an antigen-binding site. The homologous human acceptor framework regions of the non-human antibody complementarity determining regions (CDRs) This can be achieved by grafting onto the FR region (see, inter alia, WO92 / 22653 and EP0629240). To achieve this, some of the framework residues from the parent antibody (i.e., the non-human antibody) are Some substitutions (backmutations) of human framework residues of the SEQ ID NO:1 may be required. The modeling identifies amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, humanized antibodies can be used to identify non-human CDR sequences, primarily non-human amino acids. human framework regions, optionally including one or more amino acid backmutations to the amino acid sequence; The entire human constant region may include additional amino acid modifications, which may optionally be, but are not necessarily, backmutations. To obtain humanized antibodies with favorable characteristics, such as particularly useful affinity and biochemical properties to include modifications that, for example, avoid deamidation and / or improve production. Additionally, the CDR and / or framework regions may be modified to improve the affinity of the antibody, for example through affinity maturation procedures. The antibody may be modified to improve its affinity for the antigen.

[0040] As used herein, the term "human antibody" refers to an antibody that is synthesized based on human germline immunoglobulin sequences. A human antibody refers to an antibody having variable and constant regions derived from human germline immunoglobulins. Amino acid residues not encoded by the purine sequence (e.g., in vitro random or introduced by site-directed mutagenesis or by in vivo somatic mutation. However, as used herein, the term "human antibody" refers to a human antibody. CDR sequences derived from the germline of another mammalian species, such as a mouse, are placed on human framework sequences. The human monoclonal antibodies of the present invention are not intended to include antibodies that have been grafted onto a human antibody. , traditional monoclonal antibody methodologies, e.g., Kohler and Milstein ,Nature 256:495(1975) standard somatic cell hybridization techniques These can be produced by a variety of techniques, including somatic cell hybridization procedures. is preferred, but in principle other techniques for producing monoclonal antibodies are also possible, e.g. Viruses using B-lymphocyte or phage display technology using libraries of human antibody genes Human monoclonal antibodies can be used in the detection of HCo1 In mouse systems, such as mouse (see, e.g., WO03 / 059282), Instead, they use transgenic or transchromosomal mice carrying parts of the human immune system. It can be generated by

[0041] As used herein, the term "Fc region" refers to the N chain of two heavy polypeptide chains of an antibody. In the terminal to C-terminal direction, at least the hinge region, the CH2 region, and the CH3 region The Fc region of an antibody is typically glycosylated. , host tissue or cells, including various cells of the immune system (such as effector cells) and components of the complement system. The Fc region typically mediates the binding of immunoglobulins to FcRn and TA. It also binds to protein A.

[0042] As used herein, phrases such as "an amino acid corresponding to an amino acid at position ..." refer to a human I Refers to the amino acid position number in the gG1 heavy chain. The amino acid positions can be found by alignment with human IgG1. As used herein, the amino acids of the constant region sequences are referred to as E, E-, E-C, E-D, E-F, E-F, E-G, E-H ... Numbered according to the U-index numbering system (Kabat, EAet al.,1991,Sequences of proteins of immunity logical interest.5th Edition-US Departme nt of Health and Human Services,NIH publ (Described in Publication No. 91-3242, pp. 662, 680, 689) Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence An amino acid or segment can be combined with other amino acids or segments using ALIGN, Cluster, A standard sequence alignment program such as .lW, or similar, typically running with default settings The alignment was performed using a fixed ratio of at least 50% and at least 80% for human IgG1 heavy chains. %, at least 90%, or at least 95% identity to the nucleic acid sequence of the present invention. In this section, sequences or segments in sequences are aligned, thereby identifying amino acids according to the present invention. Methods for determining the corresponding positions in a sequence for an acid position are believed to be well known. do.

[0043] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody is al.,Sequences of proteins of immunology cal interest.5th Edition-US Department o f Health and Human Services,NIH publicat ion No.91-3242, pp 662, 680, 689 (1991) It corresponds to amino acids 216-230 according to the Eu numbering system in The region may also be of any of the other subtypes described herein.

[0044] As used herein, the term "CH1 region" or "CH1 domain" refers to an immunoglobulin The CH1 region of the IgG1 antibody is the CH1 region of the IgG1 antibody. Corresponding to amino acids 118 to 215 according to the Eu numbering described in abat (ibid.) However, the CH1 region may also be present in any of the other subtypes described herein. It could be either.

[0045] As used herein, the term "CH2 region" or "CH2 domain" refers to an immunoglobulin The CH2 region of the IgG1 antibody is the CH2 region of the IgG1 heavy chain. Corresponding to amino acids 231 to 340 according to the Eu numbering described in abat (ibid.) However, the CH2 region may also be associated with any of the other subtypes described herein. It could be either.

[0046] As used herein, the term "CH3 region" or "CH3 domain" refers to an immunoglobulin The CH3 region of the IgG1 antibody is the CH3 region of the IgG1 antibody. Corresponding to amino acids 341 to 447 according to the Eu numbering described in abat (ibid.) However, the CH3 region may also be associated with any of the other subtypes described herein. It could be either.

[0047] As used herein, the term "Fc-mediated effector function" refers to an Fc-mediated effector function. A polypeptide or antibody, the effector function of which is attributable to the Fc region of the polypeptide or antibody. It is intended to refer to the function that results from binding to its target or antigen on the cell membrane. Examples of mediated effector functions include (i) C1q binding, (ii) complement activation, (ii i) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (AD) CC), (v) Fc-gamma receptor (FcgR) binding, (vi) antibody-dependent, FcγR-mediated (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement-dependent (ix) complement-enhanced cytotoxicity (CDCC), (x) antibody-mediated (xi) opsonization; and (xii) (i) binding of the opsonizing antibody to a complement receptor. ) to (xi).

[0048] As used herein, "inertness" and "inert The terms "non-activated" and "non-activated" refer to antibodies that are unable to bind to any FcγR or that are not activated by any FcγR. It binds to any FcγR and induces Fc-mediated cross-linking of FcγR, and is involved in ADCC and ADC. Induce FcγR-mediated effector functions such as P and β-actin through the two Fc regions of individual antibodies. have minimal ability to induce FcγR-mediated cross-linking of target antigens and / or bind to C1q and are unable to induce complement-mediated effector functions such as CDC and CDCC. The term Fc region refers to the inactivation of the Fc region of an antibody in a monospecific or bispecific format. Antibodies can be used to test.

[0049] The term "monovalent antibody" in the context of the present invention refers to an antibody that contains one antigen-binding domain (e.g. Refers to an antibody molecule that has only one Fab arm (and one Fab arm) that can interact with an antigen. In the context of multispecific, such as bispecific, antibodies, "monovalent antibody binding" refers to the binding of a multispecific antibody. to one antigen having only one antigen-binding domain (e.g., one Fab arm) This refers to the bond between

[0050] The term "monospecific antibody" in the context of the present invention refers to an antibody that binds to one antigen, one epitope, or both. The term "monospecific, monovalent antibody" refers to an antibody that has binding specificity for only one target polypeptide. monospecific, bivalent antibodies (e.g., antibodies that bind two identical an antibody having an antigen-binding region of The antibody may be an antibody having an antigen-binding region.

[0051] The term "multispecific antibody" refers to an antibody that binds to two or more different epitopes. The term "bispecific antibody" refers to an antibody that has an antigen-binding domain that binds different epitopes. Two antigen-binding domains that bind to the polypeptide, e.g., two non-identical pairs of VH and VL domains, Has two non-identical Fab arms, or two Fab arms with non-identical CDR regions In the context of the present invention, bispecific and multispecific antibodies refer to antibodies that Such epitopes may be the same or different. If the epitopes are on different antigens, such The source may be the same or different cells, such as extracellular matrix or vesicles and soluble proteins. The antibody may be on a cell, cell type, or structure. Thus, multispecific and bispecific antibodies can be on multiple In some cases, antigens of different types, for example, may be able to cross-link two different cells.

[0052] The term "bivalent antibody" refers to an antibody having two antigen-binding regions, The regions may be identical and capable of binding to the same epitope, or they may be non-identical and capable of binding to the same epitope. Alternatively, the antibody may bind to different epitopes, which may be on different antigens. Thus, a bivalent antibody can be a monospecific or a bispecific antibody.

[0053] As used herein, the terms "amino acid" and "amino acid residue" may be used interchangeably. Amino acids are defined by their specific side chains (R groups) and Both are amines (-NH 2 ) and organic compounds containing carboxyl (-COOH) functional groups In the context of the present invention, amino acids are classified based on structural and chemical property analysis. Thus, the classes of amino acids may be reflected in one or both of the following tables: : [Table 1] [Table 2]

[0054] Substitutions of one amino acid for another are classified as conservative or non-conservative substitutions. In the context of the present invention, a "conservative substitution" refers to a substitution of one amino acid with a similar structure and / or structure. or a substitution of one amino acid residue with another amino acid having the above two chemical characteristics. Such substitutions for another amino acid residue of the same class as defined in any of the tables below: For example, a conservative substitution would be an isoleucine for leucine, since both are aliphatic, branched, and hydrophobic. Similarly, an example of a conservative substitution would be a substitution of Therefore, it is a substitution of aspartic acid with glutamic acid.

[0055] In the context of the present invention, substitutions in an antibody are made in the following order: original amino acid-number of positions-substitute amino acid It is shown as:

[0056] With reference to the well-known nomenclature for amino acids, the following formula can be used to denote any amino acid residue: A three-letter code or a one-letter code containing the code "Xaa" or "X" is used. , Xaa or X may typically represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to the following amino acid residue: glycine , alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline , tryptophan, phenylalanine, tyrosine, methionine, and cysteine It refers to one of them.

[0057] Therefore, the designation "K409R" or "Lys409Arg" indicates that the antibody It is meant to include the substitution of lysine at acid position 409 with arginine. Substitution of an amino acid in the present invention with any other amino acid may be made at the original amino acid position or, e.g., The original amino acid and / or the substituted amino acid are two or more. For modifications that may involve less than all amino acids, two or more amino acids may be denoted by "," or " For example, the lysine at position 409 can be separated by arginine, alanine, or fluorine. The substitution with phenylalanine is "Lys409Arg, Ala, Phe" or "Lys40 9Arg / Ala / Phe" or "K409R, A, F" or "K409R / A / F" or is "K409~R, A, or F." Such designations are within the context of the present invention. They may be used interchangeably and have the same meaning and purpose.

[0058] Furthermore, the term "substitution" refers to any one or more of the other 19 naturally occurring amino acids, or includes substitutions with other amino acids, such as unnatural amino acids. For example, the amino acid at position 409 The substitutions for K include each of the following substitutions: 409A, 409C, 409D, 409E, 40 9F, 409G, 409H, 4091, 409L, 409M, 409N, 409Q, 40 9R, 409S, 409T, 409V, 409W, 409P, and 409Y. The substitutions may also be K409A, K409C, etc., or K409A, C, etc., or K409A / C / etc. The same applies by analogy to each and every position mentioned herein. Thus, any one of such substitutions is specifically included herein.

[0059] As used herein, the term "host cell" refers to a cell into which a nucleic acid, such as an expression vector, has been introduced. Such terms are intended to refer not only to a particular subject cell, but also to It should be understood that the progeny of such cells are also intended to be included. Modifications can occur in subsequent generations, either by mutation or by environmental influences, so that The progeny may not, in fact, be identical to the parent cell, but are still referred to as such herein. Recombinant host cells (i.e., recombinant tandem Host cells used for the production of proteins include, for example, transfectomas, e.g. , CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells such as E. coli, and plant cells and yeast cells. Other eukaryotic hosts include bacteria.

[0060] As used herein, the term "transfectoma" refers to a cell that produces an antibody or a target antigen. Recombinant eukaryotic host cells expressing the recombinant eukaryotic vector, such as CHO cells, PER.C6 cells, NS0 cells, HE cells, The fungus may include a fungus, such as a K-293 cell, an Expi293F cell, a plant cell, or a yeast cell.

[0061] For purposes of the present invention, sequence identity between two amino acid sequences is measured over the length of the reference sequence. The EMBOSS package (EMBOSS: The European Molecular Biology) lar Biology Open Software Suite,Rice et al., 2000, Trends Genet. 16:276-277) program, preferably implemented in version 5.0.0 or later of Needleman-W The Wunsch algorithm (Needleman and Wunsch, 1970, J. The parameters used are determined using the NMR spectroscopy (Mol. Biol. 48:443-453). The data was generated with a gap open penalty of 10, a gap extension penalty of 0.5, and EB LOSUM62 (EMBOSS version of BLOSUM62) is a substitution matrix The Ne labeled "longest identity" (obtained using the -nobrief option) The output of edle is used as the percent identity and is calculated as follows: (identical residues × 100) / (length of alignment − total number of gaps in alignment) .

[0062] Retention of similar residues may also or alternatively be determined using a BLAST program (e.g., using the standard settings of BLAST). Through NCBI using OSUM62, open gap = 11, and extended gap = 1 Similarity scores, as determined by use of the BLAST 2.2.8 protocol available at Suitable variants are typically characterized by a small number of copies relative to the parent or reference sequence. 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%) This shows a similarity of 45%. [Table 3] TIFF2024116279000004.tif180152TIFF2024116279000005.tif174152TIFF2024116279000006.tif140151TIFF202 4116279000007.tif174151TIFF2024116279000008.tif140151TIFF2024116279000009.tif174151TIFF20241162790 00010.tif172152TIFF2024116279000011.tif191152TIFF2024116279000012.tif191153TIFF2024116279000013.t if186151TIFF2024116279000014.tif156152TIFF2024116279000015.tif185152TIFF2024116279000016.tif100151

[0063] Further aspects and embodiments of the invention As stated above, in a first aspect, the present invention provides a method for producing a composition comprising the steps of: (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively. A first heavy chain variable region comprising the C3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. a first light chain variable region comprising DR1, CDR2, and CDR3 sequences; A region, (ii) CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 8, and 9, respectively. A second heavy chain variable region comprising the R3 sequence, and the R4 sequence is set forth in SEQ ID NOs: 10, 11, and 12, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences as set forth above. 3. A binding region; The present invention relates to a multispecific antibody comprising the

[0064] In one embodiment, X in SEQ ID NO: 9 is H. In column number 9, X is G.

[0065] In one embodiment, the first heavy chain variable region is human or humanized. In one embodiment, the first light 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 or humanized. In another embodiment, the first heavy chain variable region and the first 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 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 humanized. is human, and the second heavy chain variable region and the second light chain variable region are human. In one embodiment, the first heavy chain variable region and the first light chain variable region are human and the second heavy chain The variable region and the second light chain variable region are humanized.

[0066] 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 the variable regions having three CDR sequences, C Each of the CDRs comprises four framework sequences: FR1, FR2, and FR3. , FR2, FR3, and FR4. This structure is preferably In one embodiment, one, two or more of the four framework sequences are also found in One, three or all may be human framework sequences.

[0067] As described above, the multispecific antibody according to the invention is capable of binding to human CD30. The antibody according to the invention comprises an antigen-binding region, the sequence of which is set out in SEQ ID NO: 39. The antigen-binding region capable of binding to human CD30 is preferably a marker for a cell, more preferably a tumor. Binding to the extracellular domain of human CD30, such as the CD30 molecule expressed on tumor cells. It is an antibody that can

[0068] As described, the antibodies have the CDR1s set forth in SEQ ID NOs: 1, 2, and 3, respectively. a first heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, and and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in and a CD30 binding region comprising:

[0069] CDR1, CDR2, and CDR3 regions can be isolated using methods known in the art. The CDRs from the variable heavy and light chain regions can be identified. Regions are annotated according to IMGT (Lefranc, M.-P., T he Immunologist,7,132-136(1999), Lefranc, Developmental and Comparative Immunology ,27(1),55-77(2003).

[0070] In one embodiment, the antigen binding region that binds to CD30 comprises The sequence SEQ ID NO: 13 or at least 90%, at least 95% identical to the sequence SEQ ID NO: 13 , or sequences having at least 97% or at least 99% amino acid sequence identity. a (first) heavy chain variable region (VH) comprising The sequence SEQ ID NO: 14 or at least 90%, at least 95% identical to the sequence SEQ ID NO: 14 , or sequences having at least 97% or at least 99% amino acid sequence identity. a (first) light chain variable region (VL); Includes.

[0071] In a further embodiment, the antibody according to the invention binds to CD30 as defined herein. The heavy chain variable (VH) region of the antigen binding region corresponds to SEQ ID NO: 13. and may contain, in total, up to 1, 2, 3, 4, or 5 amino acid substitutions.

[0072] In a further embodiment, the antibody according to the invention binds to CD30 as defined herein. The antigen binding region of the VL domain is a light chain variable region of the VL domain of the antigen binding region of the VL domain. and may contain, in total, up to 1, 2, 3, 4, or 5 amino acid substitutions.

[0073] In a further embodiment, the first heavy chain variable region comprises the sequence set forth in SEQ ID NO:13. and the first light chain variable region comprises the sequence set forth in SEQ ID NO:14.

[0074] Such antigen-binding regions capable of binding to human CD30 are, inter alia, described in No. 5,993,333, which is incorporated herein by reference. do.

[0075] The antibody according to the present invention has an equilibrium dissociation constant KD between the antigen-binding domains that bind to human CD30. and human CD30 can bind to the antibody at a concentration in the range of 0.1 to 20 nM, for example, 1.5 to 2 nM. The binding affinity is in the range of 0.5 to 5 nM, such as the monovalent binding range. This can be determined by interferometry.

[0076] In one embodiment, the antibody of the invention also binds to cynomolgus CD30 (SEQ ID NO: 40). can be combined.

[0077] As described above, the multispecific antibody according to the invention comprises an antibody capable of binding to human CD3. The present invention further relates to a method for the production of human CD3ε (epsilon)-binding domains as defined in SEQ ID NO: 22. The present invention provides an antibody capable of binding to human CD3ε (epsilon), such as CD3ε (epsilon). Such antigen-binding regions are provided for presentation on T cells, such as primary human T cells. , and is capable of binding to human CD3ε (epsilon).

[0078] As described, the antibodies have the CDR1s set forth in SEQ ID NOs: 7, 8, and 9, respectively. , CDR2, and CDR3 sequences, respectively, and a second heavy chain variable region comprising the sequences set forth in SEQ ID NO: 10, 11 and 12. and a CD3 binding domain comprising the variable region.

[0079] In one embodiment, the antigen-binding region that binds to CD3 is The sequence SEQ ID NO: 15 or at least 90%, at least 95% identical to the sequence SEQ ID NO: 15 , or sequences having at least 97% or at least 99% amino acid sequence identity. A (second) heavy chain variable region (VH) The sequence SEQ ID NO: 16 or at least 90%, at least 95% identical to the sequence SEQ ID NO: 16 , or sequences having at least 97% or at least 99% amino acid sequence identity. a (second) light chain variable region (VL); Includes.

[0080] In a further embodiment, the antibody according to the invention is a polypeptide that binds to CD3 as defined herein. The heavy chain variable (VH) region of the antigen binding region is selected from the group consisting of: , containing up to 1, 2, 3, 4, or 5 amino acid substitutions in total.

[0081] In a further embodiment, the antibody according to the invention is a polypeptide that binds to CD3 as defined herein. The antigen binding region comprises a light chain variable (VL) region, and the sequence is, when compared to SEQ ID NO: 16, , containing up to 1, 2, 3, 4, or 5 amino acid substitutions in total.

[0082] In a further embodiment, the second heavy chain variable region comprises the sequence set forth in SEQ ID NO:15. and the second light chain variable region comprises the sequence set forth in SEQ ID NO: 16. In another embodiment, X in SEQ ID NO: 15 is H. X is G.

[0083] Such antigen-binding regions capable of binding to human CD3 are, inter alia, those described by reference herein. The present invention is described in WO2015 / 001085 (Genmab), which is incorporated herein by reference. Variants of the present invention include, for example, the VH C sequence set forth in SEQ ID NO: 9, in which X is G. DR3 region and X is H, and has a lower affinity for binding to human CD3 than the parent antibody Variants include those described in WO2017 / 0094, which is incorporated herein by reference. 42 (Genmab), Example 2.

[0084] The antibody of the present invention has an equilibrium dissociation constant KD between the antigen-binding domains that bind to human CD3. and human CD3 can bind to the antibody at a concentration of 5 to 30 nM, for example, 10 to 20 nM (in the case of monovalent binding). It is within range.

[0085] In one embodiment, the antibody of the invention also binds to cynomolgus CD3 (SEQ ID NO: 43). It can be combined.

[0086] In a further embodiment, the multispecific antibody comprises: (i) a first heavy chain variable region comprising the sequence set forth in SEQ ID NO: 13 and a second heavy chain variable region comprising the sequence set forth in SEQ ID NO: 14; a first light chain variable region comprising a sequence as defined by the formula (I) and a CD30 binding region comprising the sequence as defined by the formula (I); (ii) a second heavy chain variable region comprising the sequence set forth in SEQ ID NO: 15 and a second heavy chain variable region comprising the sequence set forth in SEQ ID NO: 16; a second light chain variable region comprising the sequence set forth in claim 1; and a CD3 binding region comprising the sequence set forth in claim 1. Includes.

[0087] Antibody Format The multispecific antibodies of the present invention have two or more specificities, e.g., two or more specificities. Furthermore, the multispecific antibody may have the same or similar antigen-binding domains as those of CD3 and / or CD30. It may have more than one copy. For example, in one embodiment, the antibody binds to CD3. and having two identical antigen-binding regions capable of binding to CD3, e.g. For example, in another embodiment, the antibody comprises two antigen-binding regions that bind to CD30. For example, the antibody has two identical binding regions that bind to CD30. The additional antigen-binding region may be For example, it may be in the form of an scFv covalently linked to the constant region.

[0088] In a preferred embodiment, the multispecific antibody of the invention is a bispecific antibody. Many different formats and uses of specific antibodies are known in the art, see Kont. ermann;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 Di scov 18(8)585-608. Bispecific antibodies according to the invention The present invention may be practiced without limitation to any particular bispecific format or method of producing it. be.

[0089] Examples of bispecific antibody molecules that can be used in the present invention include (i) bispecific antibodies that contain two different antigen-binding regions. (ii) a single antibody having two arms, e.g., linked in tandem by an additional peptide linker; A single-chain antibody with specificity for two different targets via two scFvs bound to the antibody molecule. (iii) each light and heavy chain has two variable domains in tandem via short peptide bonds; Dual variable domain antibodies (DVD-Ig) containing n and Characterization of a Dual Variable e Domain Immunoglobulin (DVD-I(trademark)) Molecu le,In:Antibody Engineering,Springer Berl in Heidelberg (2010)), (iv) chemically linked bispecifics (F (v) a tetravalent bispecific fragment having two binding sites for each of the target antigens; Tandab, a fusion of two single-chain diabodies resulting in a bispecific antibody i) flexibodies, which are combinations of scFv with diabodies resulting in multivalent molecules (vii) when applied to Fab, two identical Fab fragments bound to different Fab fragments A trivalent bispecific binding protein consisting of a Fab fragment of Based on the "dimerization and docking domain" in protein kinase A, the so-called (viii) "dock-and-lock" molecules, e.g., fused to both ends of a human Fab arm; (ix) diabodies, so-called scorpion molecules, which contain two scFvs combined Includes.

[0090] Further examples of bispecific antibodies of different classes include, but are not limited to, (i) (ii) IgG-like molecules with complementary CH3 domains that force heterodimerization; Each of the two flanks is bound to a Fab fragment or Fab fragments of at least two different antibodies. (iii) a recombinant IgG-like dual targeting molecule comprising a portion of a full length IgG antibody, IgG fusion molecules fused to additional Fab fragments or portions of Fab fragments (iv) a single chain Fv molecule or a stabilized diabody comprising a heavy chain constant domain, an Fc region, or a portion thereof, Fc fusion molecules; (v) different Fab fragments, fused to a heavy chain constant domain, an Fc region, or a portion thereof; (vi) different single chain Fv molecules or different diabodies or different heavy chain antibodies The antibodies (e.g., domain antibodies, nanobodies) may be synthesized by combining heavy chain constant domains, Fc regions, or a portion thereof, fused to another protein or carrier molecule, and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies). .

[0091] Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to, However, the triomab / quadroma molecule (Trion Pharma / Fresenius Biotech, Roche, WO2011 / 069104), the so-called knob-into -Hole molecules (Genentech, WO98 / 50431), CrossMAbs (R oche, WO2011 / 117329), and electrostatic compatible molecules (Amgen, EP187 0459 and WO2009 / 089004, Chugai, US2010 / 001551 33, Oncomed, WO2010 / 129304), LUZ-Y molecule (Genent ech, Wranik et al.J.Biol.Chem.2012,287(52 ):43331-9,doi:10.1074 / jbc.M112.397869.Ep ub 2012 Nov 1), DIG-body and PIG-body molecules (Pharma bcine, WO2010 / 134666, WO2014 / 081202), strand exchange operation Domain body (SEEDbody) molecule (EMD Serono, WO2007 / 11 0205), Biclonics molecules (Merus, WO2013 / 157953), F cΔAdp molecule (Regeneron, WO2010 / 15792), bispecific IgG 1 and IgG2 molecules (Pfizer / Rinat, WO11 / 143545), Azyme etric scaffold molecules (Zymeworks / Merck, WO2012 / 058768) , mAb-Fv molecule (Xencor, WO2011028952), bivalent bispecific antibody (WO2009 / 080254), as well as DuoBody® molecules (Genma b A / S, WO2011 / 131746).

[0092] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)- Ig molecules (WO2009 / 058383), two-in-one antibodies (Genentech, B ostrom,et al 2009.Science 323,1610-1614. ), cross-linked Mab (Karmanos Cancer Center), mAb2 (FS tar, WO2008 / 003116), Zybody molecules (Zygenia, LaF leur et al.MAbs.2013 Mar-Apr;5(2):208-18 ), common light chain approach (Crucell / Merus, US7,262,028 ), kappa / lambda body™ molecules (NovImmune, WO2012 / 0230 53), and CovX-Body (CovX / Pfizer, Doppalapudi, V .R.,et al 2007.Bioorg.Med.Chem.Lett.17,5 01-506.)

[0093] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD) fusion molecules. -Ig molecule (Abbott, US7,612,181), double-domain double-headed antibody (Unilever, Sanofi Aventis, WO2010 / 0226923) , IgG-like bispecific molecule (ImClone / Eli Lilly, Lewis et al. al.Nat Biotechnol.2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Bi ol.2009 Oct 30;393(3):672-92), and BsAb molecules (Z ymogenetics, WO2010 / 111625), HERCULES molecule (Bi ogen Idec, US00 / 7951918), scFv fusion molecule (Novarti s), scFv fusion molecule (Changzhou Adam Biotech Inc, C N / 102250246), and TvAb molecules (Roche, WO2012 / 0255 25, WO2012 / 025530).

[0094] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Pea rce et al.,Biochem Mol Biol Int.1997 Sep ;42(6):1179-88), SCORPION molecule (Emergent BioS olutions / Trubion, Blankenship JW, et al.AA CR 100th Annual meeting 2009(Abstract #5 465), Zymogenetics / BMS, WO2010 / 111625), biparental compatibility retargeting technology (Fc-based DART) molecules (MacroGenics, WO2008 / 157379, WO2010 / 080538), and dual (scFv)2-Fab molecules (National Research Center for Antibody M edicine-China).

[0095] 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 (ImmunoMe dics, WO2003 / 074569, WO2005 / 004809), bivalent bispecific Sexual molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.), and Fab-Fv molecules (UCB-C elltech, WO 2009 / 040562 A1).

[0096] Examples of scFv-, diabody-based, and domain antibodies include, but are not limited to, However, bispecific T cell engager (BiTE) molecules (Micromet, WO 2005 / 061547), tandem diabody molecule (TandAb) (Affime d) Le Gall et al., Protein Eng Des Sel.200 4 Apr;17(4):357-66.), DART molecules (MacroGenics, WO2008 / 157379, WO2010 / 080538), single-chain diabody molecules (Lawrence,FEBS Lett.1998 Apr 3;425(3):47 9-84), TCR-like antibody (AIT, ReceptorLogics), human serum albumin Min scFv fusion (Merrimack, WO2010 / 059315), and COM BODY molecule (Epigen Biotech, Zhu et al. Immunol Cell Biol.2010 Aug;88(6):667-75.), dual targeting Novody (Ablynx, Hmila et al.,FASEB J.2010), and dual targeting heavy chain single domain antibodies.

[0097] In one embodiment, the bispecific antibodies of the invention have a regulated Fab-arm exchange (W Dialysis was performed using a dialysis machine, such as that described in J. D. O2011 / 131746 (Genmab). The antibody may be a antibody, a crossbody, or a bispecific antibody.

[0098] In one embodiment, the antibody of the invention is a bispecific DuoBody® molecule ( Genmab A / S, WO2011 / 131746).

[0099] The multispecific, such as bispecific, antibodies of the invention can be of any isotype. Suitable isotypes include, but are not limited to, human IgG1, IgG2, IgG Preferably, the antibody is of the IgG1 isotype, IgG2 isotype, or any of the IgG3 and IgG4 isotypes shown in the examples. The human light chain constant region may be selected to be of the human IgG1 isotype, so as to be Either kappa or lambda, or both, e.g., as set forth in SEQ ID NOs: 53 and 54. For example, in one embodiment, the light chain responsible for CD30 binding is The light chain contains a kappa constant region and is responsible for CD3 binding, the light chain contains a lambda constant region. In one embodiment, both heavy chains of the antibody of the invention are of the IgG1 isotype. In the present invention, the two heavy chains of the bispecific antibody are of IgG1 and IgG4 isotypes, respectively. Preferably, the bispecific antibody is a human IgG1 isotype, as shown in the examples. Optionally, and preferably, the heavy chain of a selected isotype and The Fc region sequence preferably has duplicated sequences in the hinge, CH2, and / or CH3 regions. Can be modified to allow for the generation of specific antibodies and / or to introduce inactivation .

[0100] In one embodiment, the multispecific antibody of the invention comprises a first and a second Fc polypeptide. The Fc region comprises:

[0101] In one embodiment, the first Fc polypeptide and the first heavy chain variable region are the same polypeptide. and the second Fc polypeptide and the second heavy chain variable region are contained within the same polypeptide. It is contained within the tide chain.

[0102] The first and second Fc polypeptides are each selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. , IgE, IgD, IgM, or IgA isotypes, or mixed isotypes. The Fc region may be of any isotype, including any human isotype. The regions are of human IgG1, IgG2, IgG3, IgG4 or mixed isotypes. In one embodiment, the Fc region is a human IgG1 Fc region.

[0103] In a further embodiment, the multispecific antibody is a full-length antibody as defined herein.

[0104] The antibodies of the present invention contain modifications in the Fc region that render the antibodies inactive or non-activating. Thus, in the antibodies disclosed herein, one or both heavy chains may comprise an anti- The antibody has an Fc domain that is mutated to a lesser extent than an identical antibody except that does not contain the modification. The antibody may be modified to induce Fc-mediated effector functions. , Fc-mediated CD69 expression on T cells (i.e., see, e.g., WO2015 / 00108 5, as a result of CD3 antibody-mediated, Fcγ receptor-dependent CD3 cross-linking. 69 expression) and binding to Fcγ receptors, which bind to C1q. In particular, the binding of FcγR to FcγR may be measured by binding to FcγR or by induction of Fc-mediated cross-linking of FcγR. The heavy chain constant sequence enhances Fc-mediated CD69 expression when compared to wild-type (unmodified) antibody. , at least 50%, at least 60%, at least 70%, at least 80%, less The Fc-mediated CD69 expression can be measured in a PBMC-based functional assay, e.g., as described in WO2015 / 0 As determined by flow cytometry as described in Example 3 of US Pat. No. 5,993,345. Modifications of the light chain constant sequence may also result in reduced binding of C1q to the antibody. Compared to the antibody, the reduction can be at least 70%, at least 80%, at least 90%, or at least and C1q binding may be at least 95%, at least 97%, or 100%, and C1q binding may be determined by ELISA. Furthermore, the Fc region can be determined by determining whether the antibody is, for example, in the linear portion of the curve. at least 50%, at least 60%, at least 70%, At least 80%, at least 90%, at least 99%, or 100% reduced Fc media The T cell proliferation may be modified to mediate PBMC-based function. It is measured in an assay.

[0105] Amino acid substitutions and combinations thereof to eliminate Fc-mediated effector functions. However, a wide range of different inactivating ligands were introduced into the constant heavy chain region of an IgG1 isotype antibody. Antibody formats have been developed (e.g., Chiu et al., Antibodies ies 2019 Dec;8(4):55, Liu et al., Antibodi es,2020 Nov 17;9(4):64;29(10):457-66, Shi elds et al.,J Biol Chem.2001 Mar 2;276(9 ):6591-604).

[0106] For example, in an IgG1 isotype antibody, examples of amino acid positions that can be modified include In one embodiment, the first and / or second F The c polypeptide is a polypeptide having an amino acid sequence at positions L234 and / or L235 in the human IgG1 heavy chain. and preferably, the substitutions are F and E, respectively. where amino acid positions are as defined by Eu numbering.

[0107] In addition to modifications at amino acid positions L234 and L235, it has been found that further positions may be modified. It will be understood that in a further embodiment, the first and second Fc polypeptides are The amino acids at positions L234 and L235 were replaced with F and E, respectively. wherein the first and / or second Fc polypeptide comprises a residue at position G236 in a human IgG1 heavy chain. and further comprising a substitution of the corresponding amino acid of .

[0108] In another embodiment, the first and second Fc polypeptides comprise L234 and L235, respectively. The first and second amino acids corresponding to the amino acid at position L235 are substituted with F and E, The Fc polypeptide has an amino acid sequence corresponding to the amino acid at position G236 in the human IgG1 heavy chain. It further includes substitution of the acid, preferably to R.

[0109] In another embodiment, the first and second Fc polypeptides comprise L234 and L235, respectively. containing substitutions of the amino acid corresponding to the amino acid at position L235 with F and E, The second Fc polypeptide corresponds to amino acid position D265 in a human IgG1 heavy chain. It further includes an amino acid substitution, preferably an A substitution.

[0110] In another embodiment, the first and second Fc polypeptides comprise L234 and L235, respectively. The first and second amino acids corresponding to the amino acid at position L235 are substituted with F and E, The Fc polypeptide has an amino acid sequence corresponding to amino acid at position D265 in the human IgG1 heavy chain. It further includes substitution of an acid, the substitution being preferably to A.

[0111] In another embodiment, one of the first and second Fc polypeptides each comprises: F, E, and R of the amino acids corresponding to the amino acids at positions L234, L235, and G236 and the other Fc polypeptide contains substitutions L234, L235E, and D, respectively. It contains substitutions of the amino acid corresponding to amino acid 265 with F, E, and A.

[0112] In a further embodiment, the first Fc polypeptide comprises L234, L235, , and the amino acid corresponding to the amino acid at position G236 is replaced by F, E, or R, The two Fc polypeptides have the amino acids at positions L234, L235E, and D265, respectively. The amino acid positions are numbered Eu and include substitutions of F, E, and A for the amino acids corresponding to As defined by the

[0113] For example, a constant region having such an Fc region substitution may be, inter alia, 44, SEQ ID NOs: 45, 46, 49, 50, 51 In one embodiment, the antibody of the invention is provided in SEQ ID NOs: 45, 46, 49, 50, 51, and 52.

[0114] In one embodiment, the multispecific or bispecific antibody of the invention comprises different first and second and an Fc region including the CH3 region of the first and second CH3 regions. The dimerization interaction is stronger than each of the homodimerization interactions of the first and second CH3 regions. For more information about these interactions and how they can be achieved, see references. WO2011 / 131746 and WO2013 / 060 are hereby incorporated by reference. 867 (Genmab). Stable, heterodimeric antibodies can be provided, for example, in the CH3 region. Based on two homodimeric starting antibodies containing only a few asymmetric mutations in The so-called Fa It can be obtained in high yields by b-arm exchange.

[0115] In a specific embodiment, the present invention provides a method for the preparation of a human Ig polypeptide comprising the steps of: the amino acid at the position corresponding to K409 in the G1 heavy chain is R; In the polypeptide, an amino acid at a position corresponding to F405 in the human IgG1 heavy chain Antibodies are provided in which the acid is L, or vice versa.

[0116] In a further embodiment, in the first Fc polypeptide, a position corresponding to K409 is R, and in the second Fc polypeptide, the amino acid at position corresponding to F405 is The amino acid in is L.

[0117] Thus, in one embodiment, in the first Fc polypeptide, a human IgG1 complex is T366, L368, K370, D399, F405, Y407, and K40 in the chain At least one of the amino acids at a position corresponding to a position selected from the group consisting of: is substituted, in the second Fc polypeptide, T3 in human IgG1 heavy chain 66, L368, K370, D399, F405, Y407, and K409 At least one of the amino acids at a position corresponding to the position selected from the and the substitutions in the first and second Fc polypeptides are not at the same position and are The acid positions are as defined by Eu numbering. For example, such Fc The constant region having the region substitutions is particularly preferred as compared to SEQ ID NO: 44, which does not have such substitutions. The sequences are provided in SEQ ID NOs: 47, 48, 49, 50, 51, and 52, which can be compared. In one embodiment, the antibody of the invention comprises SEQ ID NOs: 47, 48, 49, 50, 51, and 5 2.

[0118] Preferably, in the first Fc polypeptide, the amino acid at the position corresponding to F405 is The amino acid is L and the amino acid at the position corresponding to K409 in the second Fc polypeptide is The acid is R, or vice versa.

[0119] Thus, in one embodiment, one of the first and second Fc polypeptides is The amino acids corresponding to the amino acids at positions L234, L235, G236, and F405, respectively, are , F, E, R, and L, and the other Fc polypeptide contains substitutions of L234, , F, E, A of the amino acids corresponding to the amino acids at positions L235E, D265, and K409. , and R.

[0120] In another embodiment, one of the first and second Fc polypeptides each comprises: F, of the amino acids corresponding to the amino acids at positions L234, L235, G236, and K409; The other Fc polypeptide contains substitutions of L234, L235, L236, L237, L238, L239, L240, L241, L242, L243, L244, L245, L246, L247, L248, L249, L250, L251, L252, L253, L254, L255, L256, L257, L258, L260, L261, L262, L263, L264, L265, L266, L267, L268, L270, L271, L272, L273, L274, L275, L276, L277, L278, L279, L280, L281, L282, L283, L284, L F, E, A, and the amino acids corresponding to the amino acids at positions 35E, D265, and F405 Includes substitutions for L.

[0121] In a further embodiment, the present invention provides a method for producing a composition comprising the steps of: (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively. A first heavy chain variable region comprising the C3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. a first light chain variable region comprising DR1, CDR2, and CDR3 sequences; A region, (ii) CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 8, and 9, respectively. A second heavy chain variable region comprising the R3 sequence, and the R4 sequence is set forth in SEQ ID NOs: 10, 11, and 12, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences as set forth above. 3 binding region, Here, the multispecific antibody is a bispecific antibody and comprises a first and a second Fc polypeptide. comprising an Fc region consisting of wherein the first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain. Rarely, and wherein the second Fc polypeptide and the second heavy chain variable region are from the same polypeptide. contained within the tide chain, wherein the first Fc polypeptide has an amino acid sequence at positions L234, L235, and G236, respectively. and a second Fc polypeptide, which contains substitutions of amino acids corresponding to the amino acids F, E, and R. The peptides correspond to the amino acids at positions L234, L235, and D265, respectively. and amino acid substitutions of F, E, and A, where the amino acid positions are numbered Eu. and wherein in the first Fc polypeptide, the amino acid at the position corresponding to K409 is R and wherein in the second Fc polypeptide, the amino acid at the position corresponding to F405 is L It is.

[0122] In a further embodiment, the present invention provides a method for producing a composition comprising the steps of: (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively. A first heavy chain variable region comprising the C3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. a first light chain variable region comprising DR1, CDR2, and CDR3 sequences; A region, (ii) CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 8, and 9, respectively. A second heavy chain variable region comprising the R3 sequence, and the R4 sequence is set forth in SEQ ID NOs: 10, 11, and 12, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences as set forth above. and a multispecific antibody comprising: Here, the multispecific antibody is a bispecific antibody and comprises a first and a second Fc polypeptide. an Fc region consisting of wherein the first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain. Rarely, and wherein the second Fc polypeptide and the second heavy chain variable region are the same polypeptide. contained within the tide chain, wherein the first Fc polypeptide comprises a first Fc polypeptide having a first amino acid sequence at positions L234, L235, and D265, respectively. A second Fc polypeptide comprising the amino acid substitutions F, E, and A corresponding to the amino acids The peptides correspond to the amino acids at positions L234, L235, and D265, respectively. The amino acid substitutions are F, E, and A, where the amino acid positions are numbered Eu. and wherein in the first Fc polypeptide, the amino acid at the position corresponding to K409 is R and wherein in the second Fc polypeptide, the amino acid at the position corresponding to F405 is L It is.

[0123] In one embodiment, the antibody of the invention comprises the heavy chain sequences set forth in SEQ ID NOs: 17 and 19: and comprising or consisting of the light chain sequences set forth in SEQ ID NOs: 18 and 20.

[0124] In a further embodiment, the antibody of the invention comprises the heavy chain sequences set forth in SEQ ID NOs: 17 and 19. and the light chain sequences set forth in SEQ ID NOs: 18 and 20, The antibody is a bispecific antibody.

[0125] In one embodiment, the antibody of the invention comprises the heavy chain sequences set forth in SEQ ID NOs: 17 and 35: and comprising or consisting of the light chain sequences set forth in SEQ ID NOs: 18 and 20.

[0126] In one embodiment, the antibody of the invention comprises the heavy chain sequences set forth in SEQ ID NOs: 55 and 19, and comprising or consisting of the light chain sequences set forth in SEQ ID NOs: 18 and 20.

[0127] In one embodiment, the antibody of the invention comprises the heavy chain sequences set forth in SEQ ID NOs: 55 and 35: and comprising or consisting of the light chain sequences set forth in SEQ ID NOs: 18 and 20.

[0128] In yet a further embodiment, the antibody of the invention is bsG1-huCD3-FEALxC D30-MDX060-FEAR or bsG1-huCD3-FEALxCD30-MD In yet a further embodiment, the antibody of the invention is bsG1- huCD3-FEALxCD30-MDX060-FERR.

[0129] The constant region sequences listed in SEQ ID NOs: 44 to 52 and 55 do not include a C-terminal lysine (K). However, the naturally occurring Fc regions found in the humans from which they are derived are In the sequences shown, such C-terminal lysines are not part of an open reading frame. During cell culture production of recombinant antibodies, this terminal lysine may be present in the endogenous carboxyl It can be proteolytically cleaved by peptidases and has the same sequence but with a C-terminal lysine For purposes of antibody production, the D coding for this terminal lysine is The NA may be omitted from the sequence so that the antibody is produced without lysine. Omission of the C-terminal lysine from the sequence increases the homogeneity of the antibody relative to the presence of the C-terminal lysine. Antibodies produced from nucleic acid sequences that either do or do not encode a terminal lysine may be The degree of C-terminal lysine processing may be important when using antibodies produced in, for example, a CHO-based production system. When a sequence is found to be substantially identical in sequence and function, typically due to high ick,LWet al.Biotechnol.Bioeng.2008;100 :1132-1143). Thus, the antibodies according to the present invention can be used as described herein. It is understood that the polypeptide can be produced without encoding or having a C-terminal lysine. It is therefore understood that for manufacturing purposes, it is possible to produce antibodies without a C-terminal lysine. This can be done.

[0130] In an alternative embodiment, the multispecific antibody according to the invention comprises a classical allele comprising an Fc region. For example, in one embodiment, (i) the CD30 binding region and / or the CD3 binding region is a Fab; (ii) the CD30 binding region and / or the CD3 binding region is an scFv; (iii) Is the CD30-binding region a Fab and the CD3-binding region an scFv? or (iv) the CD30 binding region is an scFv and the CD3 binding region is a Fab.

[0131] Binding, cytotoxicity, and T cell activation A bispecific antibody as described herein capable of binding to human CD3 and human CD30. The antibodies, including specific antibodies, can advantageously target T cells to human CD30-expressing cancer cells, thereby This can induce T cell-mediated killing of the cancer cells.

[0132] As mentioned above, preferably the antibody according to the invention is inactive and furthermore the antibody Contains one or more of the following features: a) For example, for the examples described in the Examples herein, flow cytometry When tested using 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 is capable of binding to CD30-expressing human tumor cells, such as HDLM-2 cells; b) when assayed as described in the Examples herein, e.g., purified Use PBMCs, ADCC effector cells, or T cells as effector cells. can mediate concentration-dependent cytotoxicity of CD30-expressing human tumor cells, c) when assayed as described in the Examples herein, e.g., purified When PBMCs or T cells are used as effector cells, SU-DHL-1 cells, L-428 cells, KM-H2 cells, SUP-M2 cells, KI-JK cells, and HDLM- Concentration-dependent cell proliferation of one or more human CD30-expressing tumor cell lines selected from the group consisting of 2 cells. capable of mediating cytotoxicity, d) CD30, e.g., when assayed as described in the Examples herein. capable of inducing T cell proliferation in vitro in the presence of human tumor cells expressing e) SU-DHL-1, when assayed as described in the Examples herein. cells, L-428 cells, KI-JK cells, and HDLM-2 cells. and activating T cells in vitro in the presence of one or more CD30-expressing human tumor cell lines. And, f) cytokine and gram-negative antibodies when assayed as described in the Examples herein; can induce dose-dependent in vitro production of enzyme B by T cells; g) Activation of CD30 when assayed as described in the examples herein Even if expressed on a subpopulation of T cells, it cannot result in T cell fratricide. and / or h) in the presence of sCD30, when assayed as described in the examples herein. can also induce T cell-mediated cytotoxicity.

[0133] In one embodiment, the antibodies described herein are those described in the Examples herein. For example, L-428 tumor cells were used and measured by flow cytometry. less than 0.025 μg / ml, such as less than 0.020 μg / ml when assayed less than 0.030 μg / ml, less than 0.035 μg / ml, etc. 0.050 μg / ml, such as less than 0.040 μg / ml, such as less than 0.045 μg / ml EC less than l 50 Induces T cell-mediated cytotoxicity at concentrations

[0134] In further embodiments, the antibodies described herein are those described in the Examples herein. For example, L-428 tumor cells were used and analyzed by flow cytometry as described in When measured and assayed, 80% or more, such as greater than 90% or greater than 85% Maximal lysis occurs when T cell-mediated cytotoxicity is induced exceeding 100%.

[0135] In one embodiment, the antibodies described herein are those described in the Examples herein. For example, L-428 tumor cells were used and measured by flow cytometry. less than 0.035 μg / ml, such as less than 0.03 μg / ml when assayed 0.05, such as less than 0.04 μg / ml, such as less than 0.045 μg / ml ECs with CD25 expression below μg / ml 50 CD4 with concentration + Induce T cell activation In further embodiments, the antibodies described herein are As described, for example, L-428 tumor cells were used and analyzed by flow cytometry. When assayed by measuring Less than 0.001 μg / ml, such as less than 0.0009 μg / ml EC with CD25 expression less than 0.005 μg / ml, etc. 50 CD8 with concentration + T Induces cell activation.

[0136] In one embodiment, the antibodies described herein are those described in the Examples herein. For example, L-428 tumor cells were used and measured by flow cytometry. When assayed as 0.001 μg / ml, such as less than 0.0009 μg / ml 0, such as less than 0.002 μg / ml, such as less than 0.003 μg / ml EC with CD69 expression less than .004 μg / ml 50 CD4 with concentration + T cell activation In a further embodiment, the antibody described herein induces As described in the Examples, for example, L-428 tumor cells were used, and the results were analyzed by flow cytometry. 0.0, such as less than 0.0015 μg / ml when assayed by 0.0030 μg, such as less than 0.0025 μg / ml, such as less than 0.020 μg / ml EC with CD69 expression less than 0.0035 μg / ml, such as less than / ml 50 Has a concentration CD8 + Induce T cell activation.

[0137] In one embodiment, the antibodies described herein are those described in the Examples herein. For example, L-428 tumor cells were used and measured by flow cytometry. less than 0.006 μg / ml, such as less than 0.005 μg / ml when assayed less than 0.007μg / ml, less than 0.008μg / ml, etc. EC for PD-1 expression <0.01 μg / ml 50 CD4 with concentration + induces T cell activation In further embodiments, the antibodies described herein are As described in the Examples, for example, L-428 tumor cells were used and analyzed by flow cytometry. Thus, when assayed, it is less than 0.004 μg / ml, such as less than 0.003 μg / ml. less than 0.006 μg / ml, such as less than 0.005 μg / ml Which EC of PD-1 expression less than 0.007 μg / ml 50 CD8 with concentration + T thin Induces cell activation.

[0138] The assay may be performed as generally known to those skilled in the art, and may optionally include a 4:1 effector - cell to target cell (E:T) ratio and / or, optionally, a 72 hour incubation period The assay can include, for example, assaying tumor cells, preferably labeled tumor cells, e.g. For example, providing L-428 tumor cells and administering the antibodies described herein, preferably at step Add T cells, preferably labeled T cells, at a dilution of 4:1 E:T Optionally, CD4, C Stain for relevant parameters such as D8, CD25, CD69, and / or PD-1 and finally, analyzing the cells using flow cytometry such as FACS. The % live cells and / or T cell proliferation / activation can be determined by: It may be calculated from the data obtained by analyses known to those skilled in the art.

[0139] Production of antibodies of the invention Hybrid hybridomas and chemical conjugation methods (Marvin and Zhu ( 2005) Acta Pharmacol Sin 26:649) and other conventional methods. can be used to prepare multispecific, such as bispecific, antibodies of the present invention. Co-expression in a host cell of two antibodies, consisting of a light and a light chain, results in the desired bispecific antibody. In addition, a mixture of possible antibody products is generated, which can then be subjected to, for example, affinity chromatography. The product can be isolated by filtration or similar methods.

[0140] As mentioned above, upon co-expression of different antibody constructs, the formation of functional bispecific products is favored. Strategies such as those described by Lindhofer et al. (1995 J Immunol 155:219) can also be used. Fusion of rat and mouse hydridomas using the fusion technique is limited due to preferential species-restricted heavy / light chain pairing. This results in a limited number of heterodimeric proteins. Another strategy for promoting this is to incorporate corresponding nucleotides in the first and second heavy chain polypeptides. A ridge is introduced into the cavity at the interface of these two heavy chains, thus positioning the ridge within the cavity. A "knobs-into-holes" strategy to promote heterodimer formation and prevent homodimer formation The "bulge" is a small amino acid side chain with a larger side chain extending from the interface of the first polypeptide. A compensatory "cavity" of the same or similar size as the ridge is constructed by replacing the chain. creates a second polypeptide by replacing large amino acid side chains with smaller side chains. (U.S. Pat. No. 5,731,168). EP1870459 (Ch ugai and WO2009 / 089004 (Amgen) disclose different We describe another strategy to favor heterodimer formation upon co-expression of antibody domains. In the method described by et al., homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favored. As expected, both CH3 domains have one or more residues that constitute the CH3-CH3 interface. The group is replaced with a charged amino acid. WO2007 / 110205 (Merck) discloses: Differences between the IgA and IgG CH3 domains are exploited to promote heterodimerization We describe yet another strategy to

[0141] Another in vitro method for producing bispecific antibodies is described in WO2008 / 119353 (Genmab), and the bispecific antibodies were incubated under reducing conditions. During the synthesis, the "Fab arms" or " It is formed by "half molecule" exchange (exchange of a heavy chain and an associated light chain). The resulting product is The antibody is a bispecific antibody having two Fab arms which may comprise a sequence such as

[0142] A preferred method for preparing a bispecific CD3xCD30 antibody of the invention comprises the following steps: WO2011 / 131746 and WO13 / 060867 (Genmab ) a) providing a first antibody comprising an Fc region, the Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region, the Fc region comprising a second CH3 region; fruit, wherein the first antibody is a CD30 antibody and the second antibody is a CD3 antibody. or vice versa, wherein the sequences of the first and second CH3 regions are different, and thus the first and second The heterodimeric interaction between the CH3 domains is in the homodimeric phase of the first and second CH3 domains. stronger than each of the interactions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining the bispecific CDxCD30 antibody.

[0143] Similarly, the present invention provides a method for producing multispecific, e.g. bispecific, antibodies according to the present invention. The method comprises: a) a first homodimeric antibody comprising a CD30 binding region as described herein; and and a second homodimeric antibody comprising the CD3 binding region described, wherein the antibody comprises an Fc region, and optionally further features as described herein; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, and thus The heterodimeric interaction between the first and second CH3 domains is stronger than each of the dimeric interactions; b) The first antibody is combined with the second antibody to form a disulfide bond between the cysteines in the hinge region. incubating under reducing conditions sufficient to allow the compound to undergo isomerization; and c) a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody and a second antibody; and a second immunoglobulin heavy chain and a second immunoglobulin light chain of the present invention. Obtaining a dimeric multispecific antibody, This includes:

[0144] In one embodiment, the first antibody, together with the second antibody, binds to the nucleotide sequence at the hinge region. Reducing conditions sufficient to enable the cysteines present to undergo disulfide bond isomerization. and incubating the mixture under a temperature of 100° C. for 24 hours at 40° C. for 24 hours to obtain a heterodimeric antibody having a first and second antibody bond. Heterodimeric interactions of Fab-arm exchange were observed after 24 h at 37°C with 0.5 mM GSH. It is such that no conversion occurs.

[0145] Without being limited by theory, in step c), the heavy chain in the hinge region of the parent antibody is The disulfide bonds are reduced and the resulting cysteines are then substituted with cysteines from another parent antibody molecule. It can form inter-heavy chain disulfide bonds with tein residues (originally with different specificities In one embodiment of this method, the reducing conditions in step c) include a reducing agent, e.g. For example, the addition of 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-mercapto-ethanol, preferably 2-mercaptoethylamine, dithiothiazolium leitol, and tris(2-carboxyethyl)phosphine Reducing Agent. In a preferred embodiment, the reducing agent is 2-mercaptoethylamine. In some embodiments, step c) may be performed by, for example, desalting, for example by removal of the reducing agent. This includes restoring conditions that would otherwise be non-reducing or less reducing.

[0146] In a further aspect, the present invention relates to a monospecific antibody comprising: (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively. A first heavy chain variable region comprising the R3 sequence and the R4 sequence as set forth in SEQ ID NOs: 4, 5, and 6, respectively. a first light chain variable region comprising CDR1, CDR2, and CDR3 sequences; The combined area, and (ii) an Fc region consisting of a first and a second Fc polypeptide, The Fc polypeptide comprises amino acids corresponding to amino acids at positions L234 and L235, respectively. to F and E, and wherein the first and second Fc polypeptides are human further comprising an amino acid substitution corresponding to the amino acid at position G236 in the IgG1 heavy chain, and the substitution is preferably to R, where the amino acid positions are represented by the Eu numbering system The Fc region is as defined by

[0147] In one embodiment, the first heavy chain variable region comprises the sequence set forth in SEQ ID NO: 13, The first light chain variable region comprises the sequence set forth in SEQ ID NO:14.

[0148] In a further aspect, the present invention relates to a monospecific antibody comprising: (i) CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 7, 8, and 9, respectively. A second heavy chain variable region comprising the R3 sequence, and the R4 sequence is set forth in SEQ ID NOs: 10, 11, and 12, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences as set forth above. 3 binding region, and (ii) an Fc region consisting of a first and a second Fc polypeptide, The Fc polypeptide comprises amino acids corresponding to amino acids at positions L234 and L235, respectively. to F and E, and wherein the first and second Fc polypeptides are human further comprising an amino acid substitution corresponding to the amino acid at position G236 in the IgG1 heavy chain, and the substitution is preferably to R, where the amino acid positions are represented by the Eu numbering system The Fc region is as defined by

[0149] In one embodiment, X in SEQ ID NO: 9 is H. In one embodiment, the second heavy chain variable region is No. 15, and the second light chain variable region comprises the sequence set forth in SEQ ID NO: 16. Includes.

[0150] In one embodiment, the monospecific antibody is a full-length antibody. T366, L368, K370, D399, F405, Y407, and and K409, At least one amino acid at the position corresponding to F405 is substituted, preferably with L. Or, the amino acid at the position corresponding to K409 is R.

[0151] In a further aspect, the present invention relates to a method for producing a multispecific antibody, said method comprising the steps of: teeth, a) a first monospecific CD30 antibody as described herein above, and a second antibody; (i) CDR1, CDR2, and a second heavy chain variable region comprising CDR3 sequences and and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth. CD3 binding domain, and (ii) an Fc region consisting of a first and a second Fc polypeptide, 2 polypeptides, respectively, containing amino acids corresponding to amino acids at positions L234 and L235 and the substitution of F and E with the amino acid corresponding to amino acid D265 in the human IgG1 heavy chain. an Fc region comprising a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, or a second monospecific CD3 antibody as described herein above, and a first monospecific CD3 antibody comprising: providing an antibody; (i) CDR1, CDR2, and A second heavy chain variable region comprising the CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. and a second light chain variable region comprising CDR1, CDR2, and CDR3 sequences corresponding to the CDR1, CDR2, and CDR3 sequences of the first light chain variable region. 30 binding regions, and (ii) an Fc region consisting of a first and a second Fc polypeptide, 2 polypeptides, respectively, containing amino acids corresponding to amino acids at positions L234 and L235 and the substitution of F and E with the amino acid corresponding to amino acid D265 in the human IgG1 heavy chain. an Fc region comprising a substitution of 5'-amino acid A to A; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, and thus The heterodimeric interaction between the first and second CH3 domains is stronger than each of the monodimeric interactions, where preferably in the first CH3 region, The amino acid at the position corresponding to F405 is L, and in the second CH3 region , the amino acid at the position corresponding to K409 is R, or vice versa; b) The first antibody is combined with the second antibody to form a disulfide bond between the cysteines in the hinge region. incubating under reducing conditions sufficient to permit the compound to undergo bond isomerization; and hand c) a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody and a second A multispecific antibody comprising a second immunoglobulin heavy chain and a second immunoglobulin light chain of the antibody. Get a body, The method includes:

[0152] In one embodiment, the present invention provides a method for producing the multispecific antibodies described herein. The method comprises: a) a first monospecific CD30 antibody as described herein, and a second monospecific CD30 antibody comprising: providing an antibody; (i) CDR1, CDR2, and a second heavy chain variable region comprising CDR3 sequences and and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth. , the CD3 binding region, and (ii) an Fc region consisting of a first and a second Fc polypeptide, 2 polypeptides, respectively, containing amino acids corresponding to amino acids at positions L234 and L235 and the substitution of F and E with the amino acid corresponding to amino acid D265 in the human IgG1 heavy chain. an Fc region comprising a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, and thus The heterodimeric interaction between the first and second CH3 domains is stronger than each of the monodimeric interactions, where preferably in the first CH3 region, The amino acid at the position corresponding to K409 is R, and in the second CH3 region , the amino acid at the position corresponding to F405 is L; b) The first antibody is combined with the second antibody to form a disulfide bond between the cysteines in the hinge region. incubating under reducing conditions sufficient to permit the compound to undergo bond isomerization; and hand c) a first immunoglobulin heavy chain and a first immunoglobulin light chain of a first antibody and a second A multispecific antibody comprising a second immunoglobulin heavy chain and a second immunoglobulin light chain of the antibody. Get a body, This includes:

[0153] The present invention further relates to multispecific antibodies obtainable by the methods described herein.

[0154] In the above method, the first or second antibody capable of binding to CD30 and / or CD3 is The step of providing a homodimeric antibody of may comprise the steps of: - providing a cell containing an expression vector for producing one or more of said antibodies; - allowing the cells to produce one or more of said antibodies, - Obtaining one or more of said antibodies, thereby providing one or more of said antibodies.

[0155] In one embodiment of this method, the first and / or second homodimeric antibody is a full length antibody. It is a body.

[0156] The Fc regions of the first and second homodimeric antibodies are IgG1, IgG2, IgG3, or I The antibody may be of any isotype, including but not limited to gG4. In an embodiment, the Fc region of both the first and second homodimeric antibodies is IgG In another embodiment, one of the Fc regions of the homodimeric antibody is of isotype One is an IgG1 isotype and the other is an IgG4 isotype. The bispecific antibody obtained comprises an IgG1 Fc region and an IgG4 Fc region. Thus, they may have interesting intermediate properties with respect to the activation of effector functions.

[0157] In a further embodiment, one of the homodimeric starting antibodies is bound to Protein A. The product is engineered to be heterodimerized by passing it over a Protein A column. Allow the monodimeric antibody to separate from the homodimeric starting antibody and remove the flow-through. The heterodimeric antibody is eluted from the Protein A column to obtain a purified heterodimeric antibody. A composition is obtained.

[0158] As described above, the sequences of the first and second CH3 regions of the homodimeric starting antibody may be different, The heterodimeric interaction between the first and second CH3 domains then These interactions and the interactions that reach them are stronger than the homodimer interactions of the CH3 regions of Further details on how the method can be constructed are incorporated herein by reference in their entirety. , WO2011 / 131746 and WO2013 / 060867 (Genmab ) will be provided.

[0159] In particular, the stable bispecific CD3xCD30 antibody binds to CD30 and CD3, respectively. The two homodimers combine to form a single homodimer that contains only a few, fairly conservative, asymmetric mutations in the CH3 region. Based on the starting antibody, high yields can be obtained using the above-described method of the invention. The difference is that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions. means.

[0160] The multispecific, e.g. bispecific, antibodies of the invention also include a first and a second antibody in a single cell. The polypeptide may be obtained by co-expression of a construct encoding the polypeptide of the present invention.

[0161] Thus, in a further aspect, the present invention provides a nucleic acid encoding a multispecific antibody according to the invention. and a combination of nucleic acid constructs, or an expression vector comprising such a nucleic acid construct. or a combination of expression vectors.

[0162] Furthermore, the present invention relates to recombinant host cells capable of producing the multispecific antibodies according to the invention. In accordance with the present invention, the host cell comprises one or more nucleic acid constructs encoding the multispecific antibody according to the present invention. include.

[0163] The present invention therefore also relates to a method for producing a multispecific antibody according to the invention. The method comprises: (i) culturing a recombinant host cell of the invention under conditions such that an antibody is produced; (ii) isolating the multispecific antibodies produced from the culture.

[0164] In one embodiment, the method comprises the steps of: a) a first polypeptide comprising a first Fc region and a first antigen-binding region of a first antibody heavy chain; and a first nucleic acid construct encoding the first Fc region, the first Fc region comprising a first CH3 region. fruit, b) a second polypeptide comprising a second Fc region and a second antigen-binding region of a second antibody heavy chain. and a second nucleic acid construct encoding the second Fc region, the second Fc region comprising a second CH3 region. fruit, wherein the sequences of the first and second CH3 regions are different, and thus the first and second The heterodimeric interaction between the CH3 domains is in the homodimeric phase of the first and second CH3 domains. each of the interactions being stronger than the first, and a light chain sequence of a second antibody; c) co-expressing the first and second nucleic acid constructs in a host cell; and d) obtaining the heterodimeric protein from the cell culture.

[0165] Preferably, the encoded amino acid at the position corresponding to F405 is the first CH3 region. The amino acid encoded at the position corresponding to K409 is L in the second CH region. R in the 3 region, or vice versa.

[0166] Suitable expression vectors, including promoters, enhancers, etc., suitable for producing antibodies Suitable host cells are well known in the art. Exemplary host cells include yeast, bacterial, and mammalian host cells. Mammalian cells, e.g., Chinese hamster ovary cells (CHO) or human, e.g., human Examples include human embryonic kidney (HEK) cells.

[0167] The nucleic acid, or one or more nucleic acids, as defined herein may be RNA or DNA. The nucleic acid or one or more nucleic acids defined herein may be used for expression in mammalian cells. The present invention therefore further relates to a nucleic acid as defined herein. Or, one or more cells containing the nucleic acid(s) are provided.

[0168] Nucleic acids in the context of the present invention include chromosomal, non-chromosomal and synthetic nucleic acid vectors (suitable for expression The expression vector may be any suitable vector that contains a nucleic acid sequence that includes a set of regulatory elements. Examples of such vectors include derivatives of SV40, bacterial plasmids, and the like. , phage DNA, baculovirus, yeast plasmid, plasmid and phage DNA and viral nucleic acid (RNA or DNA) vectors derived from the combination of In one embodiment, the CD30 or CD3 antibody encoding nucleic acid is, for example, a linear Expression elements (e.g., Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), compact nucleic acid vectors (e.g., S6,077,835 and / or WO00 / 70087), pBR322, Plasmid vectors such as pUC 19 / 18 or pUC 118 / 119, The smallest size nucleic acid vector (e.g., Schakowski et al., Naked DNA or DNA fragments, including those described in the 1990 Ther 3,793 800 (2001) or in an RNA vector, or in a CaP04 precipitation construct (see, for example, WO2000 / 46147 , Benvenisty and Reshef, PNAS USA 83,9551 55 (1986), Wigler et al., Cell 14,725 (1978) , and Coraro and Pearson, Somatic Cell Genet. As a precipitated nucleic acid vector construct, such as that described in ICS 7,603 (1981) Such nucleic acid vectors and methods for their use are well known in the art (see, e.g., See, for example, US 5,589,466 and US 5,973,972.

[0169] In one embodiment, the vector comprises an expression vector for the expression of a CD30 antibody and / or a CD3 antibody in a bacterial cell. An example of such a vector is BlueScript (St ratagene), pIN vector (Van Heeke & Schuster, JB iol Chem 264,5503 5509(1989), pET vector (Nov Examples of expression vectors include those available from Agene, Madison WI.

[0170] The expression vector may also, or alternatively, be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include Constitutive or inducible, such as alpha factor, alcohol oxidase, and PGH Examples of vectors containing promoters include those shown in F. Ausubel et al., ed. Current Protocols in Molecular Biology,G reene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in (reviewed in Enzymol 153, 516 544 (1987)).

[0171] The nucleic acid and / or expression vector may also include a nucleic acid sequence encoding a secretion / localization sequence. This involves the transfer of polypeptides, such as nascent polypeptide chains, into the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leaders, or a signal peptide. The nucleic acid and / or expression vector may be used to direct the production of a (bispecific) antibody. Any suitable nucleic acid that facilitates expression, i.e., transcription and / or translation, of the nucleic acid, may be used to express the nucleic acid. The nucleic acid and / or vector may include any suitable promoter, enhancer, or promoter sequence. The expression promoter is associated with a number of promoters, including the strong expression promoter, and other expression promoting elements. promoter / enhancer (e.g., human CMV IE promoter / enhancer, as well as RSV, SV4 0, SL3 3, MMTV, and HIV LTR promoters), with efficient poly(A) termination sequence, origin of replication of the plasmid product in E. coli, and an antibody as a selectable marker. Examples of suitable vectors include: The nucleic acid may be an inducible promoter, as opposed to a constitutive promoter, such as CMV IE. It may also include a ter.

[0172] Composition and (medical) uses The present invention further provides a composition comprising an antibody as defined herein. Such compositions are pharmaceutical compositions, i.e., the antibody is in a pharma- ceutically acceptable carrier. Included.

[0173] Pharmaceutical compositions are described in Remington: The Science and Practice Ice of Pharmacy, 19 th Edition,Gennaro,Ed. , Mack Publishing Co., Easton, PA, 1995. The pharmaceutical compositions of the present invention can be formulated according to conventional techniques, such as those described in Dilutants, bulking agents, salts, buffers, detergents (e.g., Tween-20 or Tween-80, etc.) , non-ionic detergents), stabilizers (e.g., sugar or protein-free amino acids), preservatives The composition may include a tissue fixative, a solubilizing agent, and / or other materials suitable for inclusion in a pharmaceutical composition. do.

[0174] The pharmaceutical composition may be administered by any suitable route and mode. In certain embodiments, the pharmaceutical compositions are administered by intravenous or subcutaneous injection or infusion.

[0175] The antibody, composition or pharmaceutical composition according to the invention is preferably formulated for use as a medicament. This is for the purpose.

[0176] The antibody, composition or pharmaceutical composition according to the invention is preferably used in the treatment of a disease. It is for use.

[0177] In particular, the bispecific antibodies of the present invention may be used for the treatment of various forms of cancer.

[0178] In one aspect, the present invention provides a method for treating cancer in a subject, the method comprising: The method comprises administering a therapeutically effective amount of a multispecific antibody of the invention. The invention provides a method for treating a disorder involving cells expressing CD30 in a subject. and, the method comprises administration of a therapeutically effective amount of a multispecific antibody of the invention.

[0179] As mentioned above, suitable diseases that can be contemplated in the methods and uses according to the invention are The cancer is most preferably characterized by expression of CD30. Expression of CD30 in the cells can be determined by any method known in the art, such as PCR, immunostaining, or FACS analysis. i.e., by detecting expression of CD30 transcript and / or protein. The amount of the antibody capable of binding to human CD30 can be readily determined. The antibodies obtained may be used, for example, in immunostaining and / or FACS analysis.

[0180] In one aspect, the present invention relates to a method for treating Hodgkin's lymphoma or anaplastic large cell lymphoma. The present invention also relates to a multispecific antibody, composition or pharmaceutical composition according to the invention for use in

[0181] In a further aspect, the present invention relates to a method for treating Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma ( Multispecific antibodies, compositions or medicaments according to the invention for use in the treatment of NHL The present invention relates to a composition for

[0182] In one embodiment, the Hodgkin's lymphoma is classical Hodgkin's lymphoma (cHL). .

[0183] In one embodiment, the non-Hodgkin's lymphoma is T-cell non-Hodgkin's lymphoma (T-NHL ) or B-cell non-Hodgkin's lymphoma (B-NHL). Hodgkin's lymphoma is a T-cell non-Hodgkin's lymphoma (T-NHL).

[0184] In a further embodiment, the T-cell non-Hodgkin's lymphoma is peripheral T-cell lymphoma (PTC). In yet a further embodiment, the T cell Non-Hodgkin's lymphoma (T-NHL) is anaplastic large cell lymphoma (ALCL). In a further embodiment, the peripheral T-cell lymphoma (PTCL) is anaplastic large cell lymphoma ( ALCL).

[0185] In one embodiment, the B-cell non-Hodgkin's lymphoma (B-NHL) is mantle cell lymphoma. Myeloma (MCL).

[0186] In still further embodiments, the Hodgkin's lymphoma (HL) is relapsed or refractory Hodgkin's lymphoma. In a further embodiment, the Hodgkin's lymphoma (HL) is a CD30+ In yet a further embodiment, the Hodgkin's lymphoma (HL) is In yet a further embodiment, the patient has relapsed or refractory CD30+ Hodgkin's lymphoma. Hodgkin lymphoma (HL) is a relapsed and refractory CD30+ classical Hodgkin lymphoma .

[0187] In still further embodiments, the non-Hodgkin's lymphoma (NHL) is relapsed or refractory non-Hodgkin's lymphoma (NHL). In a further embodiment, the non-Hodgkin's lymphoma (NHL) is In yet a further embodiment, the non-Hodgkin's lymphoma is a CD30+ non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a relapsed and refractory CD30+ non-Hodgkin's lymphoma.

[0188] In a further embodiment, Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL A multispecific antibody, composition or pharmaceutical composition according to the invention for use in the treatment of is being or has been administered intravenously and / or subcutaneously, preferably subcutaneously.

[0189] In a further embodiment, a patient diagnosed with cancer is diagnosed with CD30 expression in cancer cells. When CD30 is detected, it may range from low to high. Such patients may be selected for treatment with the antibodies according to the invention. It may not always be a requirement to include such an evaluation in selecting a candidate.

[0190] The multispecific antibodies of the present invention are useful in the diagnosis and treatment of disorders involving cells expressing CD30. Antibodies have numerous in vitro and in vivo diagnostic and therapeutic utilities. For example, antibodies can be used to treat a variety of disorders. To treat, prevent, and / or diagnose a disease, cells in culture, e.g., in vitro The subject can be administered ex vivo or to a subject, for example, in vivo. As used herein, the term "subject" is intended to include human and non-human individuals. .

[0191] In one aspect, the present invention relates to a method for the treatment of a pulmonary arthritis, comprising administering to a patient a therapeutically effective amount of ... pulmonary arthritis. The present invention relates to a diagnostic composition comprising a polyspecific antibody.

[0192] In one embodiment, the diagnostic composition is a compound that would benefit from treatment with a multispecific antibody. It is a companion diagnostic used to screen and select patients with cancer.

[0193] kit The present invention also provides a kit of parts, e.g., a companion diagnostic, comprising the antibody disclosed above. As a medicine / within a patient population, the ability to respond to treatment with an antibody as defined herein above The efficacy of the antibody when used to identify patients with a predisposition to A kit for use in predicting efficacy or antitumor activity of a compound as defined above, The present invention provides a kit comprising an antibody that inhibits the IL-15 antibody and instructions for use of the kit.

[0194] In one embodiment, the present invention relates to a method for treating a multispecific CD3xCD30 antibody comprising administering to a patient a therapeutically effective amount of the multispecific CD3xCD30 antibody. and one or more reagents for detecting crosslinking of CD3-expressing cells and CD3-expressing cells, The present invention provides a kit for the diagnosis of cancer. The reagents may be, for example, fluorescent tags, enzyme tags, or other detection The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions. In other words, the enzymatic reaction produces a product that can be visualized.

[0195] In a further aspect, the present invention provides a method for the preparation of ... medicament according to any one of the embodiments disclosed herein. Upon administration of the multispecific antibody, CD30-expressing cells and CD3-expressing cells were detected in samples derived from the patient. The present invention relates to a method for detecting whether cross-linking between cells occurs, the method comprising the steps of: (i) combining a sample with a multispecific antibody according to any one of the embodiments disclosed herein; A method for enabling the formation of a complex between the bispecific antibody and a CD30-expressing cell and a CD3-expressing cell. and (ii) analyzing whether a complex is formed; This includes:

[0196] Delivery of Nucleic Acid Constructs and Delivery Vehicles In a further aspect, the present invention provides a nucleic acid encoding an antibody of the invention for in vivo expression. For in vivo expression of nucleic acid encoding an antibody, the nucleic acid may be administered Typically, the nucleic acid is administered in a form suitable for entering the cells of the subject. There are different methods for delivering nucleic acids, including both mechanical and chemical means. For example, such methods include electroporation or injecting nucleic acids onto the skin. (Patel et al., 2018, Cell Report s 25, 1982-1993). Another suitable method for administering the nucleic acid to a subject is to administer the nucleic acid in a suitable formulation. This involves administration of nucleic acid in the

[0197] Thus, the present invention also relates to a delivery vehicle comprising a nucleic acid or a combination of nucleic acids according to the present invention. In some embodiments, the delivery vehicle may be a lipid formulation. The lipid may be a particle, for example a lipid nanoparticle (LNP). The combination can be encapsulated within the particle, e.g., within the LNP. Different lipid formulations suitable for administration of nucleic acids to a subject for administration are well known to those skilled in the art. For example, The lipid formulation typically comprises a lipid, an ionizable amino lipid, a PEG lipid, a cholesterol lipid, or a glycerol lipid. rolls, or any combination thereof.

[0198] Various forms for the preparation of lipid formulations suitable for administration of nucleic acids to a subject for expression of therapeutic antibodies. Methods and methods for the preparation of lipids are well known in the art. Examples of such lipid formulations include US20 18 / 0170866(Arcturus), EP2391343(Arbutus), WO2018 / 006052 (Protiva), WO2014 / 152774 (Shi re Human Genetics), EP2972360(Translate B io), US10195156 (Moderna), and US2019 / 0022247 (Acuitas).

[0199] Thus, in a further aspect, the present invention provides (a) a preferred For use in the treatment of cancer, such as the treatment of Hodgkin's lymphoma or anaplastic large cell lymphoma The present invention relates to a nucleic acid construct according to the invention or a delivery vehicle according to the invention for use in a pharmaceutical composition comprising:

[0200] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. should not be interpreted as EXAMPLES

[0201] Example 1: Synthesis of CD3×CD30 Bispecific Antibodies by 2-MEA-Induced Fab-Arm Exchange Generate The following antibodies were used in the examples:

[0202] Humanized CD3 antibody IgG1-hu described in Example 1 of WO2015 / 001085 (Genmab) IgG1-huCD3-H1L1 is referred to herein as "Ig It is called "G1-huCD3". IgG1-huC described in Example 2 of WO2017 / 009442 (Genmab) IgG1-huCD3-H1L1-H101G is Referred to herein as "IgG1-huCD3-H101G."

[0203] CD30 antibody MDX-060, also called HuMab 5F11, is disclosed in WO2003 / 059282 (Medarex). hAC10 (or SGN-30) is disclosed in US 825 7706 and US20100239571 (Seattle Genetics) HRS-3 is disclosed in WO2016 / 0177846 (Affimed). HeFi-I, T405, T105, T408, and T215 are WO200 7 / 040653 (U.S. Government and Health).

[0204] Antibody expression The antibody sequence was cloned into the pcDNA3.3 expression vector (Invitrogen, US). and Fc silencing and / or DuoBody (registered trademark) in the Fc domain. The antibodies were expressed as IgG1, κ or IgG1, λ with or without amino acid substitutions. (See below). All antibodies were prepared essentially as described by the manufacturer. ExpiFectamine(TM) 293 (Thermo Fisher Scien Expi293F™ cells were cultured using a 30-well platelet culture system (Cat. No. A14525) (Thermo Fisher Scientific, US, cat.no.A145 27) by cotransfecting the relevant heavy and light chain expression vectors Therefore, it was produced under serum-free conditions.

[0205] Generation of bispecific antibodies Bispecific antibodies are developed using DuoBody® platform technology, i.e. WO2011 / 147986, WO2011 / 131746 and WO2013 / 06086 7 (Genmab) and Labrijn et al. .,PNAS 2013,110:5145-50,Gramer et al.,MA bs 2013,5:962-973) The clones were generated in vitro using ab-arm exchange (cFAE). Ig carrying a single mutation in the CH3 domain to allow for the generation of specific antibodies G1 molecule: In one parent IgG1 antibody, the F405L mutation (i.e., D3 antibody or a control, HIV-1 gp120-specific, antibody) and other parent IgG1 antibodies In the present study, the mutation was K409R (i.e., CD30 or control antibody). In addition, the parent IgG1 antibody binds IgG Fc receptors (Fc gamma receptors) and / or complement receptors. Factors such as C1q:L234F, L235E, D265A (FEA, US2015 / 0337049) or L234F, L235E, G236R (FER) The antibody contained substitutions that result in an Fc domain that cannot be bound to the Fc domain.

[0206] The combination of Fc silencing and DuoBody® technology mutations provides the following It was called so. L234F, L235E, D265A, and F405L:FEAL L234F, L235E, D265A, and K409R: FEAR L234F, L235E, G236R, and K409R: FERR

[0207] The heavy chain (HC) and light chain (LC) sequences of the parent antibody are set forth in the following SEQ ID NOs: [Table 4]

[0208] To generate bispecific antibodies, mix the two parent antibodies in equimolar ratio in PBS buffer (phosphate Buffered saline, 8.7 mM HPO 4 2- , 1.8 mM H 2 PO 4 - , 163.9m M Na + , 140.3 mM Cl - The mixture was mixed in a 2-mercaptoethyl Add 2-aminopropylamine-HCl (2-MEA) to a final concentration of 75 mM and incubate the reaction mixture at 31 °C for 5 min. The 2-MEA was incubated for 2 h. Small volume cut-off Slide-A-Lyzer carriage (Thermo Fisher The samples were then dialyzed into PBS buffer using a 4-phase dialysis machine (Scientific Research). Store at 37 °C overnight to allow reoxidation of disulfide bonds and formation of intact bispecific antibodies. The effectiveness of cFAE was confirmed by Gramer et al. (MAbs. 2013 Nov 1;5(6):962-973. >95% as assessed by quantitative analysis (ESI-MS).

[0209] Non-binding control antibody b12 IgG1-b12 is an HIV-1 gp120-specific antibody (Barbas, C.F.J. Mol Biol. 1993 Apr 5;230(3):812-23), and is used as a negative, non-binding control antibody in some of the examples. These sequences are referred to herein as SEQ ID NOs: 36 and 37 (FEAL), respectively, or and are included as SEQ ID NOs: 38 and 37 (FERR).

[0210] Example 2 - Human Hodgkin's Lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and T CD30 expression in LL cell lines CD30 surface expression levels were determined by quantitative flow cytometry (human IgG calibrator kit, Biocytex, cat no. CP010) was used to measure HL, ALCL, , and a panel of TLL cell lines (Table 4). 4 Cells / well ) in a polystyrene 96-well round-bottom plate (Greiner bio-one, cat. Add 50 μL of staining buffer (0.1% bovine serum albumin [ BSA, Fraction V, Roche, cat. no. 10735086001] and 0.02% PB supplemented with NaN3 [Sigma Aldrich, cat no. 13412] 10 μg / mL IgG1 in 100 mM NaCl [Lonza, cat. no. BE17-517Q] -CD30-MDX060-FERR for 30 min at 4 °C. Human IgG calibrator kit (Biocytex, cat. no. CP010) A standard curve was generated using 100 μl of ... Calibration beads containing a defined number of human IgG monoclonal antibodies were then mixed with the same R-PE conjugated Next antibody (Jackson ImmunoResearch, UK, cat.no.109 -116-098, 1:500 dilution) for 30 min at 4 °C protected from light. The cells and beads were washed with FACS buffer and analyzed using a FACSCelesta flow cytometer. The cells were analyzed by flow cytometry using a chromatograph (BD Biosciences, USA). A standard curve obtained using a human IgG calibrator kit was used to measure the Gr Bound IgG per cell was analyzed using aphPad Prism Software. 1-CD30-MDX060-FERR antibody (ABC) number interpolated and expressed on the cell surface The estimated number of CD30 molecules detected is shown.

[0211] Tables 4 and 5 show that CD30 expression above the lower limit of quantification (LLOQ) was greater in all patients except SUP-T1. The results show that this was observed in the cell lines. [Table 5]

[0212] [Table 6]

[0213] Example 3 - Human Hodgkin's Lymphoma (HL) such as Anaplastic Large Cell Lymphoma (ALCL) Cells and binding of CD3×CD30 bispecific antibodies to non-Hodgkin's lymphoma (NHL) CD3xCD30 bispecific antibody against two CD30-expressing human tumor cell lines, SU-DHL -1 (ALCL, ATCC, cat. no. ACC 356) and HDLM-2 (HL, Binding to the antibody (ATCC, cat. no. CRL-2965) was measured by flow cytometry. Analyzed.

[0214] cells (3x10 4 Cells / well were plated in polystyrene 96-well round-bottom plates (Grei 50 μL of staining buffer (Bio-One, cat. no. 650180) Serial dilutions of antibodies in the medium (ranging from 0.0046 to 10 μg / mL in 3-fold dilution steps) and After washing twice in staining buffer, the cells were transferred to 50 μL of The sections were incubated with the secondary antibody at 1:40 in staining buffer for 30 min at 4°C. R-PE-conjugated goat anti-human IgG (Jackson ImmunoRes) diluted to 0 Next, the cells were Wash twice in staining buffer and incubate with TO-PRO-3 iodide (Thermo Fisher Scientific). Scientific, cat. no. T3605, 1:8000 dilution) Resuspend in 0.1 mL of staining buffer and run on a FACSCelesta flow cytometer (BD The samples were analyzed using FSC / SSC and TOPRO-3 staining. Live cells were gated based on the absence of color. Binding curves were analyzed using GraphPad Prism V7.02 software (GraphPad Software, San Nonlinear regression (sigma with variable slope) of log-transformed data was used The analyses were performed using a four-parameter (moid dose-response) method.

[0215] result Figure 1 shows the CD3xCD30 bispecific antibody bsG1-huCD3-FEALxCD30 -MDX060-FEAR(A), bsG1-huCD3-FEALxCD30-hAC 10-FEAR(B), bsG1-huCD3-FEALxCD30-HRS-3-FE AR(C), BsG1-huCD3-FEALxCD30-HeFi-I-FEAR(D ), bsG1-huCD3-FEALxCD30-T405-FEAR(E), bsG1 -huCD3-FEALxCD30-T105-FEAR(F), BisIgG1-hu CD3-FEALxCD30-T408-FEAR(G), and bsG1-huCD3- FEALxCD30-T215-FEAR(H) SU-DHL-1 (left panel) and Dose-response binding curves to HDLM-2 (right panel) tumor cells are shown.

[0216] bsG1-huCD3-FEALxCD30-MDX0 at a concentration of 1.11 μg / mL 60-FEAR, bsG1-huCD3-FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR and bsG1-h uCD3-FEALxCD30-T105-FEAR is a monospecific, bivalent CD30 parent antibody. Body IgG1-CD30-MDX060-FEAR, IgG1-CD30-hAC10-F EAR, IgG1-CD30-HRS-3-FEAR, and IgG1-CD30-T10 Similar binding was observed when comparing the binding of 5-FEAR (Fig. 1I).

[0217] In contrast, at a concentration of 1.11 μg / mL, bsG1-huCD3-FEALxCD30 -T405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEA The binding of R and bsG1-huCD3-FEALxCD30-T215-FEAR was Monospecific, bivalent CD30 parent antibody IgG1-CD30-T405-FEAR, IgG1-C D30-T408-FEAR and IgG1-CD30-T215-FEAR, SU- This was lower than the binding to DHL-1 and HDLM-2 cells (Fig. 1I).

[0218] Overall, bsG1-huCD3-FEALxCD30 at a concentration of 1.11 μg / mL -MDX060-FEAR, bsG1-huCD3-FEALxCD30-hAC10- FEAR, bsG1-huCD3-FEALxCD30-HRS-3-FEAR, BsG 1-huCD3-FEALxCD30-HeFi-I-FEAR, and bsG1-huC Binding of D3-FEALxCD30-T105-FEAR was significantly higher than that of bsG1-hu at the same concentration. CD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALx CD30-T408-FEAR and bsG1-huCD3-FEALxCD30-T2 was higher than that of 15-FEAR (Fig. 1I).

[0219] The negative control antibody BsG1-huCD3-FEALxb12-FE was included in these experiments. AR showed no binding to SU-DHL-1 or HDLM-2 cells, and indicates that none of the HDLM-2 cells express CD3.

[0220] In conclusion, the CD30 antibody clones T405, T408, and T215 are bivalent foci. The clone MDX060, hA C10, HRS-3, and T105 express CD30 in both monovalent and bivalent formats. It demonstrated efficient binding to current tumor cells.

[0221] Figure 2 shows (A) HDLM-2(HL), (B) L-428(HL), (C) DEL(A (D) bsG1-huCD3-FEAL in KI-JK (ALCL) cells Dose-response binding curves of xCD30-MDX060-FERR are shown. -FEALxCD30-MDX060-FERR is a monospecific, bivalent CD30 parent antibody I showed similar maximum binding when compared to gG1-CD30-MDX060-FERR Negative control antibodies bsG1-huCD3-FEALxb12-FEAR, IgG1-huC D3-FEAL and IgG1-b12-FEAL did not bind to any of these cell lines. These data showed that these cells do not express CD3 on the cell surface. The data are bsG1-huCD3-FEALxCD30-MDX060-FEER HL and Efficient binding to ALCL cell lines was confirmed.

[0222] Table 6 shows the results of bsG1-huCD3-FEALx, evaluated in two independent experiments. CD30-MDX060-FEAR HDLM-2, L-428, DEL, and KI-J EC for binding to K cells 50 The value is shown. EC 50 Values ​​are 0.05-0.30 μg / The range was 1 mL. [Table 7]

[0223] Figures 8, 9, and 10 show bsG1-huCD3-FEALxCD30-MDX060- FERR CD30-expressing HL cell lines (Figure 8), ALCL cell lines (Figure 9), and NHL cell lines The dose-response binding curves of BsG1-huC to the various cell lines (Figure 10) are shown. D3-FEALxCD30-MDX060-FERR is the monovalent control antibody BsG1-b12 -FEALxCD30-MDX060-FERR showed similar maximum binding compared to The monospecific, bivalent CD30 parent antibody IgG1-CD30-MDX060-FERR was Although it showed dose-dependent binding to the cell lines, BsG1-huCD3-FEALxCD30-M The negative control antibody bsG1-h showed lower maximum binding compared to DX060-FERR. uCD3-FEALxb12-FEAR, IgG1-huCD3-FEAL, and IgG 1-b12-FEAL showed no binding to any of these cell lines, indicating that these cells These data indicate that bsG1-huCD3 does not express CD3 on the cell surface. -FEALxCD30-MDX060-FERR, HL cell lines, ALCL and CTCL Efficient binding to T-NHL, such as cell lines, and B-NHL, such as MCL cell lines, was confirmed. did.

[0224] Table 7 shows the results of bsG1-huCD3-F, evaluated in two or three independent experiments. EALxCD30-MDX060-FERR HL, T-NHL, and B-NHL cells EC for binding to strains 50 The value is shown. EC 50 Values ​​range from 0.12 to 0.35 μg / mL The range was. [Table 8]

[0225] Example 4 - In vitro T cell-mediated cytotoxicity by CD3xCD30 bispecific antibodies Induction of T cell proliferation and proliferation CD3×CD30 bispecific antibody was used to target CD30-positive tumor cell lines and T cells. The cells were used as effector cells and tested in an in vitro cytotoxicity assay. As a source of T cells, CD3 positive ADCC effector cells type IV (Clean C Cells, Montaigu, France) or purified T cells (described in Example 5 ) was used to assess CD3-dependent tumor cell killing.

[0226] SU-DHL-1(ALCL), HuT78(ALCL), HDLM-2(HL), N CEB-1 (MCL) or L540 (HL) cells were cultured at a density of 10,000 cells / well. Polystyrene 96-well round-bottom plates (Greiner bio-one, cat. Effector cells were seeded on 0.5 μM CFSE (Carboxythiophene)-treated medium. Cyfluorescein succinimidyl ester, Cell Signalling Tech hnology, Danvers, MA, cat. no. C34554) at 37°C for 20 The tumor cells were labeled for 1 min and incubated with 10:1 (ADCC effector cells) or 7:1 ( Purified T cells were added. Bispecific CD3xCD30, b12xCD30, or CD3 Serial dilutions of xb12 antibody or monospecific bivalent CD30 antibody were added (10 to 0.04 (final concentrations ranging from 1 μg / mL, 3-fold dilutions) and incubate cells at 37°C for 72 h. In some experiments, bsG1-huCD3-FEALxCD30-MDX060 -A variant of FEAR containing the H101G mutation, which has reduced affinity for CD3 The CD3 binding arm was used (WO2017 / 009442, Genmab). After washing twice in 0 μL of staining buffer, cells were stained with TO-PRO-3 iodide (Ther mo Fisher Scientific, cat.no.T3605, 1:4000 Resuspend in staining buffer containing 100 mM MgCl (diluted) and measure with a FACSCelesta flow cytometer. (BD Biosciences, USA).

[0227] 5 μM staurosporine (Sigma-Aldrich, US, cat. no. S6 The viability of tumor cell samples treated with 942) was set to 0%, and the viability of untreated tumor cell samples was set to 0%. Survival rate was set at 100%.

[0228] The "percentage of viable cells" was calculated as follows: % Viable Cells = ([cell count sample - cell count staurosporine-treated target cells] / [cell count untreated Target cells - cell number (staurosporine-treated target cells) x 100. CFSE-positive cells were Counts were made as a measure of absolute T cell numbers to assess cell proliferation.

[0229] Dose-response curves were generated using GraphPad Prism V8 software (GraphPad Nonlinear regression (possible) was performed using the FTIR FTIR Software (DeepLab, San Diego, CA, USA). The data were analyzed by sigmoidal dose-response with variable slope.

[0230] result The CD3×CD30 bispecific antibody was used to treat CD30-positive tumor cell lines, SU-DHL-1 cells and HDLM-2 cells were used as target cells, and ADCC effector type IV cells (Clea n Cells, Montaigu, France) were used as effector cells. , and tested in an in vitro cytotoxicity assay.

[0231] Figure 3 shows bsG1-huCD3-FEALxCD30-MDX060-FEAR(A). , bsG1-huCD3-FEALxCD30-hAC10-FEAR(B), bsG1 -huCD3-FEALxCD30-HRS-3-FEAR(C), BsG1-huCD 3-FEALxCD30-HeFi-I-FEAR(D), bsG1-huCD3-FE ALxCD30-T405-FEAR(E), bsG1-huCD3-FEALxCD3 0-T105-FEAR(F), bsG1-huCD3-FEALxCD30-T408 -FEAR(G), and bsG1-huCD3-FEALxCD30-T215-FEA R(H) shows dose-dependent changes in SU-DHL-1 (left panel) or HDLM-2 (right panel) cells. The results show that the antibody induced selective T cell-mediated cytotoxicity (expressed as a reduction in the % of viable cells). .

[0232] Monospecific, bivalent CD30 antibody IgG1-MDX060-FEAR(A), IgG1- CD30-hAC10-FEAR(B), IgG1-CD30-HRS-3-FEAR( C), IgG1-CD30-HeFi-I-FEAR(D), IgG1-CD30-T4 05-FEAR(E), IgG1-CD30-T105-FEAR(F), IgG1-C D30-T408-FEAR (G), and IgG1-CD30-T215-FEAR (H ) did not induce T cell-mediated cytotoxicity. The control antibody bsG1-huCD3-FE ALxb12-FEAR also inhibited T cell-mediated inhibition of SU-DHL-1 or HDLM-2 cells. It did not induce cytotoxicity.

[0233] In addition, the CD3×CD30 bispecific antibody was administered to different MCL, ALCL, and HL cells. line as target cells, as well as purified T cells or ADCC effector type IV cells The antibodies were tested in an in vitro cytotoxicity assay using as effector cells. 4A-B are bsG1-huCD3-FEALxCD30-MDX060-FEAR T cell-mediated cytotoxicity of SU-DHL-1, HuT78, or NCEB-1 cells induced by Cytotoxicity was observed in variants of this antibody that had a CD3-binding arm with reduced affinity ( bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR) and Compared to the control, the maximal T cell-mediated cytotoxicity of HDLM-2 cells was more potent than the control. The toxicity was bsG1-huCD3-FEALxCD30-MDX060-FEAR and bs By G1-huCD3-H101G-FEALxCD30-MDX060-FEAR (Figure 4B). The bivalent, monospecific antibody IgG1-huCD was included as a control. Incubation with 3-FEAL or IgG1-b12-FEAR inhibited these cells. The L540 cells did not induce any T cell-mediated cytotoxicity in the line. The toxicity was measured using bsG1-huCD3-FEALxCD30-MDX060-FEAR and b sG1-huCD3-H101G-FEALxCD30-MDX060-FEAR The control antibody b was induced at the lowest concentration tested (0.014 μg / mL, FIG. 4C). sG1-b12-FEALxCD30-MDX060-FEAR or IgG1-MDX0 60-FEAR did not induce T cell-mediated cytotoxicity of L540 cells.

[0234] In conclusion, bsG1-huCD3-FEALxCD30-MDX060-FEAR , induced potent killing of a variety of CD30-expressing MCL, ALCL, and HL tumor cell lines. BsG1-huCD3-FEA contains H at position 101 of the VH CDR3 of the CD3 arm LxCD30-MDX060-FEAR is a lower affinity CD30 containing G at position 101. Kills CD30-expressing MCL and ALCL tumor cells compared to the 3-arm variant It is more powerful in destroying

[0235] HDLM-2 cells (left panel) or NCEB-1 cells (right panel) were used as target cells. In a cytotoxicity assay using CFSE, the number of CFSE-positive cells is evaluated as a measure of absolute T cell numbers. Figure 4D shows the bsG1-huCD3-FEALxCD30-MDX060-FEAR or bsG1-huCD3-H101G-FEALxCD30-MDX060-FEAR T cell-mediated cytotoxicity of HDLM-2 and NCEB-1 cells induced by B) was associated with a dose-dependent increase in T cell numbers. In general, similar T cell numbers were observed in this assay with bsG1-huCD3-FEALx CD30-MDX060-FEAR or bsG1-huCD3-H101G-FEALx Counts were taken after incubation with CD30-MDX060-FEAR. 1μg / mL At a higher concentration than bsG1-huCD3-FEALxCD30-MDX060-FEAR In co-cultures with NCEB-1 cells incubated with IL-1, reduced T cell numbers were observed. The control antibody IgG1-b12-FEAL had no effect on T cell numbers in these experiments. It didn't have any impact.

[0236] 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. Led.

[0237] Example 5 - Human, cynomolgus, or rhesus monkey antibodies expressed in Expi293F cells Binding of CD3xCD30 bispecific antibodies to CD30 Expi29 transiently transfected with human CD30 or cynomolgus CD30 Bispecific CD3xCD30 and monospecific, bivalent CD30 antibodies to the plasma membrane of three cells Binding was analyzed by flow cytometry.

[0238] Human, cynomolgus, or rhesus monkeys in HEK-293F or HEK-293 cells Transient expression of CD30 Generate the following codon-optimized constructs for expression of various full-length CD30 variants: Human (Homo sapiens) CD30 (huCD30, Uniprot accession number Session number P28908), Macaca fasciculari s) CD30 (mfCD30, Uniprot accession number A0A2K5VW07 ) (SEQ ID NO: 40), and Macaca mulatta CD30 (mm CD30, Uniprot accession number A0A1D5RK03) (SEQ ID NO: 41) The construct contained suitable restriction sites 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 The plasmid was cloned into the mammalian expression vector pcDNA3.3 (Invitrogen). The Expi293F expression platform was then engineered to express 100% β-lactam IgG1 (IgG1)-dependent IgG1 expression vector, which was then cultured on a 100% PBS-exchanged ... (Thermo Fisher Scientific, Waltham, MA, U. In another set of experiments, the entire Full-length human CD30 or cynomolgus monkey CD30 constructs were expressed in HEK-293 cells. Ta.

[0239] To human, cynomolgus, or rhesus CD30 expressed in Expi293 cells Binding of CD3×CD30 bispecific antibodies cells (3×10 4 Cells / well were plated in polystyrene 96-well round-bottom plates (Grein 100μL staining buffer (ER Bio-One, cat. no. 650180) Incubate with 10 μg / mL of antibody (range 0.005–10 μg / mL in 3-fold dilution steps) at 4°C for 30 min. The experiments were performed in technical duplicates. After washing twice in staining buffer, the cells were The sections were incubated in 50 μL of secondary antibody at 4°C for 30 minutes. R-PE-conjugated goat anti-human IgG (Jackson ImmunoResearch, UK, cat. no. 109-116-098) The cells were washed twice in staining buffer and then resuspended in 30 μL of staining buffer containing 0.4% EDTA. The cells were resuspended in 5% CO and screened using the iQue Screener (Intellicyt Corporation). Binding curves were analyzed using GraphPad Prism V9.0. 0 Software (GraphPad Software, San Diego, CA, USA) SA) were analyzed using nonlinear regression (sigmoidal dose-response with variable slope).

[0240] To human, cynomolgus, or rhesus CD30 expressed in HEK293 cells CD3×CD30 Bispecific Antibody Binding cells (3×10 4 Cells / well were plated in polystyrene 96-well round-bottom plates (Therm o Scientific, cat. no. 163320), 50 μL staining buffer Antibody in solution (range 0.0002-50 μg / mL in 4-fold dilution steps) at 4°C for 30 min. The experiments were performed in technical duplicates. After washing twice in staining buffer, the cells were The cells were incubated in 50 μL of secondary antibody at 4° C. for 30 minutes. R-PE-conjugated goat-anti-human IgG (Jackson) diluted 1:200 in color buffer n ImmunoResearch, UK, cat.no.109-116-098) Cells were washed twice in staining buffer and stained with 0.4% EDTA and ToPro-3 viability marker (Invitrogen, cat.no.T The cells were resuspended in 30 μL of staining buffer containing 3605). The binding curves were analyzed using a . Lines were plotted using GraphPad Prism V9.0.0 software (GraphPad Nonlinear regression (variable gradient) was used to measure the mean mean and mean mean deviations. The analyses were performed using a sigmoidal dose-response distribution.

[0241] Binding of CD3xCD30 bispecific antibodies to human T cells or cynomolgus monkey PBMCs Cynomolgus monkey PBMC (Tebu-Bio, The Netherlands, cat (no. PBMCMFA-10) or purified human T cells were plated in a polystyrene 96-well plate. T cells were seeded in round-bottom plates using human donor buffy coats (Sanquin, Am The name comes from RosetteSep, a company based in St. Dam, The Netherlands. Human T cell enrichment cocktail (Stemcell Technologies, France, Cat. no. 15061) according to the manufacturer's instructions. 0 4 cells / well) in 50 µL of staining buffer with antibody IgG1-CD3 0-MDX060-FEAR, bsG1-huCD3-FEALxCD30-MDX06 Serial dilutions (3 The staining buffer was incubated with 0.0001 to 10 μg / mL of 100% IgG in 2-fold dilution steps. After washing twice with buffer, cells were incubated for 30 min at 4 °C with 50 µL of secondary R-PE-conjugated goat anti-human Incubated in IgG antibody (1:400 dilution). After washing twice in staining buffer. T cells were then cultured using the T cell marker CD3 (1:100, Miltenyi Biotec, clone 10D12, conjugated to APC), CD4 (1:50, eBioscience, Lone OKT4, conjugated to APC-Cy7), CD8 (1:100, Biolegend , clone RPA-T8, conjugated to AF700), as well as the T cell activation marker CD69 (1:50, BD Biosciences, clone AB2439, conjugated to FITC ), CD25 (1:50, eBioscience, clone BC96, PE-Cy7 conjugated), and CD279 / PD1 (1:50, BD Biosciences, Clones) The staining was performed using the Ultracomp antibody (AEH12.2H7 conjugated to BV605). Single volume (5 μL, Invitrogen, cat. no. 01-2222-42) The stained sample was used for flow cytometer compensation adjustment. Incubation at 4°C for 30 minutes After incubation, cells were washed twice with staining buffer, resuspended in 100 μL of staining buffer, and incubated for 3 h. Analysis was performed using ACS Fortessa (BD Biosciences). Data were processed using owJo (BD Biosciences).

[0242] result CD30 targeting bispecific antibody bsG1-huCD3-FEALxCD30-MDX0 60-FEAR, bsG1-huCD3-H101G-FEALxCD30-MDX06 0-FEAR and bsG1-b12-FEALxCD30-MDX060-FEAR , showed no binding to wild-type Expi293F cells (Fig. 5A ), but did bind to huCD30 (Fig. 5B) or mfCD30 (Fig. 5C) into Expi293F cells transfected with The binding of these bispecific antibodies was dose-dependent. The binding was comparable to that of the antibody IgG1-CD30-MDX060-FEAR. The negative control antibody bsG1-huCD3-FEALxb12-FEAR was expressed as wild-type or huC Binding to D30 or mfCD30 transfected Expi293F cells was demonstrated. Similarly, bsG1-huCD3-FEALxCD30-MDX060-FE RR was transfected with huCD30 (Fig. 11A) or mfCD30 (Fig. 11B). The negative control antibody bsG1-huCD3- FEALxb12-FERR was transfected with huCD30 or mfCD30. This is consistent with the CD30-targeting antibody MDX06, which showed no binding to the target HEK293 cells. 0 binds bivalent and cynomolgus CD30 expressed in HEK cells Efficient binding is demonstrated in a monovalent format.

[0243] FIG. 5D shows the CD3xCD30 bispecific antibody bsG1-huCD3-FEALxCD3 0-MDX060-FEAR and control bispecific antibody bsG1-huCD3-FEALx 1 shows that b12-FEAR bound efficiently to primary human and cynomolgus T cells. This is because these CD3-targeting bispecific antibodies are expressed endogenously on human T cells and The results show that IgG1-CD30-MD efficiently binds to both IgG1 and cynomolgus CD3. The X060 bivalent, parent antibody does not bind to human or cynomolgus T cells and does not bind to CD30. It was shown that it was not expressed on the cells.

[0244] A panel of CD3xCD30 bispecific antibodies and parental CD30 monospecific antibodies, huCD Binding to Expi293F cells transfected with either mmCD30 or mmCD30 was measured using flow cytometry. CD3xCD30 bispecific antibody bsG1-huC was assessed by immunocytometry. D3-FEALxCD30-MDX060-FEAR (Figure 6A), bsG1-huCD3 -FEALxCD30-hAC10-FEAR (Figure 6B), bsG1-huCD3-FE ALxCD30-HRS-3-FEAR (Figure 6C), bsG1-huCD3-FEALx CD30-T405-FEAR (Figure 6E), bsG1-huCD3-FEALxCD30 -T105-FEAR (Figure 6F), bsG1-huCD3-FEALxCD30-T40 8-FEAR (Figure 6G), and bsG1-huCD3-FEALxCD30-T215- FEAR (Figure 6H) showed equal binding to cells expressing huCD30 or mmCD30. Similarly, the parental, monospecific CD30 antibody clones were identified as huCD30 or mmCD3 0 expressing cells. In contrast, bsG1-huCD3-FEALx CD30-HeFi-I-FEAR and parent, monospecific CD30 antibody IgG1-CD30 -HeFi-I_FEAR showed binding to huCD30 but not to mmCD30 was not shown (Figure 6D).

[0245] Example 6 - Conformation of monospecific and bispecific non-activating antibody variants by DSF analysis Evaluation of structural stability Bivalent monospecific CD30, CD3, and bispecific CD40 carrying nonactivating mutations in the constant heavy chain region Protein stability characteristics of isomeric CD3xCD30 IgG1 antibody variants were characterized using differential scanning fluorescence The evaluation was carried out using a photometric method (DSF).

[0246] IgG1-CD30-MDX060-FEAR, IgG1-CD30-MDX060- FERR, IgG1-huCD3-FEAL, and BsG1-huCD3-FEALxC The D30-MDX060-FERR sample was dissolved in PBS pH 7.4 at a concentration of approximately 1 mg / mL. It was formulated.

[0247] To evaluate the conformational stability, DSF was introduced into the hydrophobic regions exposed during the unfolding of IgG. of exogenous dye Sypro-Orange (5000x concentrate in DMSO, Cat # Changes in fluorescence intensity caused by binding of IgG (S5692, Sigma-Aldrich) The iQ5 multicolor real-time PCR detection system (Bio-Rad) was used to detect the Sypro-Orange was diluted in PBS (Hyclone) at pH 7.4. The thermal melting curves were analyzed using the I It can be derived from measuring the increase in fluorescence during the controlled, stepwise thermal denaturation of glucagon. Therefore, duplicate samples of 5 μL of antibody solution (1 mg / mL in PBS) were Add 20 μL of diluted Syp in PBS pH 7.4 to a 96-well PCR plate. Fluorescence was measured in step increments of 0.5°C per increment and for 15 seconds. The duration and time required to record the fluorescence of all wells were measured at increasing temperatures ranging from 25°C to 95°C. The data were recorded at the temperature at which the solution was added. The samples were analyzed using HPLC ware 3.0 and the melting points were calculated from the fluorescence vs. temperature graph by the software. It was decided.

[0248] result Figure 7 and Table 8 show the melting temperature (T m )but 69.0°C, which is the same as IgG1-CD3 at pH 7.4 (64.5°C). 0-MDX060-FEAR T m This is higher than that of IgG1-CD30-MDX0 60-FERR exhibits higher conformational stability than IgG1-CD30-MDX060-FEAR. IgG1-CD30-MDX060, which contains a FER backbone, has been shown to have qualitative However, it has higher conformational stability than IgG1-CD30-MDX060, which contains a FEA backbone. It shows that the antibody has qualitative properties. BsG1-huCD3-FEALxCD30-MDX060- The melting temperature of FERR was determined to be 64.5°C, which is the melting temperature of the two parent antibodies IgG 1-huCD3-FEAL (62.5℃) and IgG1-CD30-MDX060-FE T determined for RR (69.0°C) m It is between. [Table 9]

[0249] Example 7 - bsG1-huCD3-FEALxCD30-MDX060-FERR T cell Simultaneous binding to cytoplasmic and tumor cells Tumor cells of bsG1-huCD3-FEALxCD30-MDX060-FERR and Simultaneous binding to naive T cells was studied.

[0250] Thaw frozen T cells isolated from healthy donors and incubate in 0.25 mM Celltrace Violet(Pacific Blue, Invitrogen, cat no.C3 L-428 tumor cells were labeled with 4557A) for 15 min at 37°C. 3 with FarRed (APC, Invitrogen cat no.C34564A) Labeled for 15 min at 7°C and added to T cells at a 1:1 E:T ratio. FEALxCD30-MDX060-FERR or control antibody bsG1-huCD3-FE ALxb12-FEAR, bsG1b12FEALxCD30MDX060-FERR, or serial dilutions of IgG1-b12-FEAL were added (6x10 -5 ~10μg / The ink was added to the cells at final concentrations ranging from 1 mL to 3x dilutions and incubated at 4°C for 2 hours. After incubation, the viability marker 7-AAD (BD Bioscience, cat. o 559925) was added (100-fold final dilution), and the cells were incubated with FACS Celesta Analysis was performed on a flow cytometer (BD Biosciences).

[0251] result FIG. 12 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR bsG1-huCD3-FEALxCD30-MDX060-FE to tumor T cells CD3 as a measure of RR-mediated cross-linking (co-binding) + CD30 + Double positive events (flow cytometer CellTrace Far Red and CellTrace The results show that the increase in double-positive events induces the formation of cells expressing both IL-1 and IL-2. The increase was antibody concentration dependent and showed a bell-shaped curve. FEALxb12-FEAR, bsG1b12FEALxCD30MDX060-FER R, or samples incubated with IgG1-b12-FEAL, or without antibody. No increased cross-linking of tumor cells and T cells was observed in the incubated samples. (A). Figure 12B shows the results of the CellTrace Far Red and CellTrace Tumor cells and bsG1-huCD3-FEALxCD30-MDX060-FER to naive T cells (B) shows simultaneous binding of R.

[0252] These data are from bsGlhuCD3FEALxCD30-MDX06-FERR However, CD30 + Tumor cells and CD3 + Able to simultaneously bind and cross-link T cells Shows.

[0253] Example 8 - In vitro T cell-mediated cytotoxicity by CD3xCD30 bispecific antibodies Induction of T cell activation and T cell proliferation and differentiation of Karpas-299 tumor cells by a panel of CD3×CD30 bispecific antibodies Cell-mediated cytotoxicity and associated T cell activation were evaluated. The following antibodies were evaluated: G1-huCD3-FEALxCD30-MDX060-FERR, bsG1-huCD 3-FEALxCD30-MDX060-FEAR, bsG1-huCD3-FEALx CD30-hAC10-FEAR, bsG1-huCD3-FEALxCD30-HRS -3-FEAR, bsG1-huCD3-FEALx CD30-HeFi-I-FEA R, bsG1-huCD3-FEALxCD30-T105-FEAR, bsG1-hu CD3-FEALxCD30-T405-FEAR, bsG1-huCD3-FEALx CD30-T408-FEAR and bsG1-huCD3-FEALxCD30- T215-FEAR.

[0254] T cells were cultured from healthy human donor buffy coats (Sanquin, Amsterdam, The RosetteSep™ human T cell line was obtained from Cell enrichment cocktail (Stemcell Technologies, France, cat. T cells were isolated using Cell Signaling Technology (CTS) (no. 15061) according to the manufacturer's instructions. trace Violet (Invitrogen, cat.no.C34557A, most The cells were labeled with 5 μM (final concentration) for 15 min at 37°C. , Celltrace FarRed (Invitrogen, cat.no.C34 The cells were labeled with 5x the volume of ice-cold D BSI was added and incubated at RT for 5 min. Cells were pelleted and resuspended in medium. Tumor cells were plated at a density of 50,000 cells / well in 96-well plates (Greiner -bio-one, The Netherlands, cat. no. 655180) Serial dilutions of bispecific CD3xCD30 antibodies (1,000 to 0.051 ng) were Add 100 µL of ... T cells were added to tumor cells at an effector-to-target (E:T) ratio of 4:1, and plated. The plates were incubated for 72 hours at 37°C. After washing twice with dl (staining buffer), cells were stained with the T cell marker CD4 (1:50, Bi olegend, cat.no.300521, composited with Pacific Blue), CD8 (1:100, BD Biosciences, conjugated to FITC), and T cells Activation marker CD69 (1:50, Biolegend, cat.no.310934 , conjugated to BV650), CD25 (1:100, Invitrogen, cat.no .25-0259-42, conjugated to PE-Cy7), and CD279 / PD-1 (1:5 0, Biolegend, cat.no.329930, conjugated to BV605) Staining was performed using Ultracomp beads (5 μL, Invitrogen, cat. no. Used for flow cytometer compensation adjustments, including single stained samples in After 30 min incubation at 4° C., the plates were washed twice with staining buffer and Cells were stained with 7-AAD (diluted 1:100 in staining buffer) for 10 min at 4°C. Cells were analyzed using a CS Celesta (BD Biosciences). Data were processed using lowJo (BD Biosciences).

[0255] Dose-response curves were generated using GraphPad Prism V7.02 software (GraphPad Prism V7.02). Nonlinear computation using the 3D model (Hpad Software, San Diego, CA, USA) Regression analysis (sigmoidal dose-response with variable slope) was used to generate the regression curves.

[0256] result Figure 13A and B show that all CD3xCD30 antibodies inhibited T cell proliferation in Karpas-299 cells. The CD30 clone MDX060 (bsG1- huCD3-FEALxCD30-MDX060-FERR and bsG1-huCD3- CD3xCD30 generated using FEALxCD30-MDX060-FEAR The bispecific antibody inhibited the Karpas-299 cell line in comparison to all other clones tested. In fact, the MDX060-based CD3 The xCD30 bispecific antibody is a CD30 clone, HRS-3, HeFi-I, T105, CD3xCD30 bispecific antibodies generated using T405, T408, or T215 Body (bsG1-huCD3-FEALxCD30-HRS-3-FEAR, bsG1-h uCD3-FEALxCD30-HeFi-I-FEAR, bsG1-huCD3-FE ALxCD30-T105-FEAR, bsG1-huCD3-FEALxCD30-T 405-FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and bsG1-huCD3-FEALxCD30-T215-FEAR, Fig. 13A). Significantly lower IC 50 The values ​​were also shown. MDX060-FERR and bsG1-huCD3-FEALxCD30-MDX060 - FEAR is CD30 clone hAC10, HeFi-I, T405, T408, or CD3xCD30 bispecific antibody generated using T215 (bsG1-huCD3 -FEALxCD30-hAC10-FEAR, bsG1-huCD3-FEALxCD 30-HeFi-I-FEAR, bsG1-huCD3-FEALxCD30-T405 -FEAR, bsG1-huCD3-FEALxCD30-T408-FEAR, and b sG1-huCD3-FEALxCD30-T215-FEAR, Figure 13B) 13C and D show that bsG1-huCD3-FEALxC induced higher maximal killing. D30-MDX060-FERR and bsG1-huCD3-FEALxCD30-MD X060-FEAR is CD4 + T cells (Figure 13C) or CD8 + T cells (Figure 13D) As a measure of T cell activation in the IL-16 / IL-16 mice, induction of CD25 expression was associated with other CD3xCD More effective than any of the 30 bispecific antibodies (lower EC 50 PD- Similar results were observed for the expression of CD40+ / -1 (Fig. 13E and F) and CD69 (data not shown). Two MDX060-based CD3 × CD30 antibodies containing the FEAR or FERR mutations were No differences in T cell-mediated killing or T cell activation were observed between the bispecific antibodies .

[0257] The bispecific antibody bsG1-huCD3xCD30-MX060 also demonstrated a similar activity to other T cell-mediated cell proliferation in L-428 cells compared to panel CD3xCD30 bispecific antibodies It was effective in inducing toxicity (data not shown).

[0258] The panel of CD3 × CD30 bispecific antibodies used in these experiments Mean IC of induced T cell-mediated cytotoxicity 50 Concentration and maximum solubility percent, and T ECs of cell activation (CD25 expression) 50 The concentrations are summarized in Table 9.

[0259] In conclusion, these data suggest that bsG1huCD3×CD30-MDX060 inhibits T Other CD3 × CD30 bispecific antibodies evaluated for inducing cell-mediated cytotoxicity It was demonstrated to be more effective than either of the above. [Table 10] Example 9 - With bsG1-huCD3-FEALxCD30-MDX060-FERR Induction of T cell-mediated cytotoxicity, T cell proliferation, and T cell activation in vitro T cell-mediated cytotoxicity of tumor cells and bsG1-huCD3-FEALxCD30 - MDX060-FERR-associated T cell proliferation and activation in HL and ALCL cells T cells were evaluated in a cellular line. The RosetteS ep™ Human T Cell Enrichment Cocktail (Stemcell Technologies, France, cat. no. 15061) according to the manufacturer's instructions. T cells were cultured using Celltrace Violet (Invitrogen, cat. In parallel, tumor cells were labeled with 10 μM C34557A (final concentration 5 μM) for 15 min at 37 °C. L-428, KI-JK, KM-H2, or SUP-M2, Celltrace Fa rRed (Invitrogen, cat.no.C34564A, final concentration 2 μM) Labeling was performed for 15 min at 37°C. After labeling, 5x volume of ice-cold DBSI was added and incubated at RT for 5 min. The cells were pelleted and resuspended in medium, and the tumor cells were incubated at 50,000 cells / well for 1 h. Cells were cultured in 96-well plates (Greiner-bio-one, The Netherlands) at a density of 100 cells / well. etherlands, cat.no.655180). 3-FEALxCD30-MDX060-FERR or control antibody IgG1-huCD3- FEAL, bsG1-huCD3-FEALxb12-FERR, IgG1-CD30- MDX060-FERR, bsG1-b12-FEALxCD30-MDX060-FE RR, serial dilutions of IgG1-b12-FEAL were added (1,000 to 0.051 ng / mL final concentrations, 3-fold dilutions), and the plate was incubated for 15 min at RT. T cells were added to tumor cells at an effector to target (E:T) ratio of 4:1 and plated. The cells were incubated at 37°C for 72 hours. After washing twice with 1:1000 PBS (color buffer), the cells were incubated with the T cell marker CD4 (1:50, Bioleg end, cat.no.300521, composited to Pacific Blue), CD8( 1:100, BD Biosciences, cat.no.345772, FITC conjugated), and the T cell activation marker CD69 (1:50, Biolegend, cat .no.310934, conjugated to BV650), CD25 (1:100, Invitro gen, cat. no. 25-0259-42, conjugated to PE-Cy7), and CD27 9 / PD-1(1:50, Biolegend, cat.no.329930, BV60 5) were stained with Ultracomp beads (5 μL, Invitrogen Flow cytometry was performed, including single staining samples with After 30 minutes of incubation at 4°C, the plate was stained. Wash twice with the buffer and stain the cells with 7-AAD (diluted 1:100 in staining buffer) for 1 h at 4 °C. The staining was performed for 10 minutes using FACS Celesta (BD Biosciences). Cells were analyzed using FlowJo (BD Biosciences) and data were processed using FlowJo (BD Biosciences). I understood.

[0260] The percentage of live target cells was calculated using the following formula:- live target cells % = (absolute number of live, single Celltrace FarRed-labeled cells in each condition / Live, single cells in conditions containing only target cells and T cells without the addition of any antibodies (absolute number of Celltrace FarRed-labeled cells) x 100.

[0261] T cell proliferation, CD4 + or CD8 + Celltrace Vi diluted T cells The proliferation index was assessed by gating on olet staining. The growth index was calculated using a growth modeling tool. The product peaks were automatically fitted and the growth index value was calculated. It was calculated according to the following formula:

[0262] Proliferation index = total amount of cells / amount of cells at the beginning of the culture = (G0 + G1 + G2 + G3 + G4 + G5+G6) / (G0+G1:2+G2:4+G3:8+G4:16+G5:32+G6 :64). Gn = number of cells at peak generation n (n = 0–6).

[0263] Dose-response curves were generated using GraphPad Prism V7.02 software (GraphPad Prism V7.02). Nonlinear computation using the 3D model (Hpad Software, San Diego, CA, USA) Regression analysis (sigmoidal dose-response with variable slope) was used to generate the regression curves.

[0264] result FIG. 14 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR In vitro, L-428 (HL), KM-H2 (HL), SUP-M2 (ALCL), and Induced dose-dependent T cell-mediated cytotoxicity in KI-JK (ALCL) and KI-JK (ALCL) cell lines. The results show that bsG1-huCD3-FEALx in L-428 and KI-JK cells Mean I of T cell-mediated cytotoxicity induced by CD30-MDX060-FERR The C50 concentrations are summarized in Table 10. Control antibodies IgG1-huCD3-FEAL, bsG1- huCD3-FEALxb12-FERR, IgG1-CD30-MDX060-FER R, bsG1-b12-FEALxCD30-MDX060-FERR, IgG1-b1 In cells incubated with 2-FEAL or without antibody No cytotoxicity was observed in the samples.

[0265] Figures 15, 16, 17, and 18 show bsG1-huCD3-FEALxCD30-MD T cell-mediated cytokinin synthesis in L-428 and KI-JK cells induced by X060-FERR Cytotoxicity is CD4 + and CD8 + T cell proliferation (Figure 15) and T cell activation markers This is associated with expression of CD69 (Figure 16), CD25 (Figure 17), and PD-1 (Figure 18). These experiments showed that bsG1-huCD3-FEALxCD30-MD The mean EC50 concentrations of X060-FERR-induced T cell proliferation and activation are shown in Table 1. Summarize to 0.

[0266] Therefore, bsG1-huCD3-FEALxCD30-MDX060-FERR Induces dose-dependent T cell-mediated cytotoxicity in HL and ALCL cell lines in vitro This was associated with T cell proliferation and activation.

[0267] [Table 11]

[0268] Example 10 - bsG1-huCD3-FEALxCD30-MDX060-FERR Induction of cytokine production in vitro by Induced by bsG1-huCD3-FEALxCD30-MDX060-FERR The cytokine and granzyme B production was measured by the administration of L-428 as described in Example 9. During in vitro T cell-mediated cytotoxicity experiments using target cells and healthy donor T cells, The supernatants were stored at -20°C and thawed for analysis. 4 different cytokines (CD40, IFNγ, IL-10, IL-12, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, PD The concentrations of L-1, TNFα and Granzyme B are custom made by the R&D system. The antibodies were measured using a bead-based multiplex immunoassay (LumiNex).

[0269] result The increased concentration was mainly due to bsG1-huCD3-FEALxCD30-MDX060-F Granzyme B in the supernatant from co-cultures of L-428 cells and T cells in the presence of ERR and cytokines IFNγ, IL-13, and TNFα (>2000 pg / mL). When compared with the control antibody IgG1-b12-FEAL, CD40, I L-10, IL-12, IL-1β, IL-2, IL-4, IL6, and IP-10 Moderate increases were observed in the levels of IL-8, MCP-1, and PDL1. The levels of were not modulated compared to the control antibody IgG1-b12-FEAL (Figure 19 ).

[0270] Therefore, bsG1-huCD3-FEALxCD30-MDX060-FERR T cell-mediated cytotoxicity and T cell activation were cytokine- and granzyme B-dose dependent. was associated with spontaneous production.

[0271] Example 11 - Purified T cells with varying effector to target ratios were used as effector cells The bsG1-huCD3-FEALxCD30-MDX060-FERR used Induction of T cell-mediated cytotoxicity in vitro Bispecific antibody 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 To investigate the mechanism of action of cytotoxicity in vivo, an in vitro cytotoxicity assay was performed using the CD30-positive tumor cell line L-428 as the target cell. As an example, purified T cells were used as effector cells to determine the effector-to-target cell ( The experiment was carried out using a ratio of 1:1.

[0272] T cell-mediated cytotoxicity was measured by varying the ratio of T cells to each other in a 1:1, 2:1, 4:1, or 8:1 ratio. The assay was performed essentially as described above, except that effector-to-target (E:T) cell ratios were added to tumor cells at 1:1. Evaluation was carried out as described in Example 9.

[0273] result FIG. 20A shows dose-dependent T cell-mediated cytotoxicity in bsG1-huCD3-FEAL The greatest tumor cell proliferation was induced by xCD30-MDX060-FERR at all E:T ratios. Cell killing (<20% viable tumor cells) was observed at E:T ratios of 4:1 and 8:1. In line with this, CD4 + and CD8 + T cell proliferation was highest at all E:T cell ratios. The most notable was observed at E:T ratios of 4:1 and 8:1 (Figures 20B-C). At all T ratios, the control antibody bsG1-huCD3-FEALxb12-FERR No specific T cell-mediated cytotoxicity or T cell proliferation was induced by the administration of IgG1.

[0274] Taken together, these data support the in vitro expression of bsG1-huC in L-428 tumor cells. D3-FEALxCD30-MDX060-FERR-induced T cell-mediated cytotoxicity E:T ratios of 4:1 and 8:1 were shown to be most effective.

[0275] Example 12 - bsG1-huCD3-FEALxCD30-MDX060-FERR Kinetics of in vitro T cell-mediated cytotoxicity and T cell proliferation in mice T in the presence of bsG1-huCD3-FEALxCD30-MDX060-FERR To assess the kinetics of cell-mediated tumor cell killing, the CD30-positive tumor cell line L-428 was Purified T cells were used as target cells and T cells as effector cells in a variable incubation period. During the incubation period, an in vitro cytotoxicity assay was performed.

[0276] T cell-mediated cytotoxicity, tumor cell cytotoxicity and T cell proliferation at 24 and 48 hours The assays were essentially as described in Example 9, except that the assays were performed after 10 and 72 hours. Ta.

[0277] result FIG. 21 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR It induced dose-dependent T cell-mediated cytotoxicity after 48 and 72 hours, but not after 24 hours. The results show that no significant T cell-mediated cytotoxicity was observed in the dose-dependent CD4 + Reach and CD8 + T cell proliferation was assessed after 72 hours in bsG1-huCD3-FEALxCD30- MDX060-FERR induced T cell proliferation at 24 or 48 hours. None was observed (Figures 21B and C).

[0278] Taken together, these data support the conclusion that bsG1-huCD3-FEALxCD30-MDX0 60-FERR inhibits T cell-mediated cytotoxicity of tumor cells and T cell proliferation in a time-dependent manner This indicates that the following has been induced:

[0279] Example 13 - bsG1-huCD3-FEALxCD30-MDX060 in vitro Correlation of CD30 expression levels with -FERR-induced T cell-mediated cytotoxicity Eight CD30-expressing tumor cell lines, bsG1-huCD3-FEALxCD30-MDX T cell-mediated killing by 060-FERR was measured using the in vitro cytotoxicity assay described in Example 9. In the sex-specific assay, an E:T ratio of 4:1 was used. The following cell lines were used: L-428, KM-H2, DEL, KI-JK, KARPAS-299, SUP-M2, NCEB-1 and JVM-2. The CD30 expression levels of these tumor cell lines were Assessment was by quantitative flow cytometry as detailed in Example 2.

[0280] result FIG. 22 shows that bsG1-huCD3-FEALxCD30-MDX060-FERR T cell-mediated cytotoxicity was observed in all cell lines in vitro with a maximum target cell killing of 68% to 98%. The results show that bsG1-huCD3-FEALxCD30-MDX0 induced cytotoxicity. Maximal T cell-mediated tumor cell killing by 60-FERR correlated significantly with the level of CD30 expression There was a correlation (Figure 22A).

[0281] In Figure 22B, bsG1-huCD3-FEALxCD30-MDX for each cell line EC of T cell-mediated killing in the presence of 060-FERR 50 against CD30 expression levels and shows a negative but non-significant trend.

[0282] Thus, these data support the in vitro CD30 expression levels and bsG1-huCD 3-FEALxCD30-MDX060-FERR-induced maximum T cell-mediated cytotoxicity and The correlation between the

[0283] Example 14 - BsG1-huCD3-FEALxCD30-MDX060-FERR Fratricide of activated T cells Activated CD30 + BsG1-huCD3-FEALxCD30-MDX in T cells 060-FERR-induced T cell fratricide was assessed in vitro.

[0284] 96-well plate (Greiner-bio-one, The Netherland ds, cat. no. 655180) in 100 μL of PBS with 1 μg / mL anti-human CD 3 (clone OKT3, Invitrogen, cat.no.16-0037-85) The plate was incubated at 37°C for 4 hours. The antibody solution was After removal, the wells were washed with 100 μL of PBS. T cells were cultured in healthy human donor buffs. Sanquin, Amsterdam, The Netherlands and RosetteSep™ Human T Cell Enrichment Cocktail (Stemcell Technologies, France, cat. no. 15061) as the manufacturer Purified T cells were isolated using heat-inactivated donor broth containing 10% iron as directed. - Bovine serum (DBSI, Gibco, cat. no. 20731-030) and penicillin pen / strep, Lonza, cat. no. DE17- T cell medium (25 mM HEPES and L-glutamine (Lo 603E)) supplemented with nza, cat. no. BE12-115F) 2 × 106 cells / mL Resuspend in 100 µL of T cell suspension (containing 200,000 T cells) at a concentration of was added to each well of the anti-CD3 coated plate. Anti-CD28 (clone CD28.2, Invitrogen, cat.no.16-02 89-85) and 0.05 μg / mL IL-15 (ThermoFisher, cat. Add 100 μL of T cell culture medium supplemented with 0.1% EDTA (no. PHC9151) to each well. The T cells were then incubated at 37°C for 96 hours.

[0285] After 96 hours, T cells were harvested and diluted to 2 x 10 6 Resuspend in T cell medium at a concentration of 100 cells / mL Flow cytometry analysis was performed to measure the expression of CD30 and T cell activation markers. Briefly, an aliquot of cells was diluted with PBS / 0.1% BSA / 0.02% azide (staining medium). Wash with 50 μL of 1:1000 diluted FVS510 viability dye (BD Bio Stain with 100% ethanol (Chem. Sciences, cat. no. 564406) and incubate at room temperature for 15 minutes. The cells were then washed with staining buffer and stained with CD30 (1:50, Biolegen). d, cat.no.333906, complexed to PE), T cell marker CD4 (1:50, Biolegend, cat.no.300506, conjugated to FITC), CD8(1: 100, Biolegend, cat.no.301028, compounded into AF700), and and the T cell activation marker CD69 (1:50, Biolegend, cat.no.31 0910, conjugated to APC), CD25 (1:100, Invitrogen, cat. no. 25-0259-42, conjugated to PE-Cy7), and CD279 / PD1 (1: 50, Biolegend, cat. no. 329924, complexed with BV605) The staining was performed using Ultracomp beads (5 μL, Invitrogen, cat.no For flow cytometer compensation adjustments, including single stained samples (.01-2222-42) After 30 min incubation at 4°C, the plates were washed twice with staining buffer. Cells were analyzed using FACS Celesta (BD Biosciences). Data were processed using FlowJo (BD Biosciences).

[0286] BsG1-huCD3-FEALxCD30-MDX060-FERR was activated To assess whether T cell fratricide can be induced, stimulated T cells were The cells were seeded in a 96-well plate at a density of 200,000 cells / well. μL of BsG1-huCD3-FEALxCD30-MDX060-FERR or Control antibody, i.e., bsG1-b12-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEALxb12-MDX060-FERR, or IgG1 -b12 was added to each well (0.00 in 3-fold dilution steps in T cell medium). (Final concentrations ranging from 3 to 3.3 μg / mL). Plates were incubated at 37°C for 48 h. did.

[0287] After washing twice with staining buffer, cells were stained with FVS510 viability dye, resulting in T cell markers CD4 and CD8, as well as T cell activation markers CD69, CD25, and The cells were stained for CD279 / PD1 and CD279 / PD1 using FACS Celesta (BD Biosciences). Flow cytometry analysis was performed using FlowJo (BD Biosciences) and The data were processed using the osciences.

[0288] Dose-response curves were generated using GraphPad Prism V7.02 software (GraphPad Prism V7.02). The data were generated using hPad Software (San Diego, CA, USA). .

[0289] result FIG. 23 shows that the cell markers CD25 (T cell activation) (A) and CD30 (B) in 54-63% and 21-27% of T cells, respectively, after 72 h of incubation. The expression of CD25 and CD30 was measured after 96 hours of incubation. (CD25: 80-83% and CD30: 27-33%). , BsG1-huCD3-FEALxCD30-MDX060-FERR, bsG1-b 12-FEALxCD30-MDX060-FERR, bsG1-huCD3-FEAL Increasing doses of xb12-MDX060-FERR or IgG1-b12 activated Figure 1 shows that the effect of IFN-γ on T cell survival was not associated with reduced survival of induced T cells.

[0290] Thus, CD30 expression on a subpopulation of activated T cells is consistent with BsG1-huCD T cell proliferation upon incubation with 3-FEALxCD30-MDX060-FERR It did not result in fratricide.

[0291] Example 15 - Antibody of BsG1-huCD3-FEALxCD30-MDX060-FERR Interference of sCD30 with tumor activity Shedding of cell surface CD30 and production of soluble CD30 (sCD30) were examined in 17 different blood Fluorological CD30 + was evaluated in tumor cell lines.

[0292] 25 μL of undiluted cultures collected from cell cultures 3 days after seeding the cells in fresh medium The concentration of sCD30 in the supernatant was measured using the "Human sCD30 ELISA Kit" (Invitrogen) Human CD40 was transfected using GFP-1000 (Cat. No. BMS240) according to the manufacturer's instructions. 30 was measured by ELISA assay for quantitative detection.

[0293] result FIG. 24A shows the concentration of sCD30 in cell culture supernatants. As shown, varying concentrations High levels of sCD30 were detected in cell culture supernatants from different cell lines. The concentration of sCD30 in the culture supernatant was measured by quantitative flow cytometry (human IgG calibrator kit, Biocytex, cat no. CP010) Figure 2 shows that when measured by CD30 membrane expression level, it correlated significantly with CD30 membrane expression level.

[0294] sCD30 potent BsG1-huCD3-FEALxCD30-MDX060-FE To assess whether RR-induced T cell-mediated cytotoxicity can be blocked, B T cell-mediated by sG1-huCD3-FEALxCD30-MDX060-FERR Cytotoxicity was evaluated in DEL tumor cells (ALCL) and was determined by the supernatant (129 ng / The T cell-mediated cytotoxicity assay was performed using the The procedure was carried out as described above (Example 8). 0-MDX060-FERR induced potent T cell-mediated cytotoxicity in this cell line The maximum tumor cell killing of 86% was observed in BsG1-huCD3-FEALxCD 30-MDX060-FERR exhibits potent T cell-mediated IL-1 activation in vitro in the presence of sCD30 These results show that the antibody was still able to induce cytotoxicity.

[0295] Thus, sCD30 concentrations vary between cell types and correlate with the level of CD30 expression on the cell surface. Furthermore, BsG1-huCD3-FEALxCD30-MDX060-FERR still induces potent T cell-mediated cytotoxicity in vitro in the presence of sCD30. I was able to lead.

[0296] Example 16 - bsG1- using patient-derived peripheral blood mononuclear T cells as effector cells Ex vivo cytotoxicity of huCD3-FEALxCD30-MDX060-FERR CD3×CD30 bispecific antibody was used to treat primary tumors with CD30-positive tumor cell lines as target cells. Patient-derived T cells were used as effector cells in ex vivo cytotoxicity assays. As a source of T cells, we used Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma ( Peripheral blood mononuclear cells (PBMCs, Di scovery Life Sciences, Table 11) to identify CD3-dependent tumors Cell killing was assessed.

[0297] L-428 tumor cells were stained with Celltrace FarRed (Invitrogen, Cat. no. C34564A, final concentration 2 μM) for 15 minutes at 37°C. After labeling, Then, add 5x the volume of ice-cold DBSI and incubate at room temperature for 5 min. The cells were pelleted and Resuspend the tumor cells in culture medium and plate them at a density of 50,000 cells / well in 96-well plates (G reiner-bio-one, The Netherlands, cat.no.65 bsG1-huCD3-FEALxCD30-MDX060-F Serial dilutions (1,000 to 0.051) of ERR and control antibody IgG1-b12-FEAL Add 100 µg / mL final concentration (3x dilutions) and incubate the plate at room temperature for 15 min. PBMCs were thawed, counted, and cultured in medium (RPMI1640 / 10% FBS / 1 % penicillin-streptomycin / 1% glutamate) and then diluted with 8:1 The tumor cells were then added at an effector-to-target (E:T) ratio and the plates were incubated at 37°C for 72 h. The cells were incubated with PBS / 0.1% BSA / 0.02% azide (staining buffer) for two washes. Afterwards, the cells were stained for CD3 (T cells, Invitrogen, cat. 48-003 7), CD14 (monocyte / macrophage, Biolegend, cat.301834) , CD19 (B cells, Biolegend, cat.302246), CD16 (monocytes / Macrophages, BD Biosciences, cat.no.556618), CD 56 (NK cells, BD Biosciences, cat. no. 564849), and Heterologous expression of CD66b (granulocyte, Biolegend, cat.no.305116) The cells were immunofluorescently stained with the T cell marker CD4 (1:50, Biolegen). d, cat.no.300521, composited to Pacific Blue), CD8 (1: 100, BD Biosciences, cat.no.345772, conjugated to FITC ), and the T cell activation marker CD69 (1:50, Biolegend, cat. o.310934, conjugated to BV650), CD25 (1:100, Invitrogen n, cat. no. 25-0259-42, conjugated to PE-Cy7), and CD279 / PD1 (1:50, Biolegend, cat.no.329930, complexed with BV605 The antibodies were further stained with Ultracomp beads (5 μL, Invitrogen Flow cytometry was performed using a single staining sample (cat. no. 01-2222-42) After 30 min of incubation at 4°C, the plate was washed with water and then washed with 100 mL of ... The cells were washed twice with buffer and incubated with 7-AAD (diluted 1:100 in staining buffer) for 10 min at 4°C. The cells were stained for 1 min using a FACS Celesta (BD Biosciences). Analyze cells and process data using FlowJo (BD Biosciences) Dose-response curves were calculated using GraphPad Prism V7.02 software (GraphPad Prism V7.02). AphPad Software, San Diego, CA, USA) A linear regression analysis (sigmoidal dose-response with variable slope) was used to generate the mean mean curves.

[0298] The percentage of live target cells was calculated using the following formula:- live target cells % = (absolute number of live, single Celltrace FarRed-labeled cells in each condition / Live, single cells in conditions containing only target cells and T cells without the addition of any antibodies (absolute number of Celltrace FarRed-labeled cells) x 100.

[0299] result Figure 25A shows that bsG1-huCD3-FEALxCD30-MDX060-FERR , by T cells derived from both healthy control donors and different HL and NHL patient donors. The results showed that the cytotoxicity of L-428 tumor cells was mediated by β-lactamase inhibitors at 72 hours after administration of β-lactamase inhibitors. Cytotoxicity is exemplified by upregulation of CD69, CD25, and PD-1. The control antibody IgG1-b12- was associated with T cell activation and proliferation (Fig. 25B-D). No T cell-mediated cytotoxicity was observed for FEAL. No T cell-mediated cytotoxicity of L-428 tumor cells was observed in the PBMC assay. This may be due to the low frequency of T cells within the sample (Table 11).

[0300] Together, these data suggest that peripheral blood T cells from HL and NHL patients express bsG1-h T cells of tumor cell lines in the presence of uCD3-FEALxCD30-MDX060-FERR This example illustrates that IL-1 can induce IL-1-mediated cytotoxicity.

[0301] [Table 12]

[0302] Example 17 - BsG1-huCD3-FEALxCD30-MD in SCID mice Evaluation of pharmacokinetic properties of X060-FERR 11-12 week-old, female, tumor-free SCID mice (CB-17 / IcrHan( Hsd-Prkdcscid mice, Envigo (3 mice per group) ) at 1μg (0.05mg / kg), 10μg (0.5mg / kg) or 100μg ( 5mg / kg) of BsG1-huCD3-FEALxCD30-MDX060-FERR A single dose of BsG1-huCD3-FEALxCD30-M was injected intravenously (IV). Because DX060-FERR does not cross-react with mouse proteins, the experiment demonstrated that target-mediated cross-reactivity It was designed to study antibody clearance in the absence of clearance.

[0303] 40 μL of blood samples were collected at 10 min, 4 to 6 h, 24 h, 2 days, 7 days, and 14 days after antibody administration. Blood was collected via buccal or saphenous vein puncture on days 1 and 21 after administration. A-containing vial (Sarstedt, Microvette CB300, cat. No. The plasma was collected in a 100 mL flask (16.444.100) and centrifuged at 10,000 g for 10 minutes. Transfer to a labeled Eppendorf vial and store at -80°C until plasma IgG concentration determination. did.

[0304] Human IgG concentrations were determined using a total human IgG enzyme-linked immunosorbent assay (ELISA). The antibody was incubated at 2 μg / mL overnight at 4°C in a 96-well Microlon ELISA plate. Plate (Greiner, German) coated with 100 μL of PBS (B Mouse anti-human IgG- in ioTrading, cat. no. K654F500PP Kappa clone MH16 (CLB Sanquin, The Netherlands, Cat. no. M1268 was used as the capture antibody. The plates were washed with PBSA (0.2% After blocking with PBS containing bovine serum albumin [BSA] for 1 h at room temperature (RT), Add samples, serially dilute in PBSA and incubate at RT for 1 h on a plate shaker. The plate was then blotted with 300 μL of PBST (supplemented with 0.05% Tween 20). The plates were washed three times with PBS containing 0.1% ethanol and then incubated with goat anti-human IgG immunoglobulin (Jackson, West Grace, PA, cat. no. 109-035-098, 0.2% B.S. Incubate with 1:10,000 in PBST supplemented with A for 1 h at room temperature. 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonyl) 11112422001 and 1111 Before incubation with 2597001), wash the plate three times with 300 μL of PBST. 100 μL of 2% oxalic acid (Sigma-Aldrich, cat. no. 33 The reaction was stopped by adding 506) and incubated at room temperature for 10 min. Absorbance was measured using an ELx808 absorbance microplate reader (Biotek, Winoos ki, VT) at 405 nm.

[0305] From the reference antibody human IgG1λ (Pure Protein 30C, cat. No. BP078) Generate a standard curve (concentration range: 1 mg / mL [3 μL]) and 3-fold dilutions in PBSTA. A second standard curve was generated using the injected material, with further dilutions at 1 mg / mL (3. The antibody was prepared from a dose of 5 mg / kg at a concentration of 6 μL each and diluted 3-fold in PBSTA. was diluted to .

[0306] result Calibration curves were generated from the reference standards using a four-parameter logistic regression model in Microsoft Excel. The unknowns were calculated by interpolation using a linear fit curve. Concentrations 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 last day of the study (day 21) was calculated using the formula D*1.000 / AUC. where D is the dose of injection (1 mg / kg) (Figure 26B).

[0307] BsG1-huCD3-FEALxCD30-MDX060-FERR is a mouse tandem It does not cross-react with proteins and its pharmacokinetic properties are therefore similar to those of other non-binding wild-type human IgG1 molecules. Expected human IgG plasma concentrations for all dose groups is about 100μg / mL (about 5mg / kg), 10μg / mL (about 0.5mg / kg), or 1 μg / mL (approximately 0.05 mg / kg). Samples from animals treated with 0.05 mg / kg were not measurable. The plasma clearance rate of uCD3-FEALxCD30-MDX060-FERR is usually The mean maximum human IgG plasma clearance rate was comparable to the predicted plasma clearance rate of human IgG1. Plasma concentrations (Cmax) were comparable to the expected Cmax of normal human IgG1.

[0308] Therefore, the drug BsG1-huCD3-FEALxCD30-MDX060-FERR The kinetic profile was compared to normal human IgG1 expression in non-tumor-bearing SCID mice in the absence of target binding. Similar to what would be expected for IgG1.

[0309] Example 18 - BsG1-huCD3-FEALxCD30-MDX060-FERR Assessment of C1q binding Membrane-bound BsG1-huCD3-FEALxCD30-MDX of complement protein C1q 060-FERR, or BsG1-huCD3-FEALxCD30-MDX060-F The parent antibodies from which ERR was generated, i.e., IgG1-huCD3-FEAL and IgG1- Binding to CD30-MDX060-FERR was assayed using either CD3- or CD30-expressing cells. was used for evaluation.

[0310] A. BsG1-huCD3-FEALxCD30-MDX06 bound to CD3-expressing cells C1q binding to 0-FERR Complement protein C1q, CD3-binding BsG1-huCD3-FEALxCD30-M Binding to DX060-FERR and IgG1-huCD3-FEAL was measured using stimulated human CD8 + T cells. IgG1-CD52-E430G inhibits the CD52 antibody C It has VH and VL domains based on AMPATH-1H and binds to the cell surface. A positive control with an Fc-enhanced backbone known to efficiently bind 1q As non-binding negative control antibodies, IgG1-b12-FERR and IgG1- Contains b12.

[0311] Human CD8 + T cells were cultured from buffy coats (Sanq) obtained from healthy volunteers. uin) by negative selection to obtain RosetteSep™ Human CD8 + T Cell Enrichment Cocktail (Stemcell Technologies, cat. No. 15 Purified T cells were concentrated using 023C.2) according to the manufacturer's instructions. The cells were incubated with heat-inactivated donor bovine serum containing 10% iron (DBSI, Gibco, cat. no. .20731-030) and penicillin / streptomycin (pen / strep, L onza, cat. no. DE17-603E), supplemented with T cell medium (25 mM Contains HEPES and L-glutamine (Lonza, cat. no. BE12-115F). Roswell Park Memorial Institute [RPMI]-1 The cells were resuspended in 640 medium. Anti-CD3 / CD28 beads (Dynabeads™ H uman T-Activator CD3 / CD28, ThermoFisher S (Clinical Scientific, cat. No. 11132D) were washed with PBS and re-infused with T cell medium. The beads were suspended in enriched human CD8 + T cells at a 1:1 ratio and incubated at 37°C for 5 %CO 2 The beads were then removed using a magnet and the cells were incubated at 4°C for 48 h. They were washed twice in PBS and counted again.

[0312] BsG1-huCD3-FEALxCD30-MDX060-FERR and IgG1- huCD3-FEAL, activated CD8 + Binding to T cells was confirmed by BsG1-huCD3- FEALxCD30-MDX060-FERR and IgG1-huCD3-FEAL(3 0 μg / mL), and R-phycoerythrin (PE)-conjugated goat anti-human IgG F(a b') 2 (Diluted 1:200 in GMB FACS buffer, Jackson Immun Research, cat. no. 109-116-098) was used to was confirmed by tometry.

[0313] activated CD8 + T cells were plated in round-bottom 96-well plates (30,000 cells / well). Plate and pellet the cells in 30 μL of assay medium (0.1% [w / v] bovine serum albumin (BSA) Albumin fraction V (BSA, Roche, cat. no. 10735086001) and 25 mM HEPES and L-glutamine supplemented with penicillin / streptomycin The cells were then resuspended in 50 μL of BsG1-huCD3 -FEALxCD30-MDX060-FERR, IgG1-huCD3-FEAL, I gG1-b12-FERR, IgG1-CD52-E430G, or IgG1-b12( 1.7x10 in 3-fold dilution steps in assay medium -4 -30μg / mL final concentration) Add to each well and incubate at 37°C for 15 minutes to allow the antibody to bind to the cells. This made it possible to do so.

[0314] As a source of C1q, human serum (20 μL / well, Sanquin, lot 20L) 15-02) was added to a final concentration of 20%. The cells were incubated on ice for 45 min. Then, the cells were washed twice with cold GMB FACS buffer and 50 μL of fluorescein isoform was added. thiocyanate (FITC)-conjugated rabbit anti-human C1q (final concentration of 20 μg / mL (D AKO, cat no. F0254, diluted 1:75 in GMB FACS buffer) , allophycocyanin-conjugated mouse anti-CD8 (BD Biosciences, cat. No. 555369, diluted 1:50 in GMB FACS buffer) The cells were incubated in cold GMB FACS buffer for 30 min at 4° C. in the dark. and washed twice with 2 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldric h, cat. no. 03690), and 4',6-diamidino-2-phenylindole (DAPI) Viable dye (1:5,000, BD Pharmingen, cat.no The cells were resuspended in 20 μL of GMB FACS buffer supplemented with DA.564907. C1q binding to viable cells (identified by PI exclusion) was analyzed using the iQue3 screener ( The results were analyzed by flow cytometry at IntelliCyt Corporation. The data was 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 (variable slope) was performed using GraphPad Prism software. The data were analyzed using a gmoidal dose-response method.

[0315] result FIG. 27A shows dose-dependent C1q binding to membrane-bound IgG1-CD52-E430G. Although C1q binding was observed, membrane-bound BsG1-huCD3-FEALxCD30-MD X060-FERR or IgG1-huCD3-FEAL or a non-binding control antibody It indicates that no effect was observed in the body.

[0316] B. BsG1-huCD3-FEALxCD30-MDX0 bound to CD30-expressing cells C1q binding to 60-FERR CD30 binding of complement protein C1q BsG1-huCD3-FEALxCD30-M Binding to DX060-FERR and IgG1-huCD30-MDX060-FERR , and NCEB-1 mantle cell lymphoma cells were used. As a positive control, IgG 1-7D8-E430G (anti-CD20), which does not contain an inactivating mutation and binds to C1q As a non-binding negative control antibody, IgG1-b12-FERR was included. It was.

[0317] NCEB-1 cells were cultured in 0.1% bovine serum albumin (BSA, fraction V, Roche, cat, no. 10735086001) and 1% penicillin / streptomycin ( Assay medium (RPMI- 1640(Lonza, Switzerland, cat.no.BE12-115F) Tumor cells (100,000 cells in 50 μL) were suspended at a concentration of 2 x 106 cells / mL. The cells were then cultured in a 96-well round-bottom plate (Greiner Bio, cat no. 65018 0). Next, 30 μL of BsG1-huCD3-FEALxCD30-MDX 060-FERR, IgG1-CD30-MDX060-FERR or control antibody (assay The final concentration of 5.6 × 10-5-10 μg / mL was determined by 3-fold dilution steps in 100 μg / mL culture medium. 100 μl of 10 ... This allows for the integration of

[0318] Half of the cells (40 μL of the antibody-containing cell suspension) were cultured in separate wells to confirm antibody binding. The cells were seeded on a plate containing BsG1-huCD3-FEALxCD30-MDX060- Binding of FERR and IgG1-CD30-MDX060-FERR to NCEB-1 cells In this case, R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab') 2 (FAC Diluted 1:500 in S buffer, Jackson ImmunoResearch, ca t.no.109-116-098) and confirmed by flow cytometry. It was.

[0319] Human serum (10 μL / well, Sanquin, lot 21K04-01) was added to the remaining Add 40 μL of cell suspension (20% of final concentration) and incubate on ice for 45 min. , followed by washing with FACS buffer and addition of 25 μL of FITC-conjugated rabbit anti-C1q antibody (20 Final concentration: 1 μL / mL (DAKO, cat no. F0254), diluted in FACS buffer The cells were incubated with cold FACS buffer for 30 min at 4°C in the dark. , TO-PRO™-3 Viable Dye (1:5000, ThermoFisher, c Resuspend in 30 μL of FACS buffer supplemented with iQu Measured on e3 Screener (Intellicyt Corporation) The data was 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 (variable slope) was performed using GraphPad Prism software. The data were analyzed using a gmoidal dose-response method.

[0320] result FIG. 27B shows dose-dependent C1q binding to membrane-bound IgG1-CD20-E430G. Although C1q binding was observed, membrane-bound BsG1-huCD3-FEALxCD30-MDX 060-FERR or IgG1-CD30-MDX060-FERR, or No effect was observed for the non-binding control antibody.

[0321] Therefore, these results are consistent with those of bsG1-huCD3-FEALxCD30-MDX060 We demonstrated that -FERR does not bind C1q and confirmed the functionally inactive backbone Ta.

Claims

1. (i) a CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; (ii) a CD3 binding region comprising a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; A multispecific antibody comprising:

2. 2. The multispecific antibody of claim 1 , wherein the first heavy chain variable region and / or the first light chain variable region is human.

3. 2. The multispecific antibody of claim 1 , wherein the second heavy chain variable region and / or the second light chain variable region is humanized.

4. 2. The multispecific antibody of claim 1, wherein X in SEQ ID NO: 9 is H.

5. 2. The multispecific antibody of claim 1, wherein X in SEQ ID NO: 9 is G.

6. 2. The multispecific antibody of claim 1 , wherein the first heavy chain variable region comprises the sequence set forth in SEQ ID NO: 13 and the first light chain variable region comprises the sequence set forth in SEQ ID NO:

14.

7. 2. The multispecific antibody of claim 1 , wherein the second heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15 and the second light chain variable region comprises the sequence set forth in SEQ ID NO:

16.

8. The multispecific antibody of claim 1 , wherein the multispecific antibody is a bispecific antibody.

9. 2. The multispecific antibody of claim 1, wherein the multispecific antibody comprises an Fc region consisting of a first and a second Fc polypeptide.

10. 10. The multispecific antibody of claim 9, wherein the Fc region is an IgG1 Fc region, preferably a human IgG1 Fc region.

11. The multispecific antibody of claim 9 , wherein the multispecific antibody is a full-length antibody.

12. The multispecific antibody of claim 9, comprising an inactive Fc region.

13. 10. The multispecific antibody of claim 9, wherein the first and / or second Fc polypeptide comprises a substitution of an amino acid corresponding to the amino acid at position L234 and / or L235 in a human IgG1 heavy chain, wherein said substitution is preferably to F and E, respectively, and wherein said amino acid positions are as defined by Eu numbering.

14. said first and second Fc polypeptides comprising a substitution of the amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein said first and / or second Fc polypeptides further comprise a substitution of an amino acid corresponding to amino acid at position G236 in a human IgG1 heavy chain, wherein said substitution is preferably with R; 14. The multispecific antibody of claim 12 or 13, wherein the amino acid positions are as defined by Eu numbering.

15. 15. The multispecific antibody of claim 14, wherein the first and second Fc polypeptides comprise a substitution of the amino acid corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein the first and second Fc polypeptides further comprise a substitution of the amino acid corresponding to amino acid at position G236 in a human IgG1 heavy chain, wherein the substitution is preferably with R.

16. said first and second Fc polypeptides comprising substitutions of the amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein said first and / or second Fc polypeptides further comprise a substitution of an amino acid corresponding to amino acid at position D265 in a human IgG1 heavy chain, wherein said substitution is preferably with A; 14. The multispecific antibody of claim 12 or 13, wherein the amino acid positions are as defined by Eu numbering.

17. 17. The multispecific antibody of claim 16, wherein the first and second Fc polypeptides comprise a substitution of the amino acid corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein the first and second Fc polypeptides further comprise a substitution of the amino acid corresponding to amino acid at position D265 in a human IgG1 heavy chain, wherein the substitution is preferably with A.

18. 14. The multispecific antibody of claim 12 or 13, wherein one of the first and second Fc polypeptides comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and the other Fc polypeptide comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235E, and D265 with F, E, and A, respectively, wherein the amino acid positions are as defined by Eu numbering.

19. 19. The multispecific antibody of claim 18, wherein the first Fc polypeptide and the first heavy chain variable region are comprised within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are comprised within the same polypeptide chain.

20. 20. The multispecific antibody of claim 19, wherein the first Fc polypeptide comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and the second Fc polypeptide comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235E, and D265 with F, E, and A, respectively, wherein the amino acid positions are as defined by Eu numbering.

21. 10. The multispecific antibody of claim 9, wherein in said first Fc polypeptide, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second Fc polypeptide, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein the substitutions in said first and second Fc polypeptides are not at the same position, and wherein the amino acid positions are as defined by Eu numbering.

22. 22. The multispecific antibody of claim 21 , wherein in the first Fc polypeptide the amino acid at the position corresponding to F405 is L and in the second Fc polypeptide the amino acid at the position corresponding to K409 is R, or vice versa.

23. 23. The multispecific antibody of claim 22, wherein 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.

24. (i) a CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; (ii) a CD3 binding region comprising a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; wherein the multispecific antibody is a bispecific antibody and comprises an Fc region consisting of a first and a second Fc polypeptide; wherein said first Fc polypeptide and said first heavy chain variable region are comprised within the same polypeptide chain, and said second Fc polypeptide and said second heavy chain variable region are comprised within the same polypeptide chain; wherein said first Fc polypeptide comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and said second Fc polypeptide comprises substitutions of the amino acids corresponding to amino acids at positions L234, L235, and D265 with F, E, and A, respectively, wherein said amino acid positions are as defined by Eu numbering; 2. The multispecific antibody of claim 1 , wherein in said first Fc polypeptide, the amino acid at the position corresponding to K409 is R and in said second Fc polypeptide, the amino acid at the position corresponding to F405 is L.

25. 2. The multispecific antibody of claim 1, comprising or consisting of the heavy chain sequences set forth in SEQ ID NOs: 17 and 19 and the light chain sequences set forth in SEQ ID NOs: 18 and 20.

26. 2. The multispecific antibody of claim 1, comprising or consisting of heavy chain sequences set forth in SEQ ID NOs: 17 and 19 and light chain sequences set forth in SEQ ID NOs: 18 and 20, wherein the multispecific antibody is a bispecific antibody.

27. (i) the CD30-binding region and / or the CD3-binding region is a Fab; (ii) the CD30-binding region and / or the CD3-binding region is an scFv; mosquito, (iii) the CD30-binding region is a Fab and the CD3-binding region is an scFv; or (iv) The multispecific antibody of claim 1, wherein the CD30 binding region is an scFv and the CD3 binding region is a Fab.

28. A nucleic acid construct, or a combination of nucleic acid constructs, encoding the multispecific antibody of claim 1.

29. 29. An expression vector, or a combination of expression vectors, comprising the nucleic acid construct of claim 28.

30. A delivery vehicle comprising the nucleic acid construct of claim 28.

31. 31. The delivery vehicle of claim 30, wherein the delivery vehicle is a particle.

32. 32. The delivery vehicle of claim 31 , wherein the particle is a lipid nanoparticle.

33. 33. The delivery vehicle of claim 32, wherein the lipid nanoparticle comprises a lipid, an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

34. 2. A recombinant host cell capable of producing the multispecific antibody of claim 1, wherein the host cell comprises one or more nucleic acid constructs encoding the multispecific antibody of claim 1.

35. 35. The recombinant host cell of claim 34, wherein the recombinant host cell is a CHO cell.

36. A pharmaceutical composition comprising the multispecific antibody of claim 1 and a pharmaceutically acceptable carrier.

37. 37. The multispecific antibody of claim 1, the nucleic acid construct of claim 28, the delivery vehicle of claim 30, or the pharmaceutical composition of claim 36 for use as a medicament.

38. 37. The multispecific antibody of claim 1, the nucleic acid construct of claim 28, the delivery vehicle of claim 30, or the pharmaceutical composition of claim 36 for use in the treatment of cancer.

39. 37. The multispecific antibody of claim 1, the nucleic acid construct of claim 28, the delivery vehicle of claim 30, or the pharmaceutical composition of claim 36, for use in the treatment of Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL).

40. 40. The multispecific antibody, nucleic acid construct, delivery vehicle, or pharmaceutical composition for use according to claim 39, wherein the non-Hodgkin's lymphoma is T-cell non-Hodgkin's lymphoma (T-NHL).

41. 41. The multispecific antibody, nucleic acid construct, delivery vehicle, or pharmaceutical composition for use according to claim 40, wherein the T-NHL is cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL).

42. 40. The multispecific antibody, nucleic acid construct, delivery vehicle, or pharmaceutical composition for use according to claim 39, wherein the T-cell non-Hodgkin's lymphoma (T-NHL) is anaplastic large cell lymphoma (ALCL).

43. 40. The multispecific antibody, nucleic acid construct, delivery vehicle, or pharmaceutical composition for use according to claim 39, wherein the non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma (B-NHL).

44. 38. The multispecific antibody, nucleic acid construct, delivery vehicle or pharmaceutical composition for use according to claim 37, wherein said multispecific antibody, nucleic acid construct, delivery vehicle or pharmaceutical composition is administered intravenously and / or subcutaneously, preferably subcutaneously.

45. 10. A method for producing a multispecific antibody according to claim 1, the method comprising: (i) culturing the recombinant host cell of claim 34 or 35 under conditions in which the antibody is produced; (ii) isolating the produced multispecific antibodies from the culture; and A method comprising:

46. 10. A method for producing a multispecific antibody according to claim 9, comprising: a) providing a first antibody (i) comprising a CD30 binding region as defined in claim 1, and a second antibody (ii) comprising a CD3 binding region as defined in claim 1, wherein said antibodies optionally contain the further features as defined in claim 2; wherein the first and second antibodies comprise an Fc region; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, and thus the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining said multispecific antibody comprising a first immunoglobulin heavy chain and a first immunoglobulin light chain of said first antibody and a second immunoglobulin heavy chain and a second immunoglobulin light chain of said second antibody; A method comprising:

47. A kit comprising an antibody as defined in claim 1 and instructions for use. A kit of parts, such as a kit for use as a companion diagnostic / to identify patients within a patient population who have a propensity to respond to treatment with an antibody as defined in claim 1.

48. A diagnostic composition comprising the multispecific antibody of claim 1.

49. (i) a CD30 binding region comprising a first heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a first light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; (ii) an Fc region consisting of first and second Fc polypeptides, wherein said first and second Fc polypeptides comprise substitutions of amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein said first and second Fc polypeptides further comprise a substitution of an amino acid corresponding to amino acid at position G236 in a human IgG1 heavy chain, wherein said substitution is preferably with R, and wherein said amino acid positions are as defined by Eu numbering; An antibody comprising:

50. 50. The antibody of claim 49, wherein the first heavy chain variable region comprises the sequence set forth in SEQ ID NO: 13 and the first light chain variable region comprises the sequence set forth in SEQ ID NO:

14.

51. 51. The antibody of claim 49 or 50, wherein the antibody comprises or consists of a heavy chain sequence set forth in SEQ ID NO: 17 and a light chain sequence set forth in SEQ ID NO:

18.

52. (i) a CD3 binding region comprising a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; (ii) an Fc region consisting of first and second Fc polypeptides, wherein said first and second Fc polypeptides comprise substitutions of amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and wherein said first and second Fc polypeptides further comprise a substitution of an amino acid corresponding to amino acid at position G236 in a human IgG1 heavy chain, wherein said substitution is preferably with R, and wherein said amino acid positions are as defined by Eu numbering; An antibody comprising:

53. 53. The antibody of claim 52, wherein X in SEQ ID NO: 9 is H.

54. The antibody of claim 52, wherein X in SEQ ID NO: 9 is G.

55. 53. The antibody of claim 52, wherein the second heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15 and the second light chain variable region comprises the sequence set forth in SEQ ID NO:

16.

56. 50. The antibody of claim 49, wherein the antibody is a full-length antibody.

57. 50. The antibody of claim 49, wherein at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, and preferably the amino acid at the position corresponding to F405 is L or the amino acid at the position corresponding to K409 is R.

58. 1. A method for producing a multispecific antibody, the method comprising: a) providing a first antibody of claim 49 or 55 and a second light chain variable region comprising: (i) a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a CD3 binding region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second polypeptides comprise substitutions of F and E for amino acids corresponding to amino acids at positions L234 and L235, respectively, and substitution of A for an amino acid corresponding to amino acid at position D265 in a human IgG1 heavy chain; or 53. The method of claim 52, further comprising providing a second antibody and a first antibody comprising: (i) a CD30 binding region comprising a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second polypeptides comprise substitutions of F and E for amino acids corresponding to amino acids at positions L234 and L235, respectively, and substitution of A for an amino acid corresponding to amino acid at position D265 in a human IgG1 heavy chain; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different; thus, the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions, and preferably, in the first CH3 region, the amino acid at the position corresponding to F405 is L and in the second CH3 region, the amino acid at the position corresponding to K409 is R, or vice versa; b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining the multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody. A method comprising:

59. 59. A method for producing a multispecific antibody according to claim 58, the method comprising: a) providing a first antibody according to claim 49 or 55, and a second antibody comprising: (i) a CD3 binding region comprising a second heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a second light chain variable region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; and (ii) an Fc region consisting of a first and a second Fc polypeptide, wherein the first and second polypeptides comprise substitutions of F and E for amino acids corresponding to amino acids at positions L234 and L235, respectively, and substitution of A for an amino acid corresponding to amino acid at position D265 in a human IgG1 heavy chain; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different; Thus, the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions between the first and second CH3 regions, and preferably, in the first CH3 region, the amino acid at the position corresponding to K409 is R, and in the second CH3 region, the amino acid at the position corresponding to F405 is L; b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining the multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody; A method comprising:

60. 59. A multispecific antibody obtained by the method of claim 58.

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