Antibodies capable of binding to ROR2, and bispecific antibodies that bind to both ROR2 and CD3.

Abstract

The present invention relates to antibodies that bind to ROR2, including bispecific antibodies that bind to ROR2 and CD3. The present invention further provides pharmaceutical compositions comprising said antibodies, and uses of said antibodies for therapeutic and diagnostic procedures, particularly in cancer treatment.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to antibodies that bind to ROR2, including bispecific antibodies that bind to ROR2 and CD3. The present invention further provides pharmaceutical compositions comprising the antibody, and the use of the antibody for therapeutic and diagnostic procedures, particularly in cancer treatment. [Background technology]

[0002] background ROR2 (receptor tyrosine kinase-like orphan receptor 2, NTRKR2, neurotrophic tyrosine kinase receptor-related 2) is a single-pass type I transmembrane glycoprotein belonging to the ROR subfamily of the tyrosine protein kinase family. ROR2 is a tyrosine kinase receptor that plays a crucial role in regulating skeletal and neuronal development, cell migration, and cell polarity, partly through its role in the proposed non-canonical Wnt5a signaling pathway (Oishi 2003, Genes to cells 8:6450654 (Non-patent Literature 1)). It contains an FZ (frizzled) domain, an Ig (immunoglobulin)-like C2 type domain, an extracellular kringle domain, and a cytoplasmic protein kinase domain (Masiakowski and Carroll 1992, J Biol Chem 267:26181-90 (Non-patent Literature 2)).

[0003] In normal human adult tissues, ROR2 expression is extremely limited (limited to the uterus during the menstrual cycle, the brain during post-injury repair, bone during bone formation, and the intestines as part of intestinal homeostasis (Debebe and Rathmell 2015, Pharmcol&Therap 150:143-148 (Non-Patent Literature 3), Endo 2017, Dev Dyn 247:24-32 (Non-Patent Literature 4))). However, ROR2 expression is found on human tumor cells in numerous cancer tissues, including sarcomas, uterine, pancreatic, melanoma, renal cell carcinoma, prostate cancer, colorectal cancer, head and neck squamous cell carcinoma, stromal tumors, and breast cancer tissue (see Debebe and Rathmell 2015, Pharmcol&Therap 150:143-148 (Non-Patent Literature 3) for a review).

[0004] Therefore, targeting ROR2 to treat cancer has been proposed. For example, the ROR2-specific antibody-drug conjugate CAB-ROR2-ADC / BA3021 is under development for the treatment of solid tumors and soft tissue sarcomas (Sharp et al. Proceedings of the AACR Annual Meeting 2018; Cancer Res 78(13 Suppl): abstract 833 (Non-Patent Literature 5)). In addition, ROR2-targeting chimeric antigen receptor (CAR) T cells are under development for kidney cancer (Association for Cancer Immunotherapy (CIMT) 2019 Annual Meeting, abstract 123 (Non-Patent Literature 6)).

[0005] Efforts have also been made to target T cells to ROR2. A ROR2 / CD3 bispecific molecule in scFv-Fc format based on a humanized ROR2 rabbit antibody that exhibits T cell toxicity in tumor cell lines has been described (Goydel et al 2020, J Biol Chem 295:5995-6006 (Non-Patent Literature 7)).

[0006] Despite some progress, there is still a need to develop effective and safe ROR2-targeted antibody-based cancer therapies for use in humans, such as in the treatment of cancer.

[0007] An object of the present invention is to provide an antibody comprising at least one antigen-binding region capable of binding to human ROR2. A further object of the present invention is to provide an antibody comprising two antigen-binding regions capable of binding to human ROR2. A further object of the present invention is to provide a bispecific antibody capable of binding to human ROR2 and human CD3, for example, human CD3ε (epsilon). A further object of the present invention is to provide a CD3×ROR2 bispecific antibody in IgG format, for example, in human IgG1 format. A further object of the present invention is to provide a CD3×ROR2 bispecific antibody in IgG1 format in which the Fc region is inactive. A further object of the present invention is to provide a CD3×ROR2 bispecific antibody having a plasma half-life within the range of a normal human IgG1 antibody. A further object of the present invention is to provide a ROR2 antibody and / or CD3×ROR2 bispecific antibody that is effective and safe for the treatment of cancer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Oishi 2003,Genes to cells 8:6450654 [Non-Patent Document 2] Masiakowski and Carroll 1992, J Biol Chem 267:26181-90 [Non-Patent Document 3] Debebe and Rathmell 2015,Pharmcol&Therap 150:143-148 [Non-Patent Document 4] Endo 2017, Dev Dyn 247:24-32 [Non-Patent Document 5] Sharp et al.Proceedings of the AACR Annual Meeting 2018;Cancer Res 78(13 Suppl):abstract 833 [Non-Patent Document 6] Association for Cancer Immunotherapy(CIMT)2019 Annual Meeting,abstract 123 [Non-Patent Document 7] Goydel et al 2020,J Biol Chem 295:5995-6006 [Summary of the Invention]

[0009] In one main aspect, the present invention relates to an anti-ROR2 binding antibody, particularly an antibody comprising at least one antigen-binding region capable of binding to human ROR2, wherein the antibody comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 3, 4 and 5, respectively, and light chain variable (VL) region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 7, 8 and 9, respectively.

[0010] In a further aspect, the antibody may be a bispecific antibody particularly comprising a first antigen-binding region capable of binding to human ROR2 and a second antigen-binding region capable of binding to human CD3, for example human CD3 epsilon, such as human CD3 epsilon as specified in SEQ ID NO: 21, wherein the antibody comprises VH region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 3, 4 and 5, respectively, and VL region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 7, 8 and 9, respectively.

[0011] In a further aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding region capable of binding to human ROR2 as described herein, and a second antigen-binding region capable of binding to human CD3 comprising VH regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 25, and VL regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, GTN, and SEQ ID NO: 28, respectively.

[0012] In another aspect, the present invention is a) A nucleic acid sequence encoding a heavy chain sequence of an antibody that includes an antigen-binding region capable of binding to ROR2 as defined herein, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody that includes an antigen-binding region capable of binding to ROR2 as defined herein. Regarding nucleic acid constructs that include...

[0013] In another aspect, the present invention is a) A nucleic acid sequence encoding a heavy chain sequence of an antibody that includes an antigen-binding region capable of binding to ROR2 as defined herein, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody that includes an antigen-binding region capable of binding to ROR2 as defined herein. Regarding expression vectors containing this.

[0014] In another aspect, the present invention relates to cells comprising nucleic acid constructs or expression vectors as defined herein.

[0015] In a further aspect, the present invention relates to a composition comprising an antibody in any aspect or embodiment as described herein.

[0016] In a further aspect, the present invention relates to a pharmaceutical composition comprising an antibody in any aspect or embodiment as described herein and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present invention relates to antibodies in any aspect or embodiment described herein for use as pharmaceuticals, for example, for use in the treatment of diseases.

[0018] In a further aspect, the present invention relates to a method for treating a disease or disorder, comprising the step of administering an antibody, composition, or pharmaceutical composition of any aspect or form described herein to a subject in need.

[0019] In one aspect, the present invention relates to a method for producing an antibody in any aspect or embodiment as described herein, comprising the step of culturing recombinant host cells in a culture medium under conditions suitable for antibody production.

[0020] In another aspect, the present invention relates to a kit of parts comprising an antibody as defined herein and instructions for use of the kit.

[0021] [Invention 1001] An antibody comprising at least one antigen-binding region capable of binding to human ROR2, comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9, respectively. [Invention 1002] The antibody of the present invention 1001 comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3, each containing two antigen-binding regions capable of binding to human ROR2 and having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3, each having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. [Invention 1003] An antibody according to either Invention 1001 or 1002, which is humanized from an antibody containing a VH region having the sequence shown in SEQ ID NO:2 and / or a VL region having the sequence shown in SEQ ID NO:6. [Invention 1004] a. The VH region shown in SEQ ID NO:10(HC1); b. The VH region shown in SEQ ID NO:11(HC2); c. The VH region shown in SEQ ID NO:12(HC3); d. The VH region shown in SEQ ID NO:13(HC4); e. The VH region shown in SEQ ID NO:14(HC5); f. The VH region shown in SEQ ID NO:15(HC6); g. The VH region shown in SEQ ID NO:16(HC7), or h. VH region having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 10, 11, 12, 13, 14, 15, or 16 An antibody according to the present invention, comprising a VH region having a sequence selected from the group including the above. [Invention 1005] Any antibody of the present invention, comprising a VH region having the sequence shown in SEQ ID NO:13. [Invention 1006] a. The VL region shown in SEQ ID NO:17(LC1); b. The VL region shown in SEQ ID NO:18(LC2); c. The VL region shown in SEQ ID NO:19(LC3); d. The VL region shown in SEQ ID NO:20(LC4); or e. VL regions having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 17, 18, 19, or 20. An antibody according to the present invention, comprising a VL region having a sequence selected from the group including the above. [Invention 1007] Any antibody of the present invention, comprising a VL region having the sequence shown in SEQ ID NO:19. [Invention 1008] a. VH region containing the sequence of SEQ ID NO. 10 and VL region containing the sequence of SEQ ID NO. 17; b. The VH region containing the sequence of SEQ ID NO. 10 and the VL region containing the sequence of SEQ ID NO. 18; c. VH region containing the sequence of SEQ ID NO. 10 and VL region containing the sequence of SEQ ID NO. 19; d. The VH region containing the sequence of SEQ ID NO. 10 and the VL region containing the sequence of SEQ ID NO. 20; e. The VH region containing the sequence of SEQ ID NO. 11 and the VL region containing the sequence of SEQ ID NO. 17; f. VH region containing the sequence of SEQ ID NO. 11 and VL region containing the sequence of SEQ ID NO. 18; g. The VH region containing the sequence of SEQ ID NO. 11 and the VL region containing the sequence of SEQ ID NO. 19; h. VH region containing the sequence of SEQ ID NO. 11 and VL region containing the sequence of SEQ ID NO. 20; i. The VH region containing the sequence of SEQ ID NO. 12 and the VL region containing the sequence of SEQ ID NO. 17; j. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 18; k. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 19; l. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 20; m. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 17; n. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 18; o. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 19; p. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 20; q. The VH region containing the sequence of SEQ ID NO. 14 and the VL region containing the sequence of SEQ ID NO. 17; r. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 18; s. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 19; t. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 20; u. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 17; v. The VH region containing the sequence of SEQ ID NO. 15 and the VL region containing the sequence of SEQ ID NO. 18; w. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 19; x. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 20; y. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 17; z. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 18; aa. VH region having the sequence of SEQ ID NO. 16 and VL region having the sequence of SEQ ID NO. 19; and bb. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 20 An antibody according to the present invention, comprising a VH region and a VL region having a sequence selected from the group including the above. [Invention 1009] Any antibody of the present invention comprising a VH region and a VL region having sequences of SEQ ID No. 13 and 19. [Invention 1010] An antibody according to the present invention, wherein the VH region and VL region are humanized. [Invention 1011] An antibody according to the present invention, wherein the heavy chain constant region is human IgG1. [Invention 1012] An antibody according to the present invention, wherein the light chain constant region is human kappa. [Invention 1013] Any antibody of the present invention, which is a full-length antibody such as a full-length IgG1 antibody. [Invention 1014] A monovalent antibody, which is any of the antibodies of the present invention described above. [Invention 1015] A bivalent antibody, which is any of the antibodies of the present invention described above. [Invention 1016] An antibody according to the present invention, which is a monospecific antibody. [Invention 1017] Any of the antibodies of the present invention described above, which are bispecific antibodies. [Invention 1018] Any antibody of the present invention, wherein the human ROR2 is human ROR2 with SEQ ID NO. 1. [Invention 1019] Any antibody of the present invention that can bind to the kringle domain of human ROR2. [Invention 1020] An antibody according to the present invention, which binds to an epitope or antibody-binding region on human ROR2 involving the 322nd amino acid residue of human ROR2, with a numbering indicating its position in SEQ ID NO:1. [Invention 1021] Any antibody of the present invention can bind to the human ROR2 extracellular domain with a binding affinity corresponding to a KD value of 100 nM or less, for example, 50 nM or less, 10 nM or less, 6 nM or less, or for example, 3 nM or less, for example, in the range of 100 nM to 0.1 nM, for example, 100 nM to 1 nM, for example, in the range of 50 nM to 1 nM, for example, less than about 2.5 nM, or less than about 2.0 nM, or less than about 1.5 nM, for example, about 1.1 nM. [Invention 1022] The antibody of the present invention 1021, wherein the binding affinity is optionally determined by biolayer interferometry as shown in Example 6 of this specification. [Invention 1023] a. A step of immobilizing the antibody onto an anti-human IgG Fc capture biosensor at a volume of 1 μg / mL over 600 seconds. b. A step of determining the association and dissociation of ROR2ECDHis over a period of 1,500 seconds using a 2x dilution series in the range of 100 nM to 1.56 nM. c. The process of relating the data to a buffer control (0 nM). An antibody according to either of the present inventions 1021 or 1022, wherein the binding affinity is determined using a biolayer interference method including the above. [Invention 1024] An antibody according to any of the present invention 1021 to 1023, wherein the binding affinity is determined using any of the antibodies of the present invention, which is a monospecific bivalent antibody, for example, an antibody that is full-length IgG1. [Invention 1025] An antibody comprising a first antigen-binding region capable of binding to any of the human ROR2s of the present invention, and a second antigen-binding region capable of binding to a different target. [Invention 1026] The antibody of the present invention 1025, wherein the second antigen-binding region can bind to human CD3, for example, human CD3ε (epsilon), for example, human CD3ε (epsilon) as specified in SEQ ID NO:21. [Invention 1027] The bispecific antibody of the present invention, antibody 1025 or 1026. [Invention 1028] The antigen-binding region that binds to CD3, The heavy chain variable region (VH) contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, 24, and 25, respectively. And optionally, Light chain variable regions (VLs) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:27, GTN, and SEQ ID NO:28, respectively. The antibody of the present invention 1026 or 1027, comprising the above. [Invention 1029] The antigen-binding region that binds to CD3, a. A heavy chain variable region (VH) containing the sequence of SEQ ID NO:22 or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:22. b. and a light chain variable region (VL) containing the sequence of SEQ ID NO:26 or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:26. An antibody according to any of the present invention 1026 to 1028, including the above. [Invention 1030] The antigen-binding region that binds to CD3, a. A heavy chain variable region (VH) containing the sequence of SEQ ID NO:29 or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. b. and optionally, a light chain variable region (VL) containing the sequence of SEQ ID NO:30 or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:30. An antibody according to any of the present invention 1026 to 1029, including the above. [Invention 1031] The antigen-binding region that binds to CD3, a. Heavy chain variable region (VH) containing the sequence of SEQ ID NO:29 and b. Light chain variable region (VL) containing the sequence of SEQ ID NO:30 An antibody according to any of the present invention 1026 to 1030, including the above. [Invention 1032] An antibody according to any of the present invention 1025 to 1030, having a lower human CD3ε binding affinity than an antibody having an antigen-binding region including the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, preferably such affinity is at least 1 / 5, for example at least 1 / 10, for example at least 1 / 20, for at least 1 / 30, for at least 1 / 40, for at least 1 / 45, or for example at least 1 / 50, for example at least 1 / 54. [Invention 1033] An antibody according to any of the Invention 1026 to 1032, wherein the antigen-binding region that binds to CD3 binds with an equilibrium dissociation constant KD in the range of 200 to 1000 nM, for example, in the range of 300 to 1000 nM, in the range of 400 to 1000 nM, in the range of 500 to 1000 nM, in the range of 300 to 900 nM, in the range of 400 to 700 nM, in the range of 500 to 800 nM, in the range of 500 to 700 nM, in the range of 600 to 1000 nM, in the range of 600 to 900 nM, in the range of 600 to 800 nM, or for example, in the range of 600 to 700 nM. [Invention 1034] An antibody according to any of the Invention 1026 to 1031, wherein the antigen-binding region that binds to CD3 binds with an equilibrium dissociation constant KD in the range of 1 to 100 nM, for example, in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, or for example, in the range of 10 to 20 nM. [Invention 1035] The antigen-binding region that binds to CD3 includes a heavy chain variable (VH) region containing the CDR1, CDR2, and CDR3 sequences. When the heavy chain variable (VH) region is compared with the heavy chain variable (VH) region containing the sequence shown in SEQ ID NO:29, one of the CDR sequences has an amino acid substitution at a position selected from the group consisting of positions T31, N57, H101, G105, S110, and Y114, which are numbered according to the sequence of SEQ ID NO:29, and The wild-type light chain variable (VL) region contains the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:27, GTN, and SEQ ID NO:28, respectively. Any antibody according to invention 1026 to 1034. [Invention 1036] An antibody according to any of the Invention 1026-1035, wherein the CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding domain that binds to CD3 contain at most 1, 2, 3, 4, or 5 amino acid substitutions in total when compared to the CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO:29. [Invention 1037] An antibody according to any of Invention 1026 to 1035, wherein the antigen-binding region that binds to CD3 contains a mutation in the VH region selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V. [Invention 1038] An antibody according to any of the Invention 1025 to 1036, wherein the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 31, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, sequence GTN, and sequence shown in SEQ ID NO: 28, respectively. [Invention 1039] An antibody according to any of the present invention 1026, 1027, or 1029-1038, wherein the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:32 and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:30. [Invention 1040] This is a bispecific antibody comprising a first antigen-binding region that can bind to ROR2 and a second binding region that can bind to human CD3. The first antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. including and The second antigen-binding region is The heavy chain variable (VH) regions CDR1, CDR2, and CDR3 [anti-CD3 (SP34 / humanized SP34, WO2015001085(Genmab))-VH CDR sequences] having sequences shown in NO:23, 24, and 25 respectively, and the light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:27, GTN, and SEQ ID NO:28 respectively. including, Any antibody of the present invention as described above. [Invention 1041] This is a bispecific antibody containing a first antigen-binding region that can bind to human ROR2 and a second binding region that can bind to human CD3. The first antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. including and The second antigen-binding region is A heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 31, respectively, and a light chain variable (VL) region containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, sequence GTN, and sequence shown in SEQ ID NO: 28, respectively. including, Any antibody of the present invention as described above. [Invention 1042] It comprises a first antigen-binding region that can bind to human ROR2 and a second antigen-binding region that can bind to human CD3, The first antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:29 and a VL region containing the sequence shown in SEQ ID NO:30. Any antibody of the present invention as described above. [Invention 1043] It comprises a first antigen-binding region that can bind to human ROR2 and a second antigen-binding region that can bind to human CD3, The first antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:32 and a VL region containing the sequence shown in SEQ ID NO:30. Any antibody according to the present invention 1001-1030, 1032, 1033, 1035-1039, and 1041. [Invention 1044] a) The antigen-binding region capable of binding to ROR2 is humanized, and / or b) If an antigen-binding region capable of binding to CD3 is present, it is humanized. Any antibody of the present invention as described above. [Invention 1045] It includes the first heavy chain steady region and the second heavy chain steady region, Each of the first and second heavy chain constant regions includes at least a hinge region, CH2, and CH3 regions, and in the first heavy chain constant region, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain, and in the second heavy chain constant region, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain, and the substitutions in the first and second heavy chains are not at the same position, and the amino acid positions are numbered according to Eu numbering. Any antibody of the present invention as described above. [Invention 1046] An antibody according to the present invention, wherein in the primary heavy chain, the amino acid at the position corresponding to K409 in the human IgG mono-heavy chain is R, and in the secondary heavy chain, the amino acid at the position corresponding to F405 in the human IgG mono-heavy chain is L, or vice versa. [Invention 1047] The primary and secondary heavy chains of the antibody have been modified to induce Fc-mediated effector function to a lower degree compared to the unmodified antibody. An antibody according to any of the present inventions, including the above-mentioned antibody. [Invention 1048] It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residues corresponding to positions L234 and L235 in the human IgG monohelic acid according to Eu numbering are F and E, respectively. Any antibody of the present invention as described above. [Invention 1049] It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residue at the position corresponding to position D265 in the human IgG monohelic acid according to Eu numbering is A. Any antibody of the present invention as described above. [Invention 1050] It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residues corresponding to positions L234, L235, and D265 in the human IgG monohelic acid according to Eu numbering are F, E, and A, respectively. Any antibody of the present invention as described above. [Invention 1051] It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residues at positions L234, L235, and D265 in the human IgG monohelic acid according to Eu numbering are F, E, and A, respectively, and the first heavy chain constant region further includes a K409R substitution, and the second heavy chain constant region further includes an F405L substitution. Any antibody of the present invention as described above. [Invention 1052] Any antibody of the present invention comprising a first heavy chain constant region and a second heavy chain constant region having sequences shown in SEQ ID No. 34 and 35, respectively, or a first heavy chain constant region and a second heavy chain constant region having sequences shown in SEQ ID No. 35 and 34, respectively. [Invention 1053] It is a bispecific antibody that can bind to human ROR2 and human CD3 epsilon. a. The first connecting arm that connects to ROR2 is i. VH region having amino acid sequence SEQ ID NO:13 ii. VL region having amino acid sequence SEQ ID NO:19 iii. A heavy chain constant region having the amino acid sequence (FEAR) of SEQ ID NO:34, and iv. The steady-state region of the human kappa light chain including and b. The second binding arm that binds to CD3 epsilon, i. VH region having amino acid sequence SEQ ID NO:29 ii. VL region having amino acid sequence SEQ ID NO:30 iii. A heavy chain constant region having the amino acid sequence (FEAL) of SEQ ID NO:35, and iv. Steady-state region of human lambda light chain including, Any antibody of the present invention as described above. [Invention 1054] It is a bispecific antibody that can bind to human ROR2 and human CD3 epsilon. a. The first connecting arm that connects to ROR2 is i. VH region having amino acid sequence SEQ ID NO:13 ii. VL region having amino acid sequence SEQ ID NO:19 iii. A heavy chain constant region having the amino acid sequence (FEAR) of SEQ ID NO:34, and iv. The steady-state region of the human kappa light chain including and b. The second binding arm that binds to CD3 epsilon, i. VH region having amino acid sequence SEQ ID NO:32 ii. VL region having amino acid sequence SEQ ID NO:30 iii. A heavy chain constant region having the amino acid sequence (FEAL) of SEQ ID NO:35, and iv. Steady-state region of human lambda light chain including, Any antibody of the present invention as described above. [Invention 1055] An antibody according to the present invention, comprising a lambda (λ) light chain. [Invention 1056] a. As described in Examples 6 and 10 of this specification, it can bind to ROR2-expressing human tumor cells such as HeLa, LCLC103-H, NCI-H1650, 786-), NCI-H23, or ZR-75-1 cells. b. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of HeLa cells, for example, when assayed as described in Example 11 or 12 of this specification. c. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of 786-O, LCLC-103H, NCI-H23, NCH-H1650, or ZR-75-1 cells, for example, when assayed as described in Example 12 of this specification. d. For example, when assayed as described in Example 14 of this specification, T cells can be activated in vitro in the presence of HeLa, 786-O, LCLC-103H, NCI-H23, NCH-H1650 tumor cells, and / or e. When tumor cells such as HeLa and 786-O cells are used as target cells, T cell cytokine production can be induced, for example, when the assay is performed as described in Example 13 of this specification. Any antibody of the present invention as described above. [Invention 1057] A composition comprising any antibody according to Invention 1001 to 1056. [Invention 1058] A pharmaceutical composition comprising any antibody according to invention 1001 to 1056 and a pharmaceutically acceptable carrier. [Invention 1059] An antibody according to any of invention 1001 to 1056 for use as a pharmaceutical. [Invention 1060] An antibody for use as a pharmaceutical according to Invention 1059, for use in the treatment of a disease. [Invention 1061] An antibody for pharmaceutical use of the present invention 1060, wherein the disease is cancer. [Invention 1062] An antibody for pharmaceutical use according to the present invention 1061, characterized in that cancer cells exhibit ROR2 expression on the surface of cancer cells. [Invention 1063] An antibody for pharmaceutical use, for use in cancer cells obtained from a patient, wherein ROR2 expression is determined in cancer cells obtained from the patient, according to the invention 1062. [Invention 1064] An antibody for use as a pharmaceutical according to any of the inventions 1061 to 1063, wherein cancer is a solid tumor. [Invention 1065] An antibody for use in any of the invention 1061 to 1064, wherein the cancer is selected from the group including sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer. [Invention 1066] A method for treating a disease, comprising the step of administering an antibody according to any of Invention 1001 to 1056, a composition according to Invention 1057, or a pharmaceutical composition according to Invention 1058 to a subject in need. [Invention 1067] A method according to the present invention 1066 for the treatment of cancer. [Invention 1068] The method of the present invention 1067, wherein the cancer is selected from the group including sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer. [Invention 1069] a. A nucleic acid sequence and / or encoding a heavy chain variable region sequence of an antigen-binding region that can bind to any of the ROR2s of the present invention 1001-1005, 1008, and 1009. b. A nucleic acid sequence encoding the corresponding light chain variable region sequence of the antigen-binding region that can bind to any of the ROR2 sequences 1001-1003 and 1006-1009 of the present invention. Nucleic acids, including [Invention 1070] a. A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region that can bind to ROR2 of the present invention 1009, b. A nucleic acid sequence encoding the corresponding light chain sequence of an antibody containing an antigen-binding region capable of binding to ROR2 of the present invention 1009. One or more nucleic acids, including one or more nucleic acids. [Invention 1071] a. A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region that can bind to ROR2 as shown in SEQ ID NO:13, and / or b. A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to ROR2, as shown in SEQ ID NO:19. One or more nucleic acids, including one or more nucleic acids. [Invention 1072] A nucleic acid, or one or more nucleic acids, which are RNA or DNA, according to any of the invention items 1069 to 1071. [Invention 1073] Any nucleic acid of Invention 1069-1072 or one or more nucleic acids for use in expression in mammalian cells. [Invention 1074] a) A nucleic acid sequence encoding an antibody heavy chain sequence containing an antigen-binding region that can bind to any of the ROR2s of the present invention 1069-1073, and / or b) A nucleic acid sequence encoding an antibody light chain sequence containing an antigen-binding region that can bind to any of the ROR2 sequences described in invention 1069 to 1073. An expression vector containing [the specified element]. [Invention 1075] a. A nucleic acid sequence encoding a heavy chain sequence of an antibody containing an antigen-binding region that can bind to any of the CD3s 1028-1031 and 1035-1039 of the present invention, and / or b. A nucleic acid sequence encoding an antibody light chain sequence containing an antigen-binding region that can bind to any of the CD3s 1028-1031 and 1035-1039 of the present invention. An expression vector of the present invention 1074, further comprising: [Invention 1076] A cell comprising any nucleic acid of Invention 1069 to 1073, or one or more nucleic acids, or an expression vector of Invention 1074 or 1075. [Invention 1077] The cells of the present invention 1076, which are of human origin, for example, human fetal kidney (HEK) cells, or of rodent origin, for example, Chinese hamster ovary cells (CHO cells). [Invention 1078] a. A step of providing an antibody capable of binding to ROR2, which includes an antigen-binding region capable of binding to any of the ROR2s of the present invention 1001 to 1056. b. A step of providing an antibody capable of binding to CD3, comprising an antigen-binding region capable of binding to any of the CD3s of the present invention 1026 to 1056. c. A step of incubating an antibody capable of binding to ROR2 together with an antibody capable of binding to CD3 under conditions of sufficient reduction to allow cysteine ​​in the hinge region to undergo isomerization of the disulfide bond, and d. Steps to obtain antibodies that can bind to ROR2 and CD3. A method for producing an antibody that can bind to both ROR2 and CD3 according to any of the present invention 1001 to 1056, including the above. [Invention 1079] Steps a) and / or b) are To provide cells containing an expression vector for producing one or more of the aforementioned antibodies, and To cause cells to produce one or more of the aforementioned antibodies, and subsequently, To obtain one or more types of the aforementioned antibodies, thereby providing one or more types of the aforementioned antibodies. A method for producing an antibody capable of binding to both ROR2 and CD3, further comprising the present invention 1078. [Invention 1080] A kit of parts, such as a kit for use as a companion diagnostic agent / for identifying patients in a patient population who are capable of responding to treatment with any of the antibodies of the present invention 1001 to 1056, the kit of parts comprising any of the antibodies of the present invention 1001 to 1056 and instructions for use of the kit. [Invention 1081] An anti-idiotype antibody that binds to an antigen-binding region capable of binding to any of the ROR2s specified in invention 1001 to 1056. These and other aspects and embodiments of the present invention will be described in more detail below. [Brief explanation of the drawing]

[0022] [Figure 1-1] Figure 1. Binding of the rabbit-human chimeric antibody chIgG1-ROR2-A-FEAR and its humanized variant to ROR2 expressed on the cervical cancer cell line HeLa, as determined by flow cytometry. [Figure 1-2] See the explanation in Figure 1-1. [Figure 1-3] See the explanation in Figure 1-1. [Figure 2] Binding of the bispecific CD3×ROR2 antibody and monospecific ROR2 antibody of the present invention to human or cynomolgus monkey ROR2-expressing CHO cells. Binding of bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR, chIgG1-ROR2-A-FEAR, and IgG1-ROR2-A-HC4LC3-FEAR to human ROR2 (left panel) or cynomolgus monkey ROR2 (right panel) CHO cells was determined by flow cytometry. Untransfected CHO cells were used as a negative control (not shown). [Figure 3]The ROR2 monospecific antibody and the CD3×ROR2 bispecific antibody of the present invention were conjugated to CHO cells expressing human ROR2, cynomolgus monkey ROR2, or the T322M variant of cynomolgus monkey ROR2. The conjugation of chIgG1-ROR2-A-FEAR and bsIgG1-huCD3-FEAL×chROR2-A-FEAR was determined by flow cytometry. [Figure 4] The binding of the CD3×ROR2 bispecific antibody of the present invention to CHO cells expressing the T322M variant of cynomolgus monkey ROR2. The binding of bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR, bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR to CHO cells expressing RORmf-T322M was determined by flow cytometry. [Figure 5A] Figure 5. Binding of the CD3×ROR2 bispecific antibody of the present invention to ROR2-expressing CD3-negative human tumor cell lines. (A) Binding of bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR to human tumor cell lines HeLa (cervical cancer), LCLC103-H (large cell lung cancer), NCI-H1650 (lung adenocarcinoma), 786-O (renal cell adenocarcinoma), NCI-H23 (lung adenocarcinoma) and ZR-75-1 (ductal carcinoma). Binding was determined by flow cytometry. bsIgG1-huCD3-H101G-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control antibody. [Figure 5B] Figure 5. Binding of the CD3×ROR2 bispecific antibody of the present invention to ROR2-expressing CD3-negative human tumor cell lines. (B) ROR2 expression levels on test tumor cell lines determined by QIFI analysis; results from individual analyses (n=4~6), median and range shown. sABC: Specific antibody binding ability. [Figure 6]In vitro induction of T cell-mediated cytotoxicity in co-cultures of ROR2-positive HeLa cells and healthy human donor T cells at various effector-to-target ratios (E:T) in the presence of CD3×ROR2 bispecific antibodies bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR. bsIgG1-huCD3-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control antibody. HeLa cell viability was used as a readout for T cell-mediated cytotoxicity. [Figure 7] In vitro induction of cytotoxicity in various tumor cell lines using CD3×ROR2 bispecific antibodies bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR in the presence of healthy human donor T cells. bsIgG1-huCD3-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control antibody. Tumor cell viability was used as a readout for T cell-mediated cytotoxicity. [Figure 8] Maximum T-cell-mediated tumor cell killing (2–7 donors per cell line) in the presence of bsIgG1-huCD3-FEAL×chROR2-A-FEAR or bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR for different tumor cell lines. Cell lines were ranked according to ROR2 expression levels. Maximum tumor cell killing was determined as the difference between the upper and lower limits of the dose-response curve and is shown as the mean and standard deviation (shown either above or below the mean for clarity). The vertical dotted line indicates the detection limit of the QIFI analysis for determining the ROR2 expression level. sABC: Specific antibody binding ability. [Figure 9A]The concentration of cytokine IL-6 in the supernatant of T cell-tumor cell co-culture when the concentration of the antibody bsIgG1-huCD3-FEAL×chROR2-A-FEAR or bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR was increased using T cells from two donors and 786-O cells as target cells. [Figure 9B-1] Concentrations of IFN-gamma, IL-6, IL-8, and IL-10 associated with T cell-mediated cytotoxicity (IC50 and IC90) in 50% and 90% of tumor cells, measured in the supernatant of T cell-tumor cell (HeLa or 786-O) co-cultures in the presence of bsIgG1-huCD3-FEAL×chROR2-A-FEAR or bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR. Cytokine levels were determined by multiplex U-plex assay. (A) shows dose-response curves. (B) shows geometric mean and standard deviation (error bars). Results from two T cell donors per cell line are presented. [Figure 9B-2] Refer to the explanation in Figure 9B-1. [Figure 10] Cytotoxic activity of CD3×ROR2 bispecific antibodies bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR in HeLa cells in the presence of cynomolgus monkey PBMCs as a T cell source in vitro. bsIgG1-huCD3-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control antibody. [Figure 11]Induction of T cell activation in a cynomolgus monkey PBMC population in the presence of CD3×ROR2 bispecific antibodies bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR and HeLa cells. T cell activation (percentage of CD69, CD25, or PD-1 on CD8+ cells) in the presence of antibodies and HeLa cells was determined by flow cytometry. bsIgG1-huCD3-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control antibody. [Figure 12] ROR2 mRNA expression levels in selected primary solid tumors. ROR2 mRNA levels were extracted from the Omicsoft TCGA database and visualized using Oncoland software. Signs were ranked according to the median of ROR2 mRNA expression. LIHC = hepatocellular carcinoma, whole kidney = renal cancer (combined clear cell carcinoma, renal chromophobe cell carcinoma, and renal papillary cell carcinoma), metastatic melanoma = metastatic cutaneous melanoma, primary melanoma = primary cutaneous melanoma, COAD = colon adenocarcinoma, LUAD = lung adenocarcinoma, CESC = cervical squamous cell carcinoma, BLCA = bladder urothelial carcinoma, HNSC = head and neck squamous cell carcinoma, LUSC = lung squamous cell carcinoma, OV = ovarian serous cystadenocarcinoma, BRCA = invasive breast carcinoma, PAAD = pancreatic adenocarcinoma, UCEC = endometrial carcinoma of the uterine body, UCS = uterine carcinosarcoma, SARC = sarcoma. [Modes for carrying out the invention]

[0023] Detailed description of the invention definition In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen. The antibodies of this invention comprise an Fc domain and an antigen-binding region of the immunoglobulin. Antibodies generally contain two CH2-CH3 regions and a linking region, such as a hinge region, e.g., at least an Fc region. Thus, antibodies of this invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region or "Fc" region of the antibody can mediate the binding of the immunoglobulin to host tissue or host factors, for example, various cells of the immune system (e.g., effector cells), or to components of the complement system, e.g., the first component C1q in the classical pathway of complement activation. The Fc region of immunoglobulin as used herein typically comprises at least a CH2 domain and a CH3 domain of the immunoglobulin CH, and may include a linking region, e.g., a hinge region, unless otherwise inconsistent with the context. The Fc region is typically in a dimerized form, such as by a disulfide bridge connecting two hinge regions and / or a non-covalent interaction between two CH3 regions. This dimer can be a homodimer (in which case the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (in which case the amino acid sequences of the two Fc region monomers differ by one or more amino acids). As is well known in the art, the Fc region fragment of a full-length antibody can be produced, for example, by digestion of a full-length antibody with papain. The antibodies defined herein may further include, in addition to the Fc region and antigen-binding region, one or both of the CH1 and CL regions of the immunoglobulin. The antibody may be a multispecific antibody, e.g., a bispecific antibody or a similar molecule. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be located on the same target or on different targets. If the epitopes are located on different targets, such targets may be located on the same cell or on different cells or cell types. As stated above, unless otherwise stated,Unless otherwise clearly stated in context, the term "antibody" as used herein encompasses fragments of an antibody that include at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, but are not limited to, the following: monovalent antibodies (described in WO2007059782 by Genmab); heavy chain antibodies, which consist of only two heavy chains and are naturally occurring in animals such as camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab (Roche, WO2011069104), an asymmetric bispecific antibody-like molecule with a strand-exchange engineered domain (SEED or Seed-body) (Merck, WO2007110205); and Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol). 155:219, WO2002020039); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); Dual domain double head antibody (Unilever, Sanofi Aventis, WO20100226923), Di-diabody (ImClone / Eli Lilly), Knobs-into-holes antibody format (Genentech,WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), Electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004, Chugai, US201000155133, Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences) (Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), Dual Targeting domain antibody (GSK / Domantis), Two-in-one antibody or dual-action Fab (Genentech, NovImmune, Adimab) that recognizes two targets, Cross-linked Mab (Karmanos Cancer Center), covalently fused mAb (AIMM), CovX-body (CovX / Pfizer), FynomAb (Covagen / Janssen) ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecific molecule (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p.65-74), TIG-body, DIG-body and PIG-body (Pharmabcine), Dual-affinity retargeting molecule (Fc-DART or Ig-DART, by Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybodies (Zyngenia),Common light chain approach (Crucell / Merus, US7262028) or common heavy chain approach (κλBody by NovImmune, WO2012023053), as well as fusion proteins containing polypeptide sequences fused to antibody fragments containing Fc regions, such as scFv fusions, e.g., BsAb by ZymoGenetics / BMS, HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009.393(3):p.672-92), scFv fusion by Genentech / Roche, scFv fusion by Novartis, scFv fusion by Immunomedics, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008 / 003116), and bivalent scFv fusions. It should be understood that the term antibody, unless otherwise specified, encompasses monoclonal antibodies (e.g., human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (e.g., bivalent monospecific antibodies), bispecific antibodies, antibodies of any isotype and / or allotype; antibody mixtures (recombinant polyclonals), e.g., those produced by techniques developed by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, and fusion proteins as described in WO2014 / 031646. While these various antibody fragments and formats are broadly encompassed in the meaning of antibody, they collectively and independently constitute unique features of the present invention, exhibiting diverse biological properties and utility.

[0024] The "ROR2 antibody" or "anti-ROR2 antibody" described herein is an antibody that specifically binds to the antigen ROR2, particularly to human ROR2.

[0025] As used herein, "variant" refers to a protein sequence or polypeptide sequence that differs from the parent sequence or reference sequence by one or more amino acid residues. A variant may have, for example, at least 80%, for example 90%, 95%, 97%, 98%, or 99% sequence identity with respect to the parent sequence or reference sequence. In addition to or instead of the above, a variant may differ from the parent sequence or reference sequence by 12 or fewer mutations, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, such as substitution, insertion, or deletion of amino acid residues. Therefore, as used interchangeably herein, "variant antibody" or "antibody variant" refers to an antibody that differs from the parent antibody or reference antibody by one or more amino acid residues, for example in the antigen-binding region, the Fc region, or both. Similarly, a “variant Fc region” or “Fc region variant” refers to an Fc region that differs from the parent or reference Fc region by one or more amino acid residues, and optionally, an Fc region that differs from the amino acid sequence of the parent or reference Fc region by 12 or fewer mutations, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, e.g., substitution, insertion, or deletion of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody, which, depending on the context, may be a specific isotype. A dimerized variant Fc region may be a homodimer or a heterodimer, e.g., one of the amino acid sequences of the dimerized Fc region contains mutations, while the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference) IgG CH amino acid sequences and variant IgG constant region amino acid sequences, including the Fc region amino acid sequence, are shown in Table 1.

[0026] As used herein, the terms “immunoglobulin heavy chain” or “immunoglobulin heavy chain” refer to one of the immunoglobulin heavy chains. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. As used herein, the term “immunoglobulin” refers to a structurally related group of glycoproteins consisting of two pairs of polypeptide chains, i.e., one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four of which are potentially linked to each other by disulfide bonds. The structures of immunoglobulins are well characterized (see, for example, Fundamental Immunology Ch.7 (Paul, W., 2nd ed. Raven Press, NY (1989))). Within the structure of immunoglobulins, two heavy chains are interconnected by disulfide bonds in a so-called "hinge region." Like the heavy chains, each light chain typically consists of several regions, such as a variable light chain region (abbreviated herein as VL) and a constant light chain region. The constant light chain region typically consists of a single domain CL. Furthermore, the VH and VL regions can be further subdivided into highly variable regions, also called complementarity-determining regions (CDRs), which are interspersed with highly conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, the CDR sequence is defined according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucleic Acids Res. 36, W503-508 (2008)).

[0027] As used herein, the terms “halves,” “Fab arm,” and “arm” refer to a single heavy-light chain pair. Where a bispecific antibody is described as containing a hapmon antibody “derived” from the first antibody and a hapmon antibody “derived” from the second antibody, the term “derived” indicates that the bispecific antibody was produced by recombining halves from the first and second antibodies, respectively, using any known technique, to result in a bispecific antibody. In this context, “recombinate” is not limited to any specific recombination method and therefore encompasses all bispecific antibody production methods described herein, including, for example, recombination by “Fab arm exchange” and “halves exchange,” also described in the art as the DuoBody® method, as well as recombination at the nucleic acid level and / or by the simultaneous expression of two halves in the same cell.

[0028] As used herein, the terms “antigen-binding region,” “binding region,” or “antigen-binding domain” refer to a region of an antibody that can bind to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which contain highly conserved regions called framework regions (FRs). These hypervariable regions (i.e., regions whose sequences can be hypervariable and / or which can form structurally distinct loops) can be hypervariable in their sequence. The antigen can be any molecule, such as a polypeptide, present on a cell, bacterium, or virion. The terms “antigen-binding region,” “antigen-binding site,” and “antigen-binding domain” may be used interchangeably in relation to the present invention unless there is a contextual inconsistency.

[0029] The terms "antigen" and "target" may be used interchangeably in relation to the present invention, unless there is a contextual inconsistency.

[0030] As used herein, the term "binding" typically refers to binding to a predetermined antigen or target, as determined by biolayer interferometry using an antibody as a ligand and the antigen as an analyte, with a 1E -6 M or less, for example 5E -7 M or less, 1E -7 M or less, for example 5E -8 M or less, for example 1E -8 M or less, for example 5E -9 M or less, or for example 1E -9 M or less K D d refers to the binding of an antibody with a binding affinity corresponding to , which binds to a predetermined antigen with an affinity corresponding to K D that is at least one tenth, for example at least one hundredth, for example at least one thousandth, for example at least one ten thousandth, for example at least one one hundred thousandth of its affinity for binding to a non-specific antigen that is neither the predetermined antigen nor a closely related antigen (e.g., BSA, casein).

[0031] As used herein, "K D d" (M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is obtained by dividing k d off by k a on.

[0032] As used herein, "k d off" (sec -1 ) refers to the dissociation rate constant for a particular antibody-antigen interaction. This value is also referred to as the k off off value or off-rate.

[0033] As used herein, "k a on" (M -1 ×sec -1 ) refers to the association rate constant for a particular antibody-antigen interaction. This value is also referred to as the k on on value or on-rate.

[0034] As used herein, the term "ROR2" refers to the protein named ROR2. Also known as receptor tyrosine kinase-like orphan receptor 2, NTRKR2, and neurotrophic tyrosine kinase receptor-associated 2, it is a single-pass type I transmembrane glycoprotein belonging to the ROR subfamily of the tyrosine protein kinase family. ROR2 is a tyrosine kinase receptor that plays a crucial role in regulating skeletal and neuronal development, cell migration, and cell polarity, partly through its role in the proposed non-canonical Wnt5a signaling pathway (Oishi 2003, Genes to cells 8:6450654). It contains an FZ (frizzled) domain, an Ig (immunoglobulin)-like C2 type domain, and a kringle domain in the extracellular region and a protein kinase domain in the cytoplasm (Masiakowski and Carroll 1992, J Biol Chem 267:26181-90). In humans (Homo sapiens), the ROR2 protein has the amino acid sequence shown in SEQ ID NO:1 (Uniprot accession number Q01974). In the amino acid sequence shown in SEQ ID NO:1, amino acid residues 1-31 are the signal peptide, and amino acid residues 32-420 are the mature polypeptide. In cynomolgus monkeys (Macaca fascicularis), the ROR2 protein has the amino acid sequence shown in SEQ ID NO:39 (Uniprot accession number A0A2K5UT30). In the amino acid sequence shown in SEQ ID NO:2, amino acid residues 1-34 are the signal peptide, and amino acid residues 35-420 are the mature polypeptide.

[0035] As used herein, the term "CD3" refers to the human cluster of differentialiation (CD)3 protein, which is part of the T cell co-receptor protein complex and consists of four distinct chains. Since CD3 is also found in other species, the term "CD3" is not limited to human CD3 unless otherwise specified in the context. In mammals, this complex consists of a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No. P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No. Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No. P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No. Q95LI8), and two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No. P07766; amino acid residues 1-22 are a signal peptide, and amino acid residues 23-207 are mature CD3ε polypeptides identified herein as SEQ ID NO:21; cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No. Q95LI5; or rhesus monkey CD3ε It contains UniProtKB / Swiss-Prot No. G7NCB9 and CD3ζ-chain (zeta) chains (human CD3ζ UniProtKB / Swiss-Prot No. P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No. Q09TK0). These chains associate with molecules known as T cell receptors (TCRs) to generate activation signals in T lymphocytes. The TCR molecule and the CD3 molecule together constitute a TCR complex.

[0036] The term "antibody-binding region" refers to the region of an antigen that contains the epitope to which the antibody binds. The antibody-binding region can be determined by epitope binning using biolayer interferometry, alanine scan, or shuffle assay (which uses an antigen construct in which the region of the antigen has been swapped with that of another species to determine whether the antibody still binds to that antigen). The amino acids within the antibody-binding region that are involved in the interaction with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and crystal structure analysis of the antibody bound to the antigen.

[0037] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes typically consist of surface arrangements of molecules, such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural and specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or coated by the antibody when the antibody binds to the antigen (in other words, amino acid residues that are within or very close to the footprint of the specific antibody).

[0038] As used herein, terms such as “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” and “mAb” refer to preparations of antibody molecules with a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting a single binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas, including B cells fused to immortalized cells, obtained from transgenic non-human animals such as transgenic mice having a genome containing human heavy chain transgenes and human light chain transgenes, or from transchromosomal non-human animals. Monoclonal antibodies can also be produced from recombinantly modified host cells or from systems using cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding antibodies.

[0039] As used herein, the term “isotype” refers to an immunoglobulin class encoded by a heavy chain constant region gene (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM), or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype can be combined with either a kappa (κ) light chain or a lambda (λ) light chain.

[0040] The term "full-length antibody," as used herein, refers to an antibody comprising one or two pairs of heavy and light chains, each containing all the constant and variable domains of the heavy and light chains typically found in the heavy-light chain pair of the wild-type antibody of that isotype. In a full-length variant antibody, the constant and variable domains of the heavy and light chains may particularly contain amino acid substitutions that improve the functional properties of the antibody compared to a full-length parent antibody or a full-length wild-type antibody. The full-length antibody of the present invention may be produced by a method comprising (i) cloning a CDR sequence into a suitable vector containing the full heavy chain sequence and the full light chain sequence, and (ii) expressing the full heavy chain sequence and the full light chain sequence in a suitable expression system. Producing a full-length antibody starting from either a CDR sequence or a complete variable region sequence is within the knowledge of those skilled in the art. Therefore, the method for producing the full-length antibody of the present invention is known to those skilled in the art.

[0041] As used herein, the term "human antibody" encompasses antibodies having a variable region and framework region derived from a human germline immunoglobulin sequence and a human immunoglobulin constant domain. The human antibodies of the present invention may include amino acid residues not encoded by the human germline immunoglobulin sequence (mutations, insertions, or deletions, for example, introduced by in vitro random mutagenesis or site-directed mutagenesis, or by in vivo somatic mutagenesis). However, as used herein, the term "human antibody" does not encompass antibodies in which a CDR sequence derived from the germline of another non-human species, such as mouse, is transplanted onto a human framework sequence.

[0042] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology to the human variable domain. This can be achieved by transplanting six non-human antibody complementarity-determining regions (CDRs), which together form an antigen-determining site, into a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and binding specificity of the parent antibody, it may be necessary to replace framework residues from the parent antibody (i.e., the non-human antibody) with human framework regions (revert mutations). Structural homology modeling can help identify amino acid residues in the framework region that are important for the antibody’s binding properties. Thus, a humanized antibody may contain a non-human CDR sequence, a primarily human framework region which may contain one or more amino acid reverse mutations to the non-human amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily revertant mutations, may be applied to obtain humanized antibodies with desirable characteristics such as affinity and biochemical properties.

[0043] As used herein, the term “Fc region” refers to the region of an antibody, extending from the N-terminus to the C-terminus, including at least the hinge region, the CH2 region, and the CH3 region. The Fc region of an antibody can mediate the binding of immunoglobulins to host tissues or host factors, such as various cells of the immune system (e.g., effector cells) and components of the complement system.

[0044] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 in the Eu numbering described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991). However, the hinge region may be any of the other subtypes described herein.

[0045] As used herein, the terms “CH1 region” or “CH1 domain” refer to the CH1 region of an immunoglobulin heavy chain. Therefore, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 in the Eu numbering system shown in Kabat (op. op. cit.). However, the CH1 region may be any of the other subtypes described herein.

[0046] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. Therefore, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 in the Eu numbering system shown in Kabat (op. cit.). However, the CH2 region may be any of the other subtypes described herein.

[0047] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. Therefore, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 in the Eu numbering system shown in Kabat (op. op. cit.). However, the CH3 region may be any of the other subtypes described herein.

[0048] As used herein, the term “Fc-mediated effector function” refers to a function resulting from the binding of a polypeptide or antibody to its target or antigen on the cell membrane, where the Fc-mediated effector function is attributed to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cell-mediated cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor (FcgR) binding, (vi) antibody-dependent FcγR-mediated antigen crosslinking, (vii) antibody-dependent cell phagocytosis (ADCP), (viii) complement-dependent cell-mediated cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) antibody-mediated binding of opsonized antibodies to complement receptors, (xi) opsonization, and (xii) any combination of (i) to (xi).

[0049] As used herein, the terms “inactive,” “inactive,” or “deactivated” refer to an Fc region that, at a minimum, cannot bind to any FcγR, cannot induce Fc-mediated crosslinking of FcγR, or cannot induce FcγR-mediated crosslinking of a target antigen by two Fc regions of an individual antibody, or cannot bind to C1q. The inactivity of an antibody’s Fc region can be tested using antibodies in single-specificity or bispecificity formats. An Fc region with an FEA mutation, as described later, is an example of an inactive Fc region. Thus, in certain embodiments of the present invention, the Fc region is inactive. Therefore, in certain embodiments, some or all of the Fc-mediated effector function is attenuated or absent.

[0050] When used in relation to antibodies, the term "full length" indicates that the antibody is not a fragment, but contains all of the domains of that particular isotype that are normally found in nature, such as the VH domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, VL domain, and CL domain in the case of an IgG1 antibody.

[0051] In relation to the present invention, the term "monovalent antibody" refers to an antibody molecule that can interact with a specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm). In relation to bispecific antibodies, "monovalent antibody binding" refers to the binding of a bispecific antibody to a single specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

[0052] In relation to the present invention, the term "monospecific antibody" refers to an antibody that has binding specificity to only one epitope. This antibody may be a monospecific monovalent antibody (i.e., possessing only one antigen-binding region) or a monospecific bivalent antibody (i.e., possessing two identical antigen-binding regions).

[0053] The term "bispecific antibody" refers to an antibody having two non-identical antigen-binding domains, for example, an antibody having two non-identical Fab arms or two Fab arms having non-identical CDR regions. In relation to the present invention, a bispecific antibody has specificity to at least two different epitopes. Such epitopes may be located on the same antigen or target, or on different antigens or targets. If the epitopes are located on different antigens, such antigens may be located on the same or different cells, cell types, or structures, for example, on the extracellular matrix or vesicles and soluble proteins. Therefore, a bispecific antibody can crosslink multiple antigens, for example, two different cells. Certain bispecific antibodies of the present invention can bind to ROR2 and CD3, which are typically not expressed on the same cells, and thus can crosslink two different cells, for example, tumor cells and T cells, each expressing one of these targets.

[0054] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions, either binding to one or two epitopes on a target or antigen, or to one or two epitopes on the same antigen. Therefore, a bivalent antibody can be either a monospecific bivalent antibody or a bispecific bivalent antibody.

[0055] The terms “amino acid” and “amino acid residue” may be used interchangeably herein and should not be understood restrictively. Amino acids are organic compounds containing an amine functional group (-NH2) and a carboxyl functional group (-COOH) along with a side chain (R group) specific to each amino acid. In relation to the present invention, amino acids can be classified based on their structure and chemical characteristics. Therefore, the classes of amino acids may be reflected in one or both of the following tables.

[0056] Main classification based on the structure and general chemical characterization of the R group TIFF0007923755000001.tif52128

[0057] Alternative physical and functional classification of amino acid residues TIFF0007923755000002.tif88128

[0058] The substitution of one amino acid with another can be classified as either a conservative or non-conservative substitution. In relation to the present invention, a "conservative substitution" means substituting one amino acid with another amino acid having similar structural and / or chemical characteristics, for example, substituting one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above. For example, since both leucine and isoleucine are aliphatic branched hydrophobic substances, leucine can be substituted with isoleucine. Similarly, since both aspartic acid and glutamic acid are small load residues, aspartic acid can be substituted with glutamic acid.

[0059] In relation to the present invention, substitutions in antibodies are described as follows: Original amino acid - position - substituted amino acid

[0060] In well-known amino acid nomenclature, three-letter or one-letter codes are used, including the symbol "Xaa" or "X" that indicates any amino acid residue. Thus, Xaa or X can refer to any one of the 20 natural amino acids. As used herein, the term "natural" refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the designation "K409R" or "Lys409Arg" means that the antibody comprises the substitution of lysine by arginine at position 409.

[0061] Substitution of an amino acid at a given position by any other amino acid is referred to as follows: original amino acid-position, or for example amino acid "K409"

[0062] In the case of a modification that may comprise two or more, but not all, original and / or substituted amino acids, the two or more amino acids can be separated by a "," or a " / ". For example, substitution of lysine by arginine, alanine, or phenylalanine at position 409 is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe", or "K409R,A,F" or "K409R / A / F", or "K409→R, A or F" respectively.

[0063] Such designations may be used interchangeably in the context of the present invention, and they can have the same meaning and the same purpose.

[0064] Furthermore, the term “substitution” encompasses substitutions to any one of the other 19 natural amino acids, or to other amino acids, such as non-natural amino acids. For example, substitutions of amino acid K at position 409 include each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is equivalent to the designation 409X, where X specifies any amino acid other than the original amino acid. These substitutions may also be designated as K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. This applies equally to any position mentioned herein, and any such substitution is specifically included herein.

[0065] The antibodies of the present invention may also include deletions of amino acid residues. Such deletions can be represented as "del," and include, for example, the notation K409del. In such embodiments, the 409th lysine is deleted from the amino acid sequence.

[0066] As used herein, the term “host cell” refers to the cell into which the expression vector has been introduced. Such a term should be understood to refer not only to the specific target cell but also to the offspring of such cells. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such offspring may not be identical to the parent cell, but they are still included within the scope of the term “host cell” as used herein. Examples of recombinant host cells include transfectomas such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphoid cells, as well as prokaryotic cells such as Escherichia coli (E. coli), and other eukaryotic hosts such as plant cells and fungi.

[0067] As used herein, the term "transfectoma" encompasses fungi, including recombinant eukaryotic host cells expressing antibodies or target antigens, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or yeast cells.

[0068] For the purposes of this invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later of the Needle program. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (BLOSUM62 in the EMBOSS version) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0069] Retention of similar residues can also be measured by a similarity score determined using the BLAST program (e.g., BLAST 2.2.8, available via NCBI, with standard settings of BLOSUM62, Open Gap=11, and Extended Gap=1). A suitable variant typically exhibits a similarity of at least approximately 45%, e.g., at least approximately 55%, at least approximately 65%, at least approximately 75%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or higher (e.g., approximately 99%) to the parent sequence.

[0070] As used herein, the terms “internalized” or “internalization” refer to the biological process in which molecules such as antibodies of the present invention are phagocytosed by cells and drawn into the interior of the cells. Internalization can also be referred to as “endocytosis.”

[0071] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, e.g., lymphocytes (including B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, e.g., neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells expressing FcRs are involved in the specific killing of target cells and / or the presentation of antigens to other components of the immune system, or binding to antigen-presenting cells. In some embodiments, ADCC may be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments in target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation generates C3 fragments on target cells. These C3 cleavage products can directly promote complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells, which may be antibody-binding-dependent and mediated by FcγRs expressed on effector cells. The expression of specific FcRs or complement receptors in effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon-gamma (IFNγ) and / or G CSFs. This enhanced expression increases the cytotoxic activity of FcγRI-carrying cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells.In some embodiments, antibody-driven classical complement activation generates C3 fragments on target cells. These C3 cleavage products can either directly promote phagocytosis by effector cells or indirectly enhance antibody-mediated phagocytosis.

[0072] "Effector T cells," or "Teff," refer to T lymphocytes that perform functions of the immune response, such as activating an anti-tumor immune response that can result in the killing of tumor cells and / or the elimination of tumor tissue from the body. Examples of Teff phenotypes include CD3+CD4+ and CD3+CD8+. Teff may secrete, contain, or express markers such as IFNγ, granzyme B, and ICOS. It is understood that Teff is not limited to these phenotypes.

[0073] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on its surface. C1 is a complex consisting of six recognition proteins C1q and a serine protease heterotetramer C1r2C1s2. C1 is the first protein complex in the initial events of the classical complement cascade, involving a series of cleavage reactions beginning with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, forming C5 convertase. This C5 convertase splits C5 into C5a and C5b, the last component of which is deposited on the membrane, which then triggers a late-stage complement activation event in which terminal complement components C5b, C6, C7, C8, and C9 assemble to form the membrane invasion complex (MAC). This complement cascade results in the formation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cell injury (CDC). Complement activation can be evaluated using the C1q efficacy, CDC kinetics, or CDC assay (described in WO2013 / 004842, WO2014 / 108198), or by the method of C3b and C4b cell deposition described in Beurskens et al., J.Immunol April 1, 2012 vol.188 no.7 3532-3541.

[0074] The term "treatment" refers to administering an effective amount of the therapeutically active antibody variant of the present invention for the purpose of reducing, improving, stopping or eradicating (curing) a symptom or disease condition.

[0075] The term "effective dose" or "therapeutic dose" refers to the amount of medication that is effective in achieving the desired therapeutic outcome, in the required dosage and duration. The therapeutic dose of an antibody can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the antibody's ability to elicit the desired response in the individual. Furthermore, the therapeutic dose is the amount in which the therapeutically beneficial effects of the antibody variant outweigh any toxic or adverse effects of the antibody variant.

[0076] Specific aspects of the invention antibody In the first aspect, the present invention provides an antibody comprising at least one antigen-binding region capable of binding to human ROR2, comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9, respectively. Thus, such an antibody may be monovalent, bivalent, or polyvalent with respect to ROR2.

[0077] In one embodiment of the present invention, the antibody comprises two antigen-binding regions capable of binding to human ROR2, wherein the antibody comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9, respectively. Such an antibody may be a typical bivalent antibody.

[0078] In one embodiment of the present invention, the ROR2 antibody is humanized from an antibody comprising a VH region having the sequence shown in SEQ ID NO:2 and / or a VL region having the sequence shown in SEQ ID NO:6, which are regions capable of binding to human ROR2. In one embodiment, the antibody is humanized from an antibody that is a chimeric antibody comprising a rabbit variable heavy chain (VH) shown in SEQ ID NO:2 and a light chain (VL) shown in SEQ ID NO:6, and a human constant region, such as an Ig kappa light chain and an IgG1 allotype G1m(f) heavy chain. An example of such a chimeric antibody is chIgG1-ROR2-A. This provides a chimeric antibody that has high binding to HeLa cells, binds to human ROR2 but not to human ROR1. Such an antibody is a good starting point for providing a humanized antibody that strongly binds to ROR2 and / or HeLa cells and other ROR2-expressing tumor cells.

[0079] Humanization of antibodies produced from non-human species is within the ability of a person skilled in the art, and humanization of the antibody of the present invention can be performed as shown in Example 5 herein. The non-human ROR2 antibody may be a rabbit antibody having specificity for human ROR2. Accordingly, the parent antibody to be humanized can have rabbit VH and VL regions, and can simultaneously have a human Fc region. To identify the heavy chain and light chain human sequences having the highest degree of homology for use as a human variable domain framework, the amino acid sequences of the V regions of the heavy and light chains can be compared against a database of human germline V and J segment sequences. In one aspect, the germline sequences used as the basis for humanization design are IGHV3-23*03, IGHJ2, IGKV1-39*01 and IGKJ4. Accordingly, the antibody of the present invention can have a CDR region derived from a rabbit antibody, and portions other than the CDR region among the VH region and the VL region are humanized. Furthermore, the constant regions of the heavy and light chains are preferably of human origin. The heavy chain constant region or Fc region of the antibody of the present invention is preferably a human Fc region of human immunoglobulin. This may be any human Fc region, but is preferably a human IgG such as IgG1, IgG2, IgG3 or IgG4. In a preferred embodiment, it is human IgG1. The light chain constant region may, in one aspect, be a human kappa light chain. In another aspect, it may be a human lambda light chain.

[0080] In aspects of the present invention, the antibody is a. The VH region shown in SEQ ID NO: 10 (HC1); b. The VH region shown in SEQ ID NO: 11 (HC2); c. The VH region shown in SEQ ID NO: 12 (HC3); d. The VH region shown in SEQ ID NO: 13 (HC4); e. The VH region shown in SEQ ID NO: 14 (HC5); f. The VH region shown in SEQ ID NO: 15 (HC6); g. The VH region shown in SEQ ID NO: 16 (HC7), or h. VH region having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 10, 11, 12, 13, 14, 15, or 16 It includes a VH region having a sequence selected from the group including the following.

[0081] In one embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:10.

[0082] In one embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:11.

[0083] In one embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:12.

[0084] In a preferred embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:13.

[0085] In one embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:14.

[0086] In one embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:15.

[0087] In another embodiment, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO:16.

[0088] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:10.

[0089] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:11.

[0090] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:12.

[0091] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:13.

[0092] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:14.

[0093] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:15.

[0094] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:16.

[0095] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:10.

[0096] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:11.

[0097] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:12.

[0098] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:13.

[0099] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:14.

[0100] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:15.

[0101] In another embodiment, the present invention relates to an antibody comprising a VH region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:16.

[0102] In a further embodiment of the present invention, the antibody is a. The VL region shown in SEQ ID NO:17(LC1); b. The VL region shown in SEQ ID NO:18(LC2); c. The VL region shown in SEQ ID NO:19(LC3); d. The VL region shown in SEQ ID NO:20(LC4); or e. VL regions having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 17, 18, 19, or 20. It includes a VL region having a sequence selected from the group including the following.

[0103] In a further embodiment, the present invention relates to an antibody comprising a VL region having the sequence shown in SEQ ID NO:17.

[0104] In another embodiment, the present invention relates to an antibody comprising a VL region having the sequence shown in SEQ ID NO:18.

[0105] In a particular embodiment, the present invention relates to an antibody comprising a VL region having the sequence shown in SEQ ID NO:19.

[0106] In a further embodiment, the present invention relates to an antibody comprising a VL region having the sequence shown in SEQ ID NO:20.

[0107] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:17.

[0108] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:18.

[0109] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:19.

[0110] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 90% sequence identity with respect to the sequence shown in SEQ ID NO:20.

[0111] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:17.

[0112] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:18.

[0113] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:19.

[0114] In another embodiment, the present invention relates to an antibody comprising a VL region having at least 95% sequence identity with respect to the sequence shown in SEQ ID NO:20.

[0115] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:10 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC1LC1.

[0116] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:10 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC1LC2.

[0117] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:10 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC1LC3.

[0118] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:10 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC1LC4.

[0119] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:11 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC2LC1.

[0120] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:11 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC2LC2.

[0121] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:11 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC2LC3.

[0122] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:11 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC2LC4.

[0123] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:12 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC3LC1.

[0124] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:12 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC3LC2.

[0125] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:12 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC3LC3.

[0126] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:12 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC3LC4.

[0127] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:13 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC4LC1.

[0128] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:13 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC4LC2.

[0129] In a preferred embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:13 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC4LC3. This provides a humanized antibody that has a binding affinity very similar to the parent antibody chIgG1-ROR2-A and does not induce an immune response when used as a treatment substance in humans, and is therefore safe for use in humans.

[0130] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:13 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC4LC4.

[0131] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:14 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC5LC1.

[0132] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:14 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC5LC2.

[0133] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:14 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC5LC3.

[0134] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:14 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC5LC4.

[0135] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:15 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC6LC1.

[0136] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:15 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC6LC2.

[0137] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:15 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC6LC3.

[0138] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:15 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named ROR2-A-HC6LC4.

[0139] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:16 and a VL region having the sequence SEQ ID NO:17. Such an antibody is named ROR2-A-HC7LC1.

[0140] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:16 and a VL region having the sequence SEQ ID NO:18. Such an antibody is named ROR2-A-HC7LC2.

[0141] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:16 and a VL region having the sequence SEQ ID NO:19. Such an antibody is named ROR2-A-HC7LC3.

[0142] In a further embodiment, the antibody of the present invention comprises a VH region having the sequence SEQ ID NO:16 and a VL region having the sequence SEQ ID NO:20. Such an antibody is named.

[0143] The antibodies of the present invention are characterized by having specificity for human ROR2 or the ability to bind to human ROR2. Therefore, ROR2 as used herein may be, in particular, human ROR2, for example, the mature polypeptide of SEQ ID NO:1. In a further embodiment, these antibodies do not bind to human ROR1.

[0144] In a further embodiment, the antibody of the present invention is characterized by having specificity for cynomolgus monkey ROR2 or the ability to bind to cynomolgus monkey ROR2, for example, having specificity or binding ability to both human ROR2 and cynomolgus monkey ROR2. In particular, cynomolgus monkey ROR2 may be the mature polypeptide with SEQ ID NO:39.

[0145] In certain embodiments, the antibody of the present invention is characterized by having specificity for both ROR2 and cynomolgus monkey ROR2, or by having the ability to bind to both human ROR2 and cynomolgus monkey ROR2. This provides an antibody that eliminates the need to use surrogate antibodies for nonclinical toxicity testing and enables nonclinical safety testing in appropriate toxicological species (e.g., cynomolgus monkeys) using the intended clinical candidate.

[0146] As described above, in certain embodiments, the ROR2-binding antibody of the present invention is a humanized antibody, and the VH and VL regions of the antibody of the present invention may be humanized such that the likelihood of eliciting an immune response in humans when used as a treatment substance is reduced.

[0147] The antibody of the present invention preferably has an Fc region based on human G immunoglobulin. In one embodiment, the antibody of the present invention has an Fc region based on human IgG1. In another embodiment of the present invention, the heavy chain constant region is human IgG1, which may contain amino acid substitutions as described below. In another embodiment, the heavy chain constant region is human IgG2 or based on human IgG2. In another embodiment, the heavy chain constant region is human IgG3 or based on human IgG3. In another embodiment, the heavy chain constant region is human IgG4 or based on human IgG4. The Fc region may optionally have amino acid modifications to alter the effector function of the antibody, or for other purposes, such as enabling the formation of the bispecific antibody of the present invention. Such modifications may be substitutions as further described below.

[0148] In one embodiment of the present invention, the constant region of the antibody light chain is a human kappa light chain. In another embodiment of the present invention, the constant region of the antibody light chain is a human lambda light chain.

[0149] In yet another embodiment of the present invention, the antibody is a full-length antibody, for example, a full-length IgG1 antibody, for example, an IgG1 antibody in a conventional immunoglobulin format having two binding arms (Fab region) and an Fc region, wherein the Fc region may be inactive as described herein.

[0150] In one embodiment, the antibody of the present invention is a monovalent antibody.

[0151] In another embodiment, the antibody of the present invention is a bivalent antibody.

[0152] In yet another embodiment, the antibody of the present invention is a monospecific antibody.

[0153] In another embodiment, the antibody of the present invention is a bispecific antibody.

[0154] As mentioned above, the antibody of the present invention can bind to human ROR2. In certain embodiments, human ROR2 is a mature protein with SEQ ID NO:1.

[0155] In another embodiment, the antibody provided herein can bind to the kringle domain of human ROR2. The kringle domain is amino acids 316-394 of human ROR2, as shown in SEQ ID NO:1. This provides an antibody that binds to the cell membrane-proximal domain of ROR2.

[0156] This specification also provides antibodies that bind to an epitope or antibody-binding region on human ROR2 involving the 322nd amino acid residue of human ROR2, where the numbering refers to its position in SEQ ID NO:1.

[0157] In one embodiment, the antibody of the present invention binds to the extracellular domain of human ROR2 with a binding affinity corresponding to a KD value of 100 nM or less, for example, 50 nM or less, 10 nM or less, 6 nM or less, or for example, 3 nM or less, for example, 1.5 nM or less. In another embodiment, the antibody binds with a binding affinity corresponding to a KD value in the range of 100 nM to 0.1 nM. In yet another embodiment, the antibody binds with a binding affinity corresponding to a KD value in the range of 100 nM to 1 nM. In yet another embodiment, the antibody binds with a binding affinity corresponding to a KD value in the range of 50 nM to 1 nM. In yet another embodiment, the antibody binds with a binding affinity corresponding to a KD value of less than about 2.5 nM or less than about 2.0 nM. In a preferred embodiment, the antibody of the present invention has a binding affinity to the extracellular domain of human ROR2 of less than about 1.5 nM, for example, about 1.1 nM.

[0158] Determining the affinity of an antibody to its target is within the capabilities of those skilled in the art, but the binding affinity of the antibody of the present invention to ROR2 can be determined in particular by biolayer interferometry, as optionally shown in Examples 2 or 6 herein.

[0159] Therefore, the binding affinity is a. A step of immobilizing the antibody onto an anti-human IgG Fc capture biosensor at a volume of 1 μg / mL over 600 seconds. b. A step to determine the association and dissociation of the his-tagged ROR2 extracellular domain (ROR2-ECD, G&P Biosciences, catalog number FCL0192) over a period of 1,500 seconds, using a 2-fold dilution series in the range of 100 nM to 1.56 nM. c. The process of relating the data to a buffer control (0 nM). This can be determined using biolayer interferometry, which includes [specific method / technique].

[0160] bispecific antibody Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody having specificity for two different epitopes, for example, consisting of two scFv linked in tandem by an extra peptide linker; and (iii) a dual variable domain antibody (DVD-Ig™) in which each light chain and each heavy chain contain two variable domains in tandem via short peptide bonds. (Wu et al. "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™")) TMExamples of diabolic compounds include, but are not limited to, (iv) chemically linked bispecific (Fab')2 fragments, (v) Tandab®, a fusion of two single-chain diabolic compounds that yield a tetravalent bispecific antibody having two binding sites for each target antigen, (vi) flexibody, a combination of scFv and diabolic compounds that yield a polyvalent molecule, (vii) a so-called "dock and lock" molecule (Dock-and-Lock®), based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments, (viii) a so-called scorpion molecule, for example, containing two scFv fused to both ends of a human Fab arm, and (ix) diabolic compounds.

[0161] In one embodiment, the bispecific antibody of the present invention is a diabody or crossbody, e.g., CrossMab. In a preferred embodiment, the bispecific antibody is obtained by controlled Fab arm exchange, also known as DuoBody® technology (as described, e.g., in WO2011 / 131746).

[0162] Examples of various classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules having a complementary CH3 domain to force heterodimerization, (ii) recombinant IgG-like dual-targeting molecules with at least two different antibody Fab fragments or portions of Fab fragments on each side of the molecule, (iii) IgG fusion molecules in which a full-length IgG antibody is fused to an extra Fab fragment or portion of a Fab fragment, (iv) Fc fusion molecules in which a single-chain Fv molecule or a stabilizing diabody is fused to a heavy chain constant domain, Fc region or portion thereof, (v) Fab fusion molecules in which different Fab fragments are fused to one or to a heavy chain constant domain, Fc region or portion thereof, and (vi) ScFv-based antibodies and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®) in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein or single molecule fused to a heavy chain constant domain, Fc region or portion thereof.

[0163] Examples of IgG-like molecules using complementary CH3 domain molecules include Triomab® (Trion Pharma / Fresenius Biotech, WO / 2002 / 020039), Knobs-into-Holes (Genentech, WO9850431), CrossMAb (Roche, WO2011117329), electrostatically-matched (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y (Genentech), DIG body and PIG body (Pharmabcine), and Strand Exchange Engineered Domain body (SEEDbody) (EMD Examples include, but are not limited to, Serono (WO2007110205), Biclonics (Merus), FcΔAdp (Regeneron, WO2010 / 015792), bispecific IgG1 and bispecific IgG2 (Pfizer / Rinat, WO11143545), Azymetric scaffold (Zymeworks / Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), bivalent bispecific antibody (Roche, WO2009 / 080254), and DuoBody® molecule (Genmab A / S, WO2011 / 131746). In a preferred embodiment, the bispecific antibody of the present invention is the DuoBody molecule.

[0164] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one antibody (Genentech), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybodies® (Zyngenia), common light chain approach (Crucell / Merus, US7,262,028), κλ bodies (κλBodies) (NovImmune), and CovX-body (CovX / Pfizer).

[0165] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig (trademark) (Abbott, US7,612,181), dual domain double head antibodies (Unilever, Sanofi Aventis, WO20100226923), IgG-like bispecificity (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), Hercules (Biogen Idec, US007951918), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc, CN 102250246), and TvAb (Roche, WO2012025525, WO2012025530).

[0166] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusion products (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART®) (MacroGenics, WO2008157379, WO2010 / 080538), and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0167] Examples of Fab-fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action Fab or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecificity (Biotecnol), and Fab-Fv (UCB-Celltech).

[0168] Examples of scFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE®) (Micromet), Tandem Diabody (Tandab®) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single Chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), Dual Targeting Nanobodies® (Ablynx), and Dual Targeting Heavy Chain Only Domain Antibodies.

[0169] In a further embodiment, the present invention provides an antibody comprising a first antigen-binding region capable of binding to the human ROR2 described above, which includes the VH regions CDR1, CDR2, and CDR3 of SEQ ID NO: 3, 4, and 5, and the VL regions CDR1, CDR2, and CDR3 of SEQ ID NO: 7, 8, and 9, respectively, and a second antigen-binding region capable of binding to a different target. In a particular embodiment, the second antigen-binding region can bind to human CD3, for example, human CD3ε (epsilon), for example, human CD3ε (epsilon) as specified in SEQ ID NO: 21. In a preferred embodiment, such an antibody of the present invention is a bispecific antibody. In another embodiment, such an antibody of the present invention is a multispecific antibody.

[0170] In a further embodiment, the second antigen-binding region that binds to CD3 includes a VH region containing CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, 24, and 25, respectively, and a VL region containing CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 27, GTN, and SEQ ID NO: 28, respectively.

[0171] In another embodiment, the CD3 binding region of the present invention comprises a humanized VH region and a VL region, which are humanized from a mouse anti-human CD3 antibody SP34 having a VH region and a VL region with SEQ ID NO: 22 and SEQ ID NO: 26, respectively. As described above, humanizing an antibody is within the capabilities of those skilled in the art. Some preferred embodiments of such humanized versions of the VH region and VL region of mouse SP34 are listed below.

[0172] Therefore, in one embodiment, the antigen-binding region that binds to CD3 includes a VH region having at least 80% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. In another embodiment, the antigen-binding region that binds to CD3 includes a VH region having at least 90% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VH region having at least 95% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VH region having at least 97% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VH region having at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:29. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VH region having the amino acid sequence of SEQ ID NO:29.

[0173] In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having at least 80% amino acid sequence identity with respect to the sequence of SEQ ID NO:30. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having at least 90% amino acid sequence identity with respect to the sequence of SEQ ID NO:30. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having at least 95% amino acid sequence identity with respect to the sequence of SEQ ID NO:30. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having at least 97% amino acid sequence identity with respect to the sequence of SEQ ID NO:30. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:30. In yet another embodiment, the antigen-binding region that binds to CD3 includes a VL region having the amino acid sequence of SEQ ID NO:30.

[0174] In a preferred embodiment, the antigen-binding region that binds to CD3 includes a heavy chain variable region (VH) containing the sequence of SEQ ID NO:29 and a light chain variable region (VL) containing the sequence of SEQ ID NO:30.

[0175] In another embodiment, the antibody of the present invention includes a second antigen-binding region having a lower human CD3ε binding affinity than an antibody having an antigen-binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30. In one embodiment, the lower affinity is at least 1 / 5. In another embodiment, the lower affinity is at least 1 / 10. In yet another embodiment, the lower affinity is at least 1 / 20. In one embodiment, the lower affinity is at least 1 / 30. In yet another embodiment, the lower affinity is at least 1 / 40. In one embodiment, the lower affinity is at least 1 / 45. In one embodiment, the lower affinity is at least 1 / 50. In one embodiment, the lower affinity is at least 1 / 54. This provides a CD3 binding region having a lower affinity for human CD3 compared to an antigen-binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30. If such a CD3-binding region is part of a CD3×ROR2 bispecific antibody, that bispecific antibody will have low affinity for CD3. This provides a bispecific antibody that is still effective in treating diseases such as cancer, but can be used safely with fewer side effects.

[0176] In another aspect, the present invention provides an antibody whose antigen-binding domain that binds to CD3 has an equilibrium dissociation constant KD in the range of 200 to 1000 nM. In one embodiment, it binds in the range of 300 to 1000 nM. In one embodiment, it binds in the range of 400 to 1000 nM. In one embodiment, it binds in the range of 500 to 1000 nM. In one embodiment, it binds in the range of 300 to 900 nM. In one embodiment, it binds in the range of 400 to 900 nM. In one embodiment, it binds in the range of 400 to 700 nM. In one embodiment, it binds in the range of 500 to 900 nM. In one embodiment, it binds in the range of 500 to 800 nM. In one embodiment, it binds in the range of 500 to 700 nM. In one embodiment, it binds in the range of 600 to 1000 nM. In one embodiment, it binds in the range of 600 to 900 nM. In one embodiment, it binds in the range of 600 to 800 nM. In another embodiment, it binds in the range of 600 to 700 nM. These binding affinities to CD3 are considered low binding affinities in this specification.

[0177] In another aspect, the present invention provides an antibody whose antigen-binding domain that binds to CD3 has an equilibrium dissociation constant KD in the range of 1 to 100 nM. In one embodiment, it binds in the range of 5 to 100 nM. In one embodiment, it binds in the range of 10 to 100 nM. In one embodiment, it binds in the range of 1 to 80 nM. In one embodiment, it binds in the range of 1 to 60 nM or in the range of 1 to 40 nM. In one embodiment, it binds in the range of 1 to 20 nM. In one embodiment, it binds in the range of 5 to 80 nM. In one embodiment, it binds in the range of 5 to 60 nM. In one embodiment, it binds in the range of 5 to 40 nM. In one embodiment, it binds in the range of 5 to 20 nM. In one embodiment, it binds in the range of 10 to 80 nM. In one embodiment, it binds in the range of 10 to 60 nM. In one embodiment, it binds in the range of 10 to 40 nM. In one embodiment, it binds in the range of 10 to 20 nM. These binding affinities to CD3 are considered high binding affinities in this specification. If such CD3 binding regions are part of a CD3 × ROR2 bispecific antibody, that bispecific antibody will have a higher affinity for CD3 compared to the low-affinity antibodies described herein. This gives a bispecific antibody that has higher cytotoxicity against ROR2-expressing cells and thus may have improved efficacy in treating diseases such as ROR2-expressing cancer.

[0178] The affinity of the antibody of the present invention for binding to CD3 can be determined by biolayer interferometry, in which the antibody is immobilized on a human IgG Fc capture biosensor, and the association and dissociation of CD3E27-GSKa (SEQ ID NO: 51) to the immobilized antibody is determined. Furthermore, the affinity of the antibody of the present invention for binding to CD3 can be determined by biolayer interferometry provided in Example 9 of this specification.

[0179] Antibodies that bind to CD3, particularly human CD3, with reduced affinity are provided in WO 2017 / 009442, and it should be understood that any of these antibodies can serve as a basis for generating the antibodies of the present invention, which have the ability to bind to CD3 with reduced affinity in addition to the ability to bind to ROR2.

[0180] In a particular embodiment, the antigen-binding region of an antibody that binds to CD3 includes a heavy chain variable (VH) region containing the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region of SEQ ID NO:29, wherein one of these CDR sequences contains an amino acid substitution at a position selected from the group consisting of positions T31, N57, H101, G105, S110, and Y114, numbered according to the sequence of SEQ ID NO:29, and includes a wild-type light chain variable (VL) region containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:27, GTN, and SEQ ID NO:28, respectively. CDR sequences as used herein are defined according to IMGT.

[0181] In one embodiment, the substitution within the CD3 binding region of the antibody is at position T31. In another embodiment, the substitution is at position . In another embodiment, the substitution is at position N57. In another embodiment, the substitution is at position H101. In another embodiment, the substitution is at position G105. In another embodiment, the substitution is at position S110. In another embodiment, the substitution is at position Y114.

[0182] In another embodiment of the antibody, the heavy chain variable regions CDR1, CDR2, and CDR3 of the antigen-binding region that binds to CD3 contain at most 1, 2, 3, 4, or 5 amino acid substitutions in total, compared to the CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO:29. In one embodiment, it has only one substitution in one of the CDR regions. In another embodiment, it has a total of two substitutions in one of the CDR regions or in two different regions. In another embodiment, it has a total of three substitutions in one or more of the CDR regions. In another embodiment, it has a total of four substitutions in one or more of the CDR regions. In another embodiment, it has a total of three substitutions in one or more of the CDR regions. In another embodiment, it has a total of five substitutions in one or more of the CDR regions.

[0183] In a further embodiment, the antigen-binding region of the CD3-binding antibody contains an amino acid substitution in the VH region of SEQ ID NO:29 selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V, where the numbering refers to the position of SEQ ID NO:29. In one embodiment, the substitution is T31M. In another embodiment, the substitution is T31P. In another embodiment, the substitution is N57E. In another embodiment, the substitution is H101G. In another embodiment, the substitution is H101N. In another embodiment, the substitution is G105P. In another embodiment, the substitution is S110A. In another embodiment, the substitution is S110G. In another embodiment, the substitution is Y114M. In another embodiment, the substitution is Y114M. In another embodiment, the substitution is Y114R. In yet another embodiment, the substitution is Y114V.

[0184] In one embodiment, the present invention provides an antibody in which the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 31, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, sequence GTN, and sequence shown in SEQ ID NO: 28, respectively. This bispecific antibody has a lower affinity for CD3, as described above, compared to an antibody that is identical except for having the VH-CDR3 region of SEQ ID NO: 25. This provides a bispecific CD3×ROR2 antibody with low affinity for CD3. Such an antibody is useful for treating diseases such as ROR2-expressing tumors and may have fewer side effects, such as mild cytokine release syndrome, compared to a version of the bispecific antibody with high affinity for CD3. This may have the advantage of allowing such a bispecific antibody of the present invention to be administered at higher concentrations in certain circumstances.

[0185] In one embodiment, the present invention provides an antibody in which an antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:32 and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:30. This provides a low-affinity CD3-binding arm for the bispecific antibody of the present invention.

[0186] In its primary embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding region capable of binding to ROR2 and a second binding region capable of binding to human CD3, The first antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. It includes, and the second antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in NO:23, 24, and 25 respectively, and light chain variable (VL) regions containing CDR1, CDR2, and CDR3 sequences with SEQ ID NO:27, GTN, and SEQ ID NO:28 respectively. including, We provide bispecific antibodies.

[0187] This provides a CD3×ROR2 bispecific antibody with high affinity for CD3. Such antibodies are useful in treating diseases such as ROR2-expressing tumors. This high-affinity version of the bispecific antibody may have the advantage of being able to be administered at low concentrations and / or infrequently. It may also be potent and therefore potentially more toxic compared to a low-affinity CD3×ROR2 bispecific antibody.

[0188] In another embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding region capable of binding to human ROR2 and a second binding region capable of binding to human CD3, The first antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. It includes, and the second antigen-binding region is A heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 31, respectively, and a light chain variable (VL) region containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, sequence GTN, and sequence shown in SEQ ID NO: 28, respectively. including, We provide bispecific antibodies.

[0189] This provides a CD3 × ROR2 bispecific antibody with lower affinity for CD3 compared to the variant with the VH CDR3 region of SEQ ID NO.25. Such antibodies are equally useful in treating diseases such as ROR2-expressing tumors, as mentioned above. Under certain circumstances, such bispecific antibodies may be more tolerable and safer to use in humans.

[0190] The present invention provides a bispecific antibody comprising a first antigen-binding region capable of binding to human ROR2 and a second antigen-binding region capable of binding to human CD3, wherein the first antigen-binding region comprises a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region comprises a VH region containing the sequence shown in SEQ ID NO:29 and a VL region containing the sequence shown in SEQ ID NO:30.

[0191] The present invention provides a bispecific antibody comprising a first antigen-binding region capable of binding to human ROR2 and a second antigen-binding region capable of binding to human CD3, wherein the first antigen-binding region comprises a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region comprises a VH region containing the sequence shown in SEQ ID NO:32 and a VL region containing the sequence shown in SEQ ID NO:30.

[0192] In one embodiment of the present invention, the antigen-binding region capable of binding to ROR2 is humanized. In one embodiment, the second antigen-binding region capable of binding to CD3 is humanized, if present.

[0193] In some embodiments, the antibody of the present invention includes an Fc region consisting of two heavy chain Fc sequences in addition to the antigen-binding region. The first Fc sequence and the second Fc sequence can each be any isotype, such as any human isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotype or a mixed isotype. Preferably, the Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or a mixed isotype, e.g., a human IgG1 isotype.

[0194] In certain embodiments, the antibody of the present invention comprises a primary heavy chain and a secondary heavy chain, for example, a primary heavy chain and a secondary heavy chain, each containing at least a hinge region, a CH2 region, and a CH3 region. Stable heterodimeric antibodies can be obtained in high yield from two homodimeric starting proteins containing very slight asymmetric mutations in the CH3 region, for example, by the so-called Fab arm exchange described in WO 2011 / 131746. Accordingly, in some embodiments of the present invention, the bispecific antibody comprises a first heavy chain constant region and a second heavy chain constant region, each of which comprises at least a hinge region, CH2 and CH3 regions, and when the amino acid positions in the constant regions are numbered according to Eu numbering, in the first heavy chain constant region, at least one of the amino acids at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain is substituted, and in the second heavy chain constant region, at least one of the amino acids at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain is substituted, and the substitutions in the first heavy chain and the substitutions in the second heavy chain are not at the same position.

[0195] In a preferred embodiment, the constant region of the heavy chain of the ROR2-binding antibody of the present invention contains the amino acid R at a position corresponding to K409 in the human IgG1 heavy chain. The heavy chain constant region is preferably IgG1, but it can also be other isotypes, such as IgG4. Thus, the ROR2 antibody preferably has arginine at position 409 of its heavy chain. In a preferred embodiment, the CD3-binding arm has leucine at position 405 of its heavy chain, when using the Eu numbering system.

[0196] Accordingly, in one embodiment, the present invention provides a bispecific antibody having the amino acid arginine (R) at position 409 in the first heavy chain constant region and the amino acid leucine (L) at position 405 in the second heavy chain constant region, where the numbering follows the Eu numbering system.

[0197] In another embodiment, the present invention provides a bispecific antibody having a first heavy chain constant region having the amino acid arginine (R) at position 409 and the amino acid phenylalanine (F) at position 405, and a second heavy chain constant region having the amino acid lysine (K) at position 409 and the amino acid leucine (L) at position 405.

[0198] Furthermore, the antibody of the present invention is preferably one that, when evaluated by flow cytometry or ELISA, does not bind to FcγR, and as a result does not induce FcγR-mediated effector function, including CD3 antibody-dependent FcγR-mediated CD3 crosslinking, in the absence of target (ROR2)-specific tumor cells. Furthermore, the antibody of the present invention is preferably one that, when evaluated by flow cytometry or ELISA, does not bind to C1q, and as a result cannot induce complement-dependent effector function. In a preferred embodiment, the antibody of the present invention does not bind to FcγR and does not bind to C1q.

[0199] In another embodiment, the present invention provides an antibody comprising a primary and secondary heavy chain that has been modified so that Fc-mediated effector function is induced by the antibody to a lower degree compared to the same unmodified antibody.

[0200] The antibodies of the present invention may include modifications to the Fc region to make the antibody an inactive or deactivating antibody. Accordingly, in the antibodies disclosed herein, one or both heavy chains may be modified to induce Fc-mediated effector function by the antibody to a lower degree compared to an antibody that is identical except in that it contains unmodified primary and secondary heavy chains. Fc-mediated effector function can be measured by determining Fc-mediated CD69 expression on T cells (i.e., CD69 expression as a result of CD3 antibody-mediated Fcγ receptor-dependent CD3 crosslinking), determining binding to the Fcγ receptor, determining binding to C1q, or determining the induction of Fc-mediated crosslinking of FcγR. In particular, the heavy chain constant sequence may be modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type (unmodified) antibody, where Fc-mediated CD69 expression is determined by a PBMC-based functional assay, for example, as described in Example 3 of WO2015001085. Modification of the heavy chain constant sequence and light chain constant sequence may also result in a reduction of C1q binding to the antibody. Compared to an unmodified antibody, the reduction may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and C1q binding can be determined by ELISA. Furthermore, the Fc region may be modified to reduce antibody-mediated Fc-mediated T cell proliferation by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to the unmodified antibody, and T cell proliferation is measured in a PBMC-based functional assay.

[0201] Examples of amino acid positions that can be modified include, for example, positions L234 and L235 in IgG1 isotype antibodies. Accordingly, in one embodiment, the present invention provides an antibody comprising a first heavy chain and a second heavy chain, wherein in both the first heavy chain constant region and the second heavy chain constant region, the amino acid residues at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to Eu numbering are F and E, respectively.

[0202] In addition, the D265A amino acid substitution can reduce binding to all Fcγ receptors and interfere with ADCC (Shields et al., 2001, J. Biol. Chem. (276): 6591-604). Therefore, in another embodiment, the antibody comprises a primary and a secondary heavy chain in which the amino acid residue at the position corresponding to position D265 in the human IgG1 heavy chain according to Eu numbering is A, in both the primary and secondary constant regions of the primary and secondary heavy chains.

[0203] In another embodiment, the antibody comprises a primary heavy chain and a secondary heavy chain, wherein in both the primary and secondary heavy chain constant regions, the amino acid residues at positions L234, L235, and D265 in the human IgG monoheavy chain according to Eu numbering are F, E, and A, respectively. This provides an antibody having an inactive Fc region.

[0204] In another embodiment, the present invention provides an antibody comprising a first heavy chain and a second heavy chain, wherein in both the first and second heavy chain constant regions, the amino acid residues at positions L234, L235, and D265 in the human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, the first heavy chain constant region further contains R at position 409, and the second heavy chain constant region further contains L at position 405. This provides an antibody that induces Fc-mediated effector function to a lower degree compared to the same unmodified antibody. The amino acids at positions 409 and 405 are useful in the process of producing bispecific antibodies using the DuoBody® method, also known as the controlled Fab arm exchange method. See Example 10. In this application, antibodies having a combination of three amino acid substitutions L234F, L235E, and D265A, and in addition having the K409R mutation or F405L mutation disclosed above, are named with the suffix "FEAR" or "FEAL," respectively.

[0205] The amino acid sequence of the wild-type IgG1 heavy chain constant region is identified herein as SEQ ID NO:33. In accordance with the embodiments disclosed above, the antibody of the present invention may comprise an IgG1 heavy chain constant region having the amino acid sequence shown in SEQ ID NO:38, which retains the F405L substitution, and / or an IgG1 heavy chain constant region having the amino acid sequence shown in SEQ ID NO:49, which retains the K409R substitution.

[0206] The amino acid sequence of the IgG1 heavy chain constant region containing the L234F, L235E, and D265A substitutions is identified herein as SEQ ID NO: 50. The amino acid sequence of the IgG1 heavy chain constant region containing the L234F, L235E, D265A, and F405L substitutions is identified herein as SEQ ID NO: 35. The amino acid sequence of the IgG1 heavy chain constant region containing the L234F, L235E, D265A, and K409R substitutions is identified herein as SEQ ID NO: 34.

[0207] Accordingly, the present invention provides an antibody comprising a first heavy chain constant region and a second heavy chain constant region having sequences shown in SEQ ID No. 34 and 35, respectively, or a first heavy chain constant region and a second heavy chain constant region having sequences shown in SEQ ID No. 35 and 34, respectively.

[0208] In another embodiment, the antibody is a bispecific antibody capable of binding to human ROR2 and human CD3 epsilon, a. The first connecting arm that connects to ROR2 is i. VH region having amino acid sequence SEQ ID NO:13 ii. VL region having amino acid sequence SEQ ID NO:19 iii. A heavy chain constant region having the amino acid sequence (FEAR) of SEQ ID NO:34, and iv. The steady-state region of the human kappa light chain Includes, b. The second binding arm that binds to CD3 epsilon, i. VH region having amino acid sequence SEQ ID NO:29 ii. VL region having amino acid sequence SEQ ID NO:30 iii. A heavy chain constant region having the amino acid sequence (FEAL) of SEQ ID NO:35, and iv. Steady-state region of human lambda light chain including, It is a bispecific antibody.

[0209] In another embodiment, the antibody is a bispecific antibody capable of binding to human ROR2 and human CD3 epsilon, a. The first connecting arm that connects to ROR2 is i. VH region having amino acid sequence SEQ ID NO:13 ii. VL region having amino acid sequence SEQ ID NO:19 iii. A heavy chain constant region having the amino acid sequence (FEAR) of SEQ ID NO:34, and iv. The steady-state region of the human kappa light chain Includes, b. The second binding arm that binds to CD3 epsilon, i. VH region having amino acid sequence SEQ ID NO:32 ii. VL region having amino acid sequence SEQ ID NO:30 iii. A heavy chain constant region having the amino acid sequence (FEAL) of SEQ ID NO:35, and iv. Steady-state region of human lambda light chain including, It is a bispecific antibody.

[0210] In one embodiment, the antibody of the present invention comprises a lambda (λ) light chain. In another embodiment, the antibody of the present invention comprises a kappa light chain. The human kappa light chain preferably has the sequence shown in SEQ ID NO:36. The human lambda light chain preferably has the sequence shown in SEQ ID NO:37.

[0211] In certain embodiments, the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain, such as an antibody having a heavy chain and a lambda light chain that include a binding region capable of binding to CD3, and a heavy chain and a kappa light chain that include a binding region capable of binding to ROR2.

[0212] The ability of a CD3 × ROR2 bispecific antibody to induce T cell activation in vitro in the presence of ROR2-expressing tumor cells such as HeLa cells is as follows: i) A step to prepare T cells isolated from healthy human donor buffy coats, ii) A step of preparing a sample set in which each sample contains the T cells and ROR2-expressing tumor cells, and the ratio of T cells to tumor cells in the sample is 8:1. iii) Adding antibody to the sample set at concentrations ranging from 0.0005 ng / mL to 10,000 ng / mL (e.g., a 5-fold dilution series), and incubating the sample at 37°C for 72 hours. iv) Collecting 150 μL of supernatant containing T cells from each sample, and incubating it with fluorescently labeled antibodies against T cell markers such as CD3, CD4, and CD8, and fluorescently labeled antibodies against T cell activation markers such as CD69, CD25, and CD279 / PD1-B at 4°C for 30 minutes to stain the T cells with the antibodies, and v) Steps to analyze the sample by flow cytometry. This can be determined by a procedure that includes [this].

[0213] The ability of CD3 × ROR2 bispecific antibodies to induce cytotoxicity in ROR2-expressing tumor cells is, i) A step to prepare T cells isolated from healthy human donor buffy coats, ii) A step of preparing a test sample set and a control sample, in which each sample contains the T cells and ROR2 tumor cells adhered to the bottom of a 96-well tissue culture plate, and the ratio of T cells to tumor cells in the sample is 8:1. iii) Add antibody to the test sample set at concentrations ranging from 0.0005 ng / mL to 10,000 ng / mL (e.g., a 5-fold dilution series), while the control sample is left untreated or incubated with 16 μg / mL phenylarsine oxide (PAO), and all samples are incubated at 37°C for 72 hours. iv) Incubating adherent cells at 37°C for 4 hours in a 10% (w / w) Alamar Blue solution in RPMI-1640 medium supplemented with 10% (w / w) iron supplement donor bovine serum with iron and penicillin / streptomycin. v) Measure the absorbance of the cells. Set the absorbance of cells incubated with PAO as 0% viability, and the viability of untreated cells as 100% viability, and calculate the percentage of viable cells. The process of calculating as TIFF0007923755000003.tif9128 This can be determined by a procedure that includes [this].

[0214] In a particular embodiment, the antibody of the present invention is a. As described in Examples 3, 7 and 12 of this specification, it can bind to ROR2-expressing human tumor cells such as HeLa, LCLC103-H, NCI-H1650, 786-O, NCI-H23, or ZR-75-1 cells. b. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of HeLa cells when assayed, for example, as described in Example 13 or 14 of this specification. c. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of 786-O, LCLC-103H, NCI-H23, NCH-H1650, or ZR-75-1 cells when assayed, for example, as described in Example 14 of this specification. d. For example, when assayed as described in Example 16 of this specification, T cells can be activated in vitro in the presence of HeLa tumor cells, and / or e. When tumor cells such as HeLa and 786-O cells are used as target cells, T cell cytokine production can be induced, for example, when assayed as described in Example 15 of this specification.

[0215] nucleic acid construct A further aspect of the present invention is, a. A nucleic acid sequence and / or encoding the heavy chain sequence of an antibody containing an antigen-binding region that can bind to the ROR2 defined above. b. A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to the ROR2 defined above. The present invention provides nucleic acid constructs containing the following:

[0216] Nucleic acid constructs are a. A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region capable of binding to the CD3 defined above, and / or b. A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to the CD3 defined above. It may further include the following.

[0217] A further aspect of the present invention is, a. A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region capable of binding to ROR2, as defined in SEQ ID NO:13. b. Nucleic acid sequence encoding the corresponding light chain sequence of an antibody containing an antigen-binding region capable of binding to ROR2, as defined by SEQ ID NO:19. The present invention provides one or more nucleic acids, including the following:

[0218] In a further aspect of the present invention, the nucleic acid is RNA or DNA.

[0219] The nucleic acids of the present invention may be nucleic acids for use in expression in mammalian cells.

[0220] Expression vector Another aspect of the present invention provides an expression vector comprising nucleic acids encoding the heavy chain sequence and / or light chain sequence of the antibody of the present invention. In particular, the expression vector is a) A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region capable of binding to the ROR2 defined above, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to the ROR2 defined above. It may include.

[0221] The expression vector is, a) A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region capable of binding to the CD3 defined above, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to the CD3 defined above. It may further include the following.

[0222] In a further embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, heavy chain, or both the light and heavy chains of an antibody, such as a human IgG1,κ monoclonal antibody.

[0223] In relation to the present invention, the expression vector can be any suitable vector, such as a chromosomal vector, a non-chromosomal vector, and a synthetic nucleic acid vector (a nucleic acid sequence containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the anti-ROR2 antibody-coding nucleic acid may be contained in, for example, a naked DNA or RNA vector containing linear expression elements (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in US6, 077, 835 and / or WO00 / 70087), a plasmid vector, e.g., pBR322, pUC19 / 18, or pUC118 / 119, or a "midge" minimal-size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaP04 precipitated construct (e.g., WO00 / 46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell This includes those described in 14,725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7,603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0224] In one embodiment, the vector is suitable for the expression of anti-ROR2 antibodies in bacterial cells. Examples of such vectors include BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison, Wisconsin).

[0225] In addition to or instead of the above, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inductive promoters such as alpha factor, alcohol oxidase, and PGH (F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516 544 (1987)).

[0226] Nucleic acid constructs and / or vectors may also include nucleic acid sequences encoding secretory / localization sequences that can direct polypeptides, such as nascent polypeptide chains, into the pericellular lumen or into the cell culture medium. Such sequences are known in the art and include secretory leader or signal peptides, organelle target sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transition sequences, chloroplast transition sequences), and membrane localization / fixation sequences (e.g., membrane permeation arrest sequences, GPI fixation sequences).

[0227] In the expression vector of the present invention, the anti-ROR2 antibody-coding nucleic acid may contain or be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid production in E. coli, antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also include inductive promoters as well as constitutive promoters such as CMV IE (as will be apparent to those skilled in the art, such terms are actually descriptors of the degree of gene expression under certain conditions).

[0228] In one embodiment, an expression vector encoding an anti-ROR2 antibody is placed in and / or delivered to a host cell or host animal by a viral vector.

[0229] Cells and host cells In a further aspect, the present invention provides cells comprising the nucleic acid construct or expression vector defined herein. It should be understood that the cells may be obtained by transfecting a host cell with the nucleic acid construct or expression vector, for example, a recombinant host cell.

[0230] The host cells may be of human origin, such as human fetal kidney (HEK) cells, or HEK / Expi cells. Alternatively, the host cells may be of rodent origin, such as Chinese hamster ovary cells, or CHO / N50 cells. Furthermore, the host cells may be of bacterial origin.

[0231] The cell may contain a nucleic acid sequence encoding the antibody or a portion thereof of the present invention, which is stably integrated into the cell's genome. Alternatively, the cell may contain a plasmid, cosmid, phagemid, or linear expression element containing an unintegrated nucleic acid, such as a sequence encoding the expression of the anti-ROR2 antibody or a portion thereof of the present invention. In particular, the host cell may contain a plasmid, cosmid, phagemid, or linear expression element containing an unintegrated nucleic acid, such as a sequence encoding the expression of the anti-ROR2 antibody or a portion thereof.

[0232] composition A further aspect of the present invention provides a composition comprising an antibody, for example, the bispecific antibody defined above. The composition may be a pharmaceutical composition comprising an antibody or bispecific antibody and a pharmaceutically acceptable carrier.

[0233] Pharmaceutical compositions may be formulated using carriers, excipients and / or diluents, and any other components suitable for the pharmaceutical composition, such as known adjuvants, in accordance with the prior art, for example, as disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, should be suitable for the antibody or antibody conjugate and the chosen mode of administration of the present invention. The suitability of carriers and other components of the pharmaceutical composition is determined on the basis that they do not have a significant negative impact on the desired biological properties of the selected compound or pharmaceutical composition of the present invention (e.g., the impact on antigen binding is substantial [e.g., relative inhibition of 10% or less, relative inhibition of 5% or less]).

[0234] The pharmaceutical compositions of the present invention may include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0235] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including factors well known in the medical field, such as the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, and the age, sex, weight, condition, overall health status, and medical history of the patient being treated.

[0236] pharmaceutically acceptable carriers include any suitable solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption retarders that are physiologically compatible with the compounds of the present invention.

[0237] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline solution, phosphate-buffered saline solution, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters (e.g., ethyl oleate), and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0238] Pharmaceutically acceptable carriers include sterile aqueous solutions or sterile aqueous dispersions, and sterile powders for in-situ preparation of sterile injection solutions or sterile injection dispersions. The use of such media and active substances for pharmaceutically active substances is known in the art. Any conventional medium or active substance can be considered for use in the pharmaceutical compositions of the present invention, provided that it is not incompatible with the active compound.

[0239] The pharmaceutical compositions of the present invention may also include pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0240] The pharmaceutical compositions of the present invention may also contain sugars, polyalcohols, isotonic agents such as mannitol, sorbitol, glycerol, or sodium chloride.

[0241] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for a selected route of administration, such as preservatives, humectants, emulsifiers, dispersants, buffers, etc., which can enhance the shelf life or potency of the pharmaceutical composition. The compounds of the present invention may be prepared using a carrier that protects the compound from rapid release, such as a controlled-release formulation including, for example, implants, transdermal patches, or microencapsulated delivery systems. Such carriers may contain gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, either alone or in combination with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0242] In one embodiment, the compounds of the present invention may be formulated to ensure proper distribution in vivo. Examples of pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or sterile aqueous dispersions, and sterile powders for the in-situ preparation of sterile injection solutions or sterile injection dispersions. The use of such media and active agents for pharmaceutically active substances is known in the art. Any conventional medium or active agent may be used in the pharmaceutical compositions of the present invention, provided that it is not incompatible with the active compound. Other active or therapeutic compounds may also be incorporated into the compositions.

[0243] Pharmaceutical compositions for injection are typically sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations. Carriers can be aqueous or non-aqueous solvents or dispersions containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in dispersion systems, and the use of surfactants. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (e.g., glycerol, mannitol, sorbitol), or sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as monostearates and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination thereof, as needed, from among the components listed above, and then performing microfiltration sterilization. Generally, dispersion systems can be prepared by incorporating the active compound into a sterilization medium containing a basic dispersion medium and other necessary components, such as those selected from those listed above. For sterilization powders for preparing sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying, which yield the powders from solutions of the active component and any other desired components that have been pre-sterilized and filtered.

[0244] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination thereof of the components listed above as needed, and then performing microfiltration sterilization. Generally, dispersion systems can be prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other necessary components selected from those listed above. For sterile powders for preparing sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying, which yield the powders from solutions of the active component and any other desired components that have been pre-sterilized and filtered.

[0245] The pharmaceutical compositions of the present invention may contain one antibody or a bispecific antibody of the present invention, a combination of the antibody and the bispecific antibody of the present invention with another therapeutic compound, or a combination of compounds of the present invention.

[0246] Pharmaceutical compositions can be administered by any suitable route and manner. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0247] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally, i.e., by means of administration other than enteral and topical administration, usually by injection, which include injections and infusions in the epidermis, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, orbital, cardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal regions. In particular, the pharmaceutical compositions of the present invention may be administered by intravenous or subcutaneous injection or infusion.

[0248] Uses and therapeutic applications The present invention further provides antibodies, such as bispecific antibodies as defined herein, for pharmaceutical use. The anti-ROR2 antibodies of the present invention can be used in the treatment or prevention of diseases or disorders involving cells that express ROR2, particularly on the surface of cells. In particular, the bispecific antibodies of the present invention, i.e., antibodies comprising antigen-binding regions capable of binding to ROR2 and CD3, can be useful in therapeutic situations where specific targeting and T-cell-mediated killing of ROR2-expressing cells are desired, and may be more efficient than conventional anti-ROR2 antibodies in certain signs and situations.

[0249] In one embodiment, antibodies, such as the bispecific antibodies of the present invention, are disclosed herein for use in the treatment of cancer. Antibodies, such as bispecific antibodies, may be used in the treatment of cancer characterized in the expression of ROR2 in at least a portion of tumor cells. In one embodiment, the antibodies of the present invention are for use in the treatment of cancer that is a solid tumor.

[0250] Cancer may be selected from a group that includes, in particular, sarcomas, fibrosarcomas, gastrointestinal stromal tumors, leiomyosarcomas, rhabdomyosarcomas, liposarcomas, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer.

[0251] In addition, the present invention relates to the use of the antibodies of the present invention for producing pharmaceuticals such as pharmaceuticals for treating cancers selected from the group including sarcomas, fibrosarcomas, gastrointestinal stromal tumors, leiomyosarcomas, rhabdomyosarcomas, liposarcomas, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer.

[0252] In a further aspect, the present invention provides a method for treating a disease, comprising the step of administering an antibody, such as a bispecific antibody, or composition, such as a pharmaceutical composition, to a subject in need.

[0253] In a particular embodiment of the present invention, the method is a method for treating cancer. The method of the present invention is particularly, a) A step of selecting subjects suffering from cancer including tumor cells expressing ROR2 and / or cancer known to express ROR2, and b) The step of administering the antibody of the present invention, for example, a bispecific antibody, or a pharmaceutical composition to the subject. It may include.

[0254] Cancer may be selected from a group that includes, in particular, sarcomas, fibrosarcomas, gastrointestinal stromal tumors, leiomyosarcomas, rhabdomyosarcomas, liposarcomas, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer.

[0255] The drug regimen in the above-described treatment and usage methods is adjusted to obtain the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, or the drug may be administered in several divided doses over time, or the dose may be increased or decreased as required by the treatment situation. Parenteral compositions may be formulated into dosing units to facilitate administration and ensure uniformity of the dosage.

[0256] The effective dosage and regimen for antibodies depend on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary and non-limiting range for the therapeutically effective dose of the compounds of the present invention is about 0.001 to 10 mg / kg, for example about 0.001 to 5 mg / kg, for example about 0.001 to 2 mg / kg, for example about 0.001 to 1 mg / kg, for example about 0.001, about 0.01, about 0.1, about 1 or about 10 mg / kg. Another exemplary and non-limiting range for the therapeutically effective dose of the antibodies of the present invention is about 0.1 to 100 mg / kg, for example about 0.1 to 50 mg / kg, for example about 0.1 to 20 mg / kg, for example about 0.1 to 10 mg / kg, for example about 0.5, for example about 0.3, about 1, about 3, about 5 or about 8 mg / kg.

[0257] A physician with ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can gradually increase the dose of the antibody used in the pharmaceutical composition, starting from a level lower than the level required to achieve the desired therapeutic effect, until the desired effect is achieved. Generally, an appropriate daily dose of the antibody of the present invention would be the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Administration can be, for example, parenteral, intravenous, intramuscular, or subcutaneous.

[0258] Antibodies may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when cancer is in remission.

[0259] The antibodies of the present invention can also be administered in combination therapy, that is, in combination with other therapeutic agents related to the disease or condition being treated. In one embodiment, the antibody-containing pharmaceutical is a pharmaceutical intended for use in combination with one or more further therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents.

[0260] Antibody production Furthermore, methods for producing antibodies such as the bispecific antibodies of the present invention are also provided herein.

[0261] a. A step of culturing host cells containing an expression vector as defined herein, and b. Process of purifying antibodies from culture medium A method for producing the antibody of the present invention, including the present invention, is provided.

[0262] In another aspect of the present invention, the antibody includes a binding region that can bind to ROR2 and a binding region that can bind to CD3, a. A step of providing an antibody capable of binding to ROR2, comprising an antigen-binding region capable of binding to ROR2 as defined above in this specification. b. A step of providing an antibody capable of binding to CD3, comprising an antigen-binding region capable of binding to CD3 as defined above in this specification. c. A step of incubating an antibody capable of binding to ROR2 together with an antibody capable of binding to CD3 under conditions of sufficient reduction to allow cysteine ​​in the hinge region to undergo isomerization of the disulfide bond, and d. Steps to obtain antibodies that can bind to ROR2 and CD3. It can be produced using methods that include

[0263] In a further embodiment, steps a) and / or b) described above are: To provide cells containing an expression vector for producing one or more types of antibodies, and To cause cells to produce one or more types of the antibody, and subsequently, To obtain one or more types of the antibody, thereby providing one or more types of the antibody. A method for producing an antibody that can bind to both ROR2 and CD3, further comprising [the specified element].

[0264] kit The present invention further provides a kit of parts comprising the antibody disclosed above, for example, a kit for use as a companion diagnostic for identifying patients in a patient population who are likely to respond to treatment with the antibody specified above herein, or for predicting the efficacy or antitumor activity of the antibody or immunoconjugate or ADC, the kit comprising the antibody specified above and instructions for use of the kit.

[0265] Anti-idiotype antibodies In a further aspect, the present invention relates to an anti-idiotype antibody that binds to an antibody comprising at least one antigen-binding region capable of binding to ROR2, i.e., to the antibody of the present invention as described herein. In a particular embodiment, the anti-idiotype antibody binds to an antigen-binding region capable of binding to ROR2.

[0266] Anti-idiotype (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of the anti-ROR2 monoclonal antibody with the monoclonal antibody from which the anti-Id antibody is to be prepared. The immunized animals can typically recognize and respond to the idiotype determinants of the immunized antibody by producing antibodies against these idiotype determinants (anti-Id antibodies). Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0267] Anti-Id antibodies can also be used as "immunogens" to induce an immune response in yet another animal that produces so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may be epitopeically identical to the original monoclonal antibody that induced the anti-Id antibody. Therefore, by using an antibody against the idiotype determinant of the monoclonal antibody, it is possible to identify other clones that express antibodies with identical specificity. Anti-Id antibodies can be modified (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, for example, by the techniques described in other sections herein with respect to the ROR2-specific antibody of the present invention. For example, a monoclonal anti-Id antibody can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will typically contain anti-anti-Id antibodies with binding properties similar, if not identical, to the original / parental anti-ROR2 antibody.

[0268] array (Table 1) TIFF0007923755000004.tif187160TIFF0007923755000005.tif240160TIFF0007923755000006.tif240160TIFF00079237550 00007.tif244160TIFF0007923755000008.tif243160TIFF0007923755000009.tif243160TIFF0007923755000010.tif255158 [Examples]

[0269] Example 1 - Generation of rabbit-human chimeric antibodies specific to human ROR2 Expression construct We generated constructs encoding various full-length ROR2 variants: human ROR2 (Uniprot accession number Q01974; SEQ.ID NO:1), cynomolgus monkey ROR2 (ROR2mf; Uniprot accession number A0A2K5UT30; SEQ.ID NO:39), and cynomolgus monkey ROR2 in which the 322nd threonine is replaced by methionine (ROR2mf-T322M;; SEQ.ID NO:41).

[0270] We also generated a construct that encodes the full-length human ROR1 (Uniprot accession number Q01973; SEQ.ID NO:40).

[0271] In addition, we also generated constructs encoding shuffled variants of the Ig-like domain, frizzled-like cysteine-rich domain (CRD), and kringle domain of ROR2 and ROR1: As further explained in Table 2, ROR112 contains the Ig-like domain and CRD of ROR1 and the kringle domain of ROR2 (;SEQ.ID NO:42). ROR121 contains the Ig-like domain of ROR1, the CRD of ROR2, and the kringle domain of ROR1 (;SEQ.ID NO:43). ROR122 contains the Ig-like domain of ROR1 and the CRD and kringle domain of ROR2 (;SEQ.ID NO:44). ROR211 contains the Ig-like domain of ROR2 and the CRD and kringle domain of ROR1 (;SEQ.ID NO:45). ROR221 contains the Ig-like domain and CRD of ROR2 and the kringle domain of ROR1 (;SEQ.ID NO:45).

[0272] (Table 2) Shuffled variants of the Ig-like domain, frizzled-like cysteine-rich domain (CRD), and kringle domain of ROR2 and ROR1 TIFF0007923755000011.tif55160

[0273] The construct contained appropriate cloning restriction sites and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999;234(2):187-208). The full-length construct and the ECD construct were cloned into a mammalian expression vector pSB containing a Sleeping Beauty inverted terminal repeat sequence adjacent to an expression cassette consisting of a CMV promoter and an HSV-TK polyA signaling molecule.

[0274] Transient expression in HEK-293F cells or CHO cells Membrane (full-length ROR2 and ROR1, SEQ.ID.No. 1, 39, 40, and 41) proteins were transiently expressed in Freestyle 293-F cells (HEK293F, HEK-293 subclone adapted for suspension growth and Freestyle medium of known composition; Invitrogen, catalog no. R790-07) using 293fectin (Invitrogen, catalog no. 12347-019) essentially as described by the manufacturer, or in Freestyle CHO-S cells (CHO) (Life Technologies, catalog no. R800-07) using Freestyle Max reagent (Life Technologies, catalog no. 16447100) essentially as described by the manufacturer.

[0275] Rabbit immunization Rabbit immunization was performed at mAbDiscovery GmbH (Neuried, Germany). Rabbits were repeatedly immunized with a mixture of HEK cells overexpressing either human ROR1 (SEQ.ID.NO.40) or human ROR2 (SEQ.ID.NO.1). Blood was collected from these animals, and B lymphocytes were isolated. Using MAB Discovery's proprietary process, single B cells were fractionated into microtiter plate wells and further proliferated. The supernatant of these single B cells was analyzed for specific binding to CHO-S cells transiently expressing human ROR2 (CHO-ROR2) or cynomolgus monkey ROR2 (CHO-mfROR2).

[0276] Recombinant Chimeric Antibody Production After analyzing the results of the primary screening, primary hits were selected for sequencing, recombinant mAb production, and purification. The regions encoding the variable heavy chain (VH) and light chain (VL) were synthesized and cloned into mammalian expression vectors containing sequences encoding the human constant region (Ig kappa chain and IgG1 allotype G1m(f) heavy chain).

[0277] Recombinant rabbit-human chimeric antibodies, containing both the rabbit variable region and the human constant region, were produced in HEK293 cells by transiently co-transfecting expression vectors encoding the heavy chain (HC) and the light chain (LC) using an automated procedure on the Tecan Freedom Evo platform. Immunoglobulins were purified from the cell supernatant using affinity purification (protein A) on a Dionex Ultimate 3000 HPLC system.

[0278] The produced chimeric monoclonal antibodies (mAbs) were re-analyzed for binding to CHO-ROR2 cells or CHO-mfROR2 cells. A total of 51 antibodies were identified that bound to both human ROR2 and cynomolgus monkey ROR2 on the CHO transfectant. These were further analyzed for binding to the human ROR2-positive cervical cancer cell line HeLa (determined by flow cytometry using the method described later). ROR2 binding affinity was determined using ROR2ECD-His (determined by biolayer interferometry using the method described later), and a panel of eight antibodies that showed binding in at least one assay was obtained. These eight antibodies are listed in Table 3 (from Example 2) and Table 4 (from Example 3) below.

[0279] Example 2 - Determination of ROR2 binding affinity of rabbit-human chimeric antibodies using biolayer interferometry. The target binding affinity of rabbit-human chimeric antibodies was determined by label-free biolayer interferometry (BLI) using an Octet HTX instrument (ForteBio). The experiment was performed at 30°C with shaking at 1,000 RPM.

[0280] An anti-human IgG Fc capture (AHC) biosensor (ForteBio, catalog number 18-5060) was preconditioned by exposing it to 10 mM glycine (Riedel-de Haen, catalog number 15527) buffer pH 1.7 for 5 seconds, followed by neutralization in sample diluent (ForteBio, catalog number 18-1048) for 5 seconds. Both steps were repeated 5 times. Next, the AHC sensor was loaded with antibody (2.5 μg / mL in sample diluent) for 600 seconds. After baseline measurement (300 seconds) with sample diluent, the association (1,000 seconds) and dissociation (1,000 seconds) of a commercially available his-tagged ROR2 extracellular domain (ROR2-ECD, G&P Biosciences, catalog number FCL0192) were determined using a concentration range of 6.25–400 nM by a 2-fold dilution step with sample diluent. The calculation used was the calculated molecular mass of ROR2 ECD, based on its amino acid sequence, which was 42.7 kDa. For each antibody, a reference sensor incubated with sample diluent instead of antigen was used. The AHC sensor was regenerated by exposure to 10 mM glycine buffer pH 1.7 for 5 seconds, followed by neutralization in sample diluent for 5 seconds. Both steps were repeated twice. Subsequently, the sensor was reloaded with antibody for the next cycle of kinetic measurement.

[0281] Data was acquired using Data Acquisition Software v8.1.0.42 (ForteBio) and analyzed using Data Analysis Software v8.1 (ForteBio). Data traces were corrected for each antibody by subtracting the mean response of the reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and interstep correction alignment for dissociation and Savitzky-Golay filtering were applied. Data were fitted using a 1:1 global full-fit model with window of interest of 1,000 seconds and 200 seconds for association and dissociation times, respectively.

[0282] Table 3 shows the association rate constants k for human ROR2-ECD for the panel of the eight rabbit-human chimeric antibodies listed above. a (1 / Ms), dissociation rate constant k d (1 / s) and equilibrium dissociation constant K D (nM) is shown.

[0283] (Table 3) Binding affinity of rabbit-human chimeric ROR2 antibody to recombinant human ROR2-ECD (G&P Biosciences) as determined by label-free biolayer interferometry. TIFF0007923755000012.tif93128

[0284] Example 3 - Binding of rabbit-human chimeric ROR2 antibody to ROR2 expressed on the cervical cancer cell line HeLa. Using the ROR2-expressing cervical adenocarcinoma cell line HeLa (ATCC, catalog number CCL-2), the binding of a rabbit-human chimeric ROR2 antibody to ROR2 expressed on human tumor cells was determined by flow cytometry. To confirm that binding to HeLa cells is dependent on ROR2 expression, a single-stranded guide RNA targeting the human ROR2 gene exclusively was used in a gene editing technology (Cellecta, USA) based on the CRISPR (clustered regularly interspaced short palindrome repeat)-related nuclease Cas9. HeLa cells in which ROR2 expression was suppressed using TIFF0007923755000013.tif4128 were used.

[0285] cells (1×10 5 Cells (per well) were incubated in 100 μL of PBS / 0.1% BSA / 0.02% azide (FACS buffer) in a 96-well polystyrene round-bottom plate (Greiner bio-one, catalog no. 650180) with serial dilutions of antibody (ranging from 0.01 to 10 μg / mL in 3- or 4-fold dilution steps) at 4°C for 30–60 minutes. The experiment was technically repeated twice. After washing twice in FACS buffer, cells were incubated in 50 μL of secondary antibody (R-phycoerythrin [PE] conjugate goat anti-human IgG F(ab')2; 1:200 dilution in FACS buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania, catalog no. 109-116-098) at 4°C for 30 minutes. Cells were washed twice in FACS buffer, resuspended in 30 μL of FACS buffer containing Topro-3 (1:10,000 dilution), and analyzed using an iQue screener (Intellicyt Corporation, USA). Binding curves were analyzed using nonlinear regression (variable slope sigmoid dose-response) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA).

[0286] Of the eight rabbit-human chimeric antibodies in the panel above, seven showed low binding to HeLa cells (maximum MFI less than 5,000), while one antibody, chIgG1-ROR2-A, showed high binding to HeLa cells (maximum MFI greater than 20,000) (Table 4). Since chIgG1-ROR2-A did not bind to HeLa cells in which ROR2 was specifically inactivated, it was shown that chIgG1-ROR2-A is ROR2 specific.

[0287] (Table 4) Binding of rabbit-human chimeric ROR2 antibody to HeLa cells TIFF0007923755000014.tif69128

[0288] chIgG1-ROR2-A showed very slight binding to the ROR1-expressing cell line Calu-1. This binding is due to a single-stranded guide RNA that targets the human ROR1 gene exclusively. The loss of ROR1 expression using TIFF0007923755000015.tif4128 did not have any effect. Since the binding of chIgG1-ROR2-A was reduced by the loss of ROR2 expression in all cases, it was shown that low ROR2 expression in the Calu-1 cell line is the cause of residual binding of chIgG1-ROR2-A to the Calu-1 cell line.

[0289] In conclusion, chIgG1-ROR2-A was the only antibody in the chimeric ROR2-specific antibody panel that showed high binding to ROR2-positive tumor cells. The binding was demonstrated to be ROR2-specific.

[0290] Example 4 - Binding of chIgG1-ROR2-A to CHO cells expressing ROR1 / 2 shuffle protein To investigate the ROR2 domain involved in the binding of ROR2-specific antibody A, we explored the binding of chIgG1-ROR2-A to CHO cells transfected to transiently express shuffled variants of the Ig-like domains, CRD, and kringle domains of ROR2 and ROR1. ROR112 contains the Ig-like domain and CRD of ROR1 and the kringle domain of ROR2. ROR121 contains the Ig-like domain, the CRD of ROR2, and the kringle domain of ROR1. ROR122 contains the Ig-like domain of ROR1 and the CRD and kringle domain of ROR2. ROR211 contains the Ig-like domain of ROR2 and the CRD and kringle domain of ROR1. ROR221 contains the Ig-like domain and CRD of ROR2 and the kringle domain of ROR1.

[0291] Binding was determined by flow cytometry using a cell imaging screening system (CellInsight, Thermo Fisher) according to the manufacturer's recommendations. Briefly, CHO cells expressing shuffle constructs ROR112, ROR121, ROR122, ROR211, or ROR221 (3,000 cells / well in a 384-well plate) were incubated with antibody or control sample at 37°C / 5%CO2 for 18 hours, washed, and incubated with Alexa488-labeled detection antibody for 4 hours. Hoechst dye was added, fluorescence images were collected, and total spot intensity (RFU) was measured. As shown in Table 5, chIgG1-ROR2-A bound to cells expressing ROR112 and ROR122, but not to cells expressing ROR121, ROR211, or ROR221. This indicates that the kringle domain of ROR2 is involved in the binding of chIgG1-ROR2-A.

[0292] (Table 5) Binding of chIgG1-ROR2-A to CHO cells expressing ROR1 / 2 shuffle protein. Present: RFU above 12,000; Absent: RFU = 0. TIFF0007923755000016.tif17135

[0293] Example 5 - Humanization of rabbit chimeric antibodies Generation of humanized antibody sequences The humanized antibody sequence derived from the antibody chIgG1-ROR2-A was generated at Abzena (Cambridge, UK). The humanized antibody sequence was generated using germline humanization (CDR transplantation) technology. The humanized V region genes were designed based on human germline sequences with the closest homology to the VH and Vκ amino acid sequences of the rabbit antibody. A series of seven VH and four Vκ(VL) germline humanized V region genes were designed and named according to Table 6 below.

[0294] TIFF0007923755000017.tif93160

[0295] To identify amino acids in the V-region framework that may be important for antibody binding properties, we created and analyzed structural models of rabbit antibody V-regions using Swiss PDB. We focused on these amino acids for incorporation into one or more variant CDR-transplanted antibodies.

[0296] To identify the human heavy and light chain sequences with the highest degree of homology for use as a human variable domain framework, the V region amino acid sequences of the heavy and light chains were compared with databases of human germline V and J segment sequences. Table 7 shows the germline sequences used as the basis for humanization design.

[0297] (Table 7) Sequences of the nearest human germline V and J segments TIFF0007923755000018.tif40133

[0298] Next, the CDR was transplanted onto the framework, and a series of humanized heavy and light chain V regions were designed by, if necessary, reverting residues identified in structural modeling that could have significant implications for antibody binding properties to rabbit residues. Then, the variant sequence with the lowest occurrence rate of potential T cell epitopes was selected using Abzena's proprietary in silico technology iTope® and TCED® (T Cell Epitope Database) (Perry, LCA, Jones, TD and Baker, MP New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9(6):385-396, Bryson, CJ, Jones, TD and Baker, MP Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24(1):1-8). Finally, the nucleotide sequences of the designed variants were codon-optimized.

[0299] Table 1 shows the variable region sequence of the humanized ROR2 antibody.

[0300] The sequences of the variable regions of the obtained heavy and light chains were synthesized, and each possible combination of heavy and light chains was cloned into an expression vector containing a human IgG1 heavy chain and an expression vector containing a human kappa or lambda light chain, each containing the following amino acid mutations, collectively referred to as FEAR: L234F, L235E, D265A (FEA mutation, for silencing of FcγR and C1q binding; Engelberts et al, 2020, EBioMedicine 52:102625) and K409R(R), with amino acid position numbers according to Eu numbering (corresponding to SEQ ID NO. 34).

[0301] Example 6 - Determination of ROR2 binding affinity of humanized variant of chIgG1-ROR2-A using biolayer interferometry. To determine the affinity of the humanized variant of chIgG1-ROR2-A for human ROR2 compared to the rabbit-human chimeric version, a BLI instrument similar to that described in Example 2 was used. The following modifications were made: AHC sensor preconditioning was repeated twice, the antibody concentration was set to 1 μg / mL, the association measurement was 1500 seconds, the dissociation measurement was 1500 seconds, and the analyte (ROR2-ECD) used was in the concentration range of 1.56 to 100 nM. Data traces were corrected for each antibody by subtracting the reference sensor. Data analysis was performed using data analysis software v9.0.0.12 (ForteBio) with a 1:1 model and global full fit, using an association time of 1500 seconds and a dissociation time of 200 seconds.

[0302] Table 8 shows the association rate constants k for human ROR2-ECD for the rabbit-human chimeric antibody chIgG1-ROR2-A (with the Fc mutation FEAR) and its humanized variant. a (1 / Ms), dissociation rate constant k d (1 / s) and equilibrium dissociation constant K D (M) indicates.

[0303] (Table 8) Binding affinity of the rabbit-human chimeric antibody chIgG1-ROR2-A and its humanized variant to recombinant human ROR2-ECD (G&P Biosciences), as determined by label-free biolayer interferometry. TIFF0007923755000019.tif31128TIFF0007923755000020.tif243122TIFF0007923755000021.tif81128

[0304] These data show that the variant IgG1-ROR2-HC4LC3 has a binding affinity that is virtually comparable to that of the parent antibody chIgG1-ROR2-A.

[0305] Example 7 - Binding of the humanized variant of chIgG1-ROR2-A to ROR2 expressed on the cervical cancer cell line HeLa. Using the HeLa cervical adenocarcinoma cell line expressing ROR2, the binding of humanized variants of chIgG1-ROR2-A to ROR2 expressed on human tumor cells was determined by flow cytometry. Figure 1 shows that the rabbit-human chimeric antibody chIgG1-ROR2-A-FEAR and all humanized variants of this antibody showed dose-dependent binding to HeLa cells.

[0306] Example 8 - Humanized CD3 antibody for generating CD3×ROR2 bispecific antibody The generation of the humanized antibody IgG1-huCD3-H1L1 (the sequences of its variable heavy and variable light chain regions are shown herein as SEQ ID NO: 29 and 30) is described in Example 1 of WO2015 / 001085. IgG1-huCD3-H1L1 is referred to herein as "IgG1-huCD3". The antibody IgG1-huCD3-H1L1-FEAL is a variant of it that has three amino acid substitutions in the Fc region (L234F, L235E, D265A; FEA), in addition to the amino acid substitution (F405L) that enables the generation of a bispecific antibody by controlled Fab arm exchange, as described below. Such mutations have been shown to have no effect on the target binding of the antibody into which they are introduced (see, e.g., WO 2014 / 108483 and Engelberts et al., 2020, EBioMedicine 52:102625). Fc regions containing FEA mutations are inactive Fc regions, meaning they cannot induce Fc-mediated antibody effector function through binding of FcγR or C1q.

[0307] The production of the humanized antibody IgG1-huCD3-H1L1-H101G (the sequences of its variable heavy chain region and variable light chain region are shown herein in SEQ ID NO: 32 and 30) is described in Example 2 of WO2017 / 009442. IgG1-huCD3-H1L1-H101G will be referred to as "IgG1-huCD3-H101G". This variant contains the substitution H101G (IMGT numbering) in the variable heavy chain region sequence (compare SEQ ID NO. 29 and 32) and has the same light chain as IgG1-huCD3-H1L1. The antibody IgG1-huCD3-H101G-FEAL is a variant of this antibody that has constant region amino acid substitutions L234F, L235E, D265A (FEA) and F405L (Eu numbering).

[0308] Example 9 - Determination of CD3 binding affinity using biolayer interferometry The binding affinity of IgG1-huCD3-FEAL and IgG1-huCD3-H101G-FEAL was determined as described in Example 7 of WO2017 / 009442.

[0309] In short, the binding affinity of selected CD3 antibodies in the IgG1-huCD3-FEAL format to recombinant soluble CD3ε (CD3E27-GSKa) (mature protein, SEQ ID NO: 21) was determined using biolayer interferometry with the ForteBio Octet HTX (ForteBio). hIgG (1 μg / mL) was loaded onto an anti-human Fc capture biosensor (ForteBio, catalog no. 18-5060) for 600 seconds. After baseline measurement (200 seconds), the association (1000 seconds) and dissociation (2000 seconds) of CD3E27-GSKa were determined using a 3-fold dilution step (sample diluent, ForteBio, catalog no. 18-5028) with a CD3E27-GSKa concentration range of 27.11 μg / mL to 0.04 μg / mL (1000 nM to 1.4 nM). The theoretical molecular mass of CD3E27-GSKa, based on its amino acid sequence, i.e., 27.11 kDa, was used for the calculations. Experiments were performed at 30°C with shaking at 1000 rpm. Each antibody was tested in at least two independent experiments. Data were analyzed using ForteBio data analysis software v8.1 with a 1:1 model and global full fit, with an association time of 1000 seconds and a dissociation time of 100 seconds. Data traces were corrected by subtracting the reference curve (measurements with antibody on biosensor and sample diluent only), the Y-axis was aligned to the last 10 seconds of the baseline, and inter-process corrections and Savitzky-Golay filtering were applied. Data traces with a response <0.05 nm were excluded from the analysis.

[0310] Table 9 shows the association rate constant k for recombinant CD3ε, determined by biolayer interferometry. a (1 / Ms), dissociation rate constant k d (1 / s) and equilibrium dissociation constant K D (M) is shown. IgG1-huCD3-FEAL is recombinant CD3ε, and IgG1-huCD3-H101G-FEAL(K D Relatively high binding affinity (K) compared to 683nM D It showed a mass of 15 nM.

[0311] (Table 9) Binding affinity of monospecific bivalent CD3 antibody to recombinant CD3ε as determined by label-free biolayer interferometry. TIFF0007923755000022.tif41128

[0312] Example 10 - Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific antibodies were generated in vitro using the DuoBody® platform technology, namely WO2011131746 and WO2013060867 (Genmab), and the 2-MEA-induced Fab arm exchange described by Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50, Gramer et al., MAbs 2013, 5:962-973). To enable the production of bispecific antibodies by this method, specific point mutations were preserved in the CH3 domain; that is, one parental IgG1 antibody preserved the F405L mutation (i.e., the CD3 antibody in this application), and the other parental IgG1 antibody preserved the K409R mutation (i.e., the humanized IgG1-ROR2 or control HIV-1 gp120-specific antibody in this application), thereby generating IgG1 molecules. In addition to these mutations, the parental IgG1 antibodies contained substitutions L234F, L235E, and D265A(FEA).

[0313] To generate bispecific antibodies, two parent antibodies are mixed in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4). 2- , 1.8 mM H2PO4 - , 163.9 mM Na + and 140.3 mM Cl - Equal masses were mixed in pH 7.4. 2-mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM, and the reaction mixture was incubated at 31°C for 5 hours. To allow for reoxidation of the interchain disulfide bonds and formation of intact bispecific antibodies, 2-MEA was removed by dialysis to PBS buffer using a Slide-A-Lyzer carriage (Thermo Fisher Scientific) with a cutoff molecular weight of 10 kDa, according to the manufacturer's protocol.

[0314] In the following examples, the following ROR2 antibodies based on the rabbit-chimeric antibody chIgG1-ROR2-A or the humanized variant IgG1-ROR2-A-HC4LC3 were used as parent antibodies to generate bispecific antibodies.

[0315] ROR2 antibody chIgG1-ROR2-A-FEAR (having VH and VL sequences as shown in SEQ ID NO:2 and SEQ ID NO:6).

[0316] IgG1-ROR2-A-HC4LC3-FEAR (having VH and VL sequences as shown in SEQ ID NO:13 and SEQ ID NO:19).

[0317] The annotation "IgG1" indicates that a full-length antibody of the IgG1 isotype was produced, and the annotation "FEAR" indicates that the heavy chain constant region contains amino acid substitutions L234F, L235E, D265A, and F409R (SEQ ID NO. 34). The light chain constant region was kappa-type (SEQ ID NO. 36).

[0318] CD3 antibody In the following examples, the following CD3 antibodies were used as parent antibodies to generate bispecific antibodies. IgG1-huCD3-FEAL (having VH and VL sequences as shown in SEQ ID NO:29 and SEQ ID NO:30). IgG1-huCD3-H101G-FEAL (having the VH and VL sequences shown in SEQ ID NO:32 and SEQ ID NO:30).

[0319] The annotation "IgG1" indicates that a full-length antibody of the IgG1 isotype was produced, and the annotation "FEAL" indicates that the heavy chain constant region contains amino acid substitutions L234F, L235E, D265A, and F405L (SEQ ID NO. 35). The light chain constant region was in the lambda form (SEQ ID NO. 37).

[0320] bispecific antibody By combining the aforementioned CD3 antibody and ROR2 antibody, a bispecific antibody having one antigen-binding domain capable of binding to CD3 and one antigen-binding domain capable of binding to human ROR2 was generated, thereby obtaining a bispecific antibody for isotype IgG1 annotated as bsIgG1. bsIgG1-huCD3-FEAL×chROR2-A-FEAR (having a ROR2-binding arm based on a rabbit-human chimera) bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR (having a humanized ROR2 binding arm) bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR

[0321] In addition, it is a bispecific control antibody having one antigen-binding region that can bind to human CD3 and one antigen-binding region that can bind to HIV gp120 (derived from antibody b12; Barbas, CF et al., 1993. J Mol Biol. 230(3): p.812-23). ​​Since the HIV gp120 protein is not present in any of the assays described herein, the Fab arm that binds to the HIV gp120-specific antigen-binding region is considered a non-binding control arm. bsIgG1-huCD3-FEAL×b12-FEAR (The b12 arm has VH and VL sequences as shown in SEQ ID NO:47 and SEQ ID NO:48) bsIgG1-huCD3-H1010G-FEAL×b12-FEAR

[0322] Example 11 - Conjugation of ROR2 monospecific antibody A and CD3×ROR2 bispecific antibody to CHO cells expressing human or cynomolgus monkey ROR2 or a variant of cynomolgus monkey ROR2 with the T322M mutation. First, the binding of a bispecific CD3×ROR2 antibody, which has either huCD3 or huCD3-H101G as its CD3-binding arm, and a monospecific ROR2 antibody to CHO cells expressing human ROR2 (but not human CD3) was essentially performed as described above, in 3×10⁻⁶ units. 4 The binding range was determined by flow cytometry using transfected cells / well and antibody concentrations ranging from 0.00013 to 10 μg / mL. chIgG1-ROR2-A-FEAR, bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR, bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR all showed similar binding ranges to human ROR2-expressing CHO cells.

[0323] Next, the binding of bispecific CD3×ROR2 antibodies and monospecific ROR2 antibodies to human or cynomolgus monkey ROR2-expressing CHO cells was performed in 5×10⁻⁶ units. 4The determination was made using transfected cells / well and an antibody concentration range of 0.01–10 μg / mL. Figure 2 shows that bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR, chIgG1-ROR2-A-FEAR, and IgG1-ROR2-A-HC4LC3-FEAR all bound to human ROR2 expressed in CHO cells. chIgG1-ROR2-A-FEAR and IgG1-ROR2-A-HC4LC3-FEAR bound to cynomolgus monkey ROR2 expressed on CHO cells, but the binding of the bispecific bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR was reduced. In conclusion, monoclonal bivalent ROR2 antibodies efficiently bound to human and cynomolgus monkey ROR2, while bispecific antibodies containing a single ROR2-specific binding domain showed reduced binding to cynomolgus monkey ROR2, but not to human ROR2.

[0324] As shown above, the binding domain of chIgG1-ROR2-A requires a kringle domain. The kringle domain sequences of human ROR2 and cynomolgus monkey ROR2 differ by one amino acid at position 322, which is T322 in cynomolgus monkeys and M322 in human ROR2. The binding of chIgG1-ROR2-A-FEAR and bsIgG1-huCD3-FEAL×chROR2-A-FEAR to CHO cells expressing human ROR2 (SEQ ID NO:1), cynomolgus monkey ROR2 (ROR2mf, SEQ ID NO:39), or RORmf-T322M (SEQ ID NO:41) was determined by flow cytometry. Figure 3 shows that both chIgG1-ROR2-A-FEAR and bsIgG1-huCD3-FEAL×chROR2-A-FEAR bound to human ROR2, while the binding of bsIgG1-huCD3-FEAL×chROR2-A-FEAR to ROR2mf was reduced compared to the binding of chIgG1-ROR2-A-FEAR. The binding of bsIgG1-huCD3-FEAL×chROR2-A-FEAR recovered to the same extent as the binding of chIgG1-ROR2-A-FEAR to CHO cells expressing ROR2mf-T322M. This indicates that residue 322 of the mature human ROR2 protein is involved in the binding of both chIgG1-ROR2-A-FEAR and bsIgG1-huCD3-FEAL×chROR2-A-FEAR.

[0325] Further experiments revealed that bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR, bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR all exhibit equivalent binding to RORmf-T322M (Figure 4).

[0326] Thus, according to the binding analysis studies described above, the results obtained using the chimeric variant of the antibody ROR2-A (chIgG1-ROR2-A or chIgG1-ROR2-A-FEAR) or the bispecific antibodies derived from the chimeric variant (bsIgG1-huCD3-FEAL×chROR2-A-FEAR or bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR) also apply to the humanized variant of this antibody (IgG1-ROR2-A-HC4LC3-FEAR) or the bispecific antibodies derived from the humanized variant (bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR or bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR). Therefore, amino acid residue M322 in the kringle domain of the mature human ROR2 protein (SEQ ID NO:1) is involved in the binding of these ROR2-binding antibodies.

[0327] Example 12 - Binding of bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR to ROR2-expressing human tumor cell lines The binding of bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR to ROR2-expressing human tumor cell lines HeLa, LCLC103-H (large cell lung cancer; DSMZ, catalog number ACC-384), NCI-H1650 (lung adenocarcinoma; ATCC, catalog number CRL-5883), 786-O (renal cell adenocarcinoma; ATCC, catalog number CRL-1932), NCI-H23 (lung adenocarcinoma; ATCC, catalog number CRL-5800), and ZR-75-1 (ductal carcinoma; ATCC, catalog number CRL-1500) was determined in vitro. ROR2 expression levels were determined according to the manufacturer's instructions by quantitative flow cytometry (Human IgG calibrator, BioCytex) using bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR to detect ROR2. Binding was 3 × 10⁻⁶. 4 Tumor cells were analyzed using the flow cytometry method described above, with tumor cells per well and antibody concentrations ranging from 0.014 to 30 μg / mL. bsIgG1-huCD3-H1010G-FEAL×b12-FEAR, which can bind to CD3 but not to ROR2, was used as a negative control.

[0328] Figure 5 shows that bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR binds to tumor cell lines in a dose-dependent manner, and the highest maximum binding determined by MFI (Figure 5A) corresponds to the highest target expression determined by semi-quantitative flow cytometry (Figure 5B).

[0329] Example 13 - Different Effectors: In vitro induction of T cell-mediated cytotoxicity by CD3 × ROR2 bispecific antibody in co-culture of ROR2-positive tumor cells (HeLa) and healthy human donor T cells at a target ratio. To determine the efficiency of T cell-mediated tumor cell killing in the presence of the bispecific CD3×ROR2 antibodies bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, in vitro cytotoxicity assays were performed using ROR2-positive HeLa cells as target cells (T) and purified T cells as effector cells (E) at various effector-to-target cell (E:T) ratios.

[0330] T cells were obtained from healthy human donor buffy coats (Sanquin, Amsterdam, Netherlands) and purified by using RosetteSep® human T cell enrichment cocktail (Stemcell Technologies, France, catalog no. 15061) according to the manufacturer's instructions. HeLa cells (16,000 cells / well) were seeded into flat-bottom 96-well plates (Greiner-bio-one, Netherlands, catalog no. 655180) and allowed to adhere at 37°C for 4 hours. T cells were added to tumor cells in E:T ratios of 1:1, 2:1, 4:1, 8:1, 12:1, or 16:1. Serial dilutions of bsIgG1-huCD3-FEAL×chROR2-A-FEAR, bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, or bsIgG1-huCD3-FEAL×b12-FEAR were added (final concentrations ranging from 10,000 to 0.0005 ng / mL; 5-fold dilution), and the plates were incubated at 37°C for 72 hours. The plates were then washed three times with PBS, and adherent cells were incubated with 150 μl / well of 10% alamarBlue® solution (Invitrogen, catalog no. DAL1100) at 37°C for 4 hours to determine tumor cell viability. As a positive control for cytotoxicity, cells were incubated with 16 μg / mL of phenylarsine oxide (PAO; Sigma-Aldrich, catalog no. P3075; dissolved in dimethyl sulfoxide [DMSO; Sigma-Aldrich, catalog no. D2438]). AlamarBlue fluorescence, as a measure of metabolic activity in tumor cell cultures and therefore of tumor cell viability, was measured at 615 nm (OD615) using an EnVision plate reader (PerkinElmer). The absorbance of PAO-treated tumor cell samples was set to 0% viability, and the absorbance of untreated tumor cell samples was set to 100% viability. The "viability rate" was calculated as follows: Surviving cells % = ([Absorbance of sample - Absorbance of PAO-treated target cells] / [Absorbance of untreated target cells - Absorbance of PAO-treated target cells]) × 100%

[0331] Dose-response curves and IC50 values ​​were generated using nonlinear regression analysis with GraphPad Prism V7.02 software (GraphPad Software, San Diego, California, USA) (variable slope sigmoid dose-response).

[0332] Figure 6 shows that dose-dependent T cell-mediated cytotoxicity was observed at all E:T ratios, and maximum tumor cell killing (less than 10% surviving tumor cells) was observed at E:T ratios greater than 2:1. Maximum cytotoxic activity (<10% surviving tumor cells) was achieved with both bsAb variants, but with bsIgG1-huCD3-FEAL×chROR2-A-FEAR at lower concentrations compared to bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR. The bispecific control antibody bsIgG1-huCD3-FEAL×b12-FEAR, which binds to CD3 but not ROR2, did not induce T cell-mediated cytotoxicity. Furthermore, when a ROR2-negative cell line (HT-29; colorectal adenocarcinoma; ATCC, catalog number HTB-38) was used as the target cell line, no T cell-mediated cytotoxicity was observed (data omitted). These data demonstrate that bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR can induce T cell-mediated cytotoxicity of ROR2-expressing HeLa cells. bsIgG1-huCD3-FEAL×chROR2-A-FEAR showed efficacy at lower concentrations than bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, but both reached the same maximum cytotoxic effect at a given E:T ratio.

[0333] Example 14 - In vitro induction of cytotoxicity in various ROR2-positive tumor cell lines using a CD3 × ROR2 bispecific antibody in the presence of healthy human donor T cells. The bispecific antibodies bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR were used to determine T cell-mediated killing of various ROR2-expressing tumor cell lines in the in vitro cytotoxicity assay described above, using an 8:1 E:T ratio. The following cell lines were used: HeLa, LCLC103-H, NCI-H1650, 786-O, NCI-H23, and ZR-75-1 (see above for further information on tumor cell lines).

[0334] Figure 7 shows that both bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR induced dose-dependent T cell-mediated cytotoxicity in HeLa, LCLC103-H, NCI-H1650, 786-O, NCI-H23, and ZR-75-1 cells in vitro. Since tumor cell killing occurred at lower concentrations with bsIgG1-huCD3-FEAL×chROR2-A-FEAR compared to bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR (Table 10), bsIgG1-huCD3-FEAL×chROR2-A-FEAR was shown to be more potent in tumor cell killing than bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR. Maximum tumor cell killing was comparable between bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR. When studied using T cells from different donors, no correlation was observed between the degree of T cell-mediated cytotoxicity and ROR2 expression levels in the above panel of cell lines (Figure 8).

[0335] (Table 10) In vitro induction of cytotoxicity in various tumor cell lines by CD3×ROR2 bispecific antibody in the presence of healthy human donor T cells: IC50 value is the geometric mean of IC50 of evaluable dose-response curves (indicating the number of donors). TIFF0007923755000023.tif162166

[0336] Example 15 - In vitro induction of cytokine production by CD3 × ROR2 bispecific antibody in the presence of ROR2-positive tumor cells. This experiment was conducted to demonstrate that the CD3×ROR2 bispecific antibody of the present invention activates T cells and induces cytokine production in the presence of ROR2-expressing target cells.

[0337] As described above, 150 μL of supernatant was transferred from wells incubated with HeLa and 786-O cells for T cell-mediated cytotoxicity mediated by the CD3×ROR2 bispecific molecule to a U-bottom 96-well culture plate (CellStar, catalog number 650180) to determine cytokine levels. After centrifuging the plate at 4°C for 3 minutes (300×g) to remove cells, 75 μL of supernatant was transferred to a new plate for cytokine production measurement using Mesoscale Discovery U-plex multiplex ELISA (MeSo Scale Discovery, USA, catalog number K15049K).

[0338] Of the 10 cytokines analyzed, significant increases were observed primarily in IFN-gamma, IL-6, IL-8, and IL-10 (>100 pg / ml). Levels of IL-4, IL-13, IL-1 beta, IL-2, IL-12p70, and TNF-alpha were generally below 100 pg / ml. Figure 9A shows the levels of IL-6 in the supernatant of T cell-tumor cell co-cultures when the concentration of the antibody bsIgG1-huCD3-FEAL×chROR2-A-FEAR or bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR was increased using T cells from two donors and 786-O cells as tumor cells. Figure 9B shows the levels of IFN-gamma, IL-6, IL-8, and IL-10 at antibody concentrations (IC50 and IC90) that induce T cell-mediated cytotoxicity in 50% and 90% of tumor cells, using HeLa cells or 786-O cells as tumor cells. Cytokine production levels differed between donors and target tumor cell lines. For bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR, cytokine levels were similar at concentrations associated with 50% or 90% cytotoxicity. This data indicates that both bsIgG1-huCD3-FEAL×chROR2-A-FEAR and bsIgG1-huCD3-H101G-FEAL×chROR2-A-FEAR induce cytokine production in vitro in the presence of ROR2-positive tumor cells.

[0339] Example 16 - Ability of CD3 × ROR2 bispecific antibody to induce cytotoxic activity and activation of cynomolgus monkey T cells in vitro in the presence of ROR2-positive tumor cells (HeLa cells). To determine the efficiency of tumor cell killing by peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys in the presence of the bispecific CD3×ROR2 antibodies, bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR, an in vitro cytotoxicity assay was performed using HeLa cells as target cells and an 8:1 PBMC:target cell ratio, essentially as described above. Cynomolgus monkey PBMCs were obtained from Zen-Bio (USA). Flow cytometry analysis of PMBCs showed that approximately 65% ​​of the cells were CD3+(T) cells. This experiment was designed to confirm that the CD3×ROR2 bispecific antibody can activate and engage cynomolgus monkey T cells as effector cells, and therefore cynomolgus monkeys can be considered a suitable species for evaluating the (non-clinical) safety of the bispecific antibody of the present invention.

[0340] To measure T cell activation, 150 μL of supernatant was transferred to a 96-well plate after 72 hours of incubation and centrifuged. Cells were then treated with T cell markers CD3 (1:100; Miltenyi Biotech, clone 10D12, APC conjugate; catalog number 130-091-998), CD4 (1:50; eBioscience, clone OKT4, APC-Cy7 conjugate; catalog number 47-0048-42), CD8 (1:100; Biolegend, clone RPA-T8, AF700 conjugate; catalog number 301028) and T cell activation marker CD69 (1:50; BD Cells were stained with Bioscience clone FN50, FITC conjugate (catalog no. 555530), CD25 (1:100; eBioscience clone BC96, PE-Cy7 conjugate: catalog no. 25-0259-42), and CD279 / PD1 (1:50; Biolegend clone EH12.2H7, BV605 conjugate: catalog no. 340560). Single-stained samples with Ultracomp beads (5 μL, Invitrogen, catalog no. 01-2222-42) were used for compensation preparation of the flow cytometer. After incubation at 4°C for 30 minutes, the plates were washed three times with PBS / 0.1% BSA / 0.02% azide (staining buffer). Cells were resuspended in 80 μL of staining buffer and analyzed using FACS Fortessa (BD Biosciences). The data was processed using FlowJo (BD Biosciences).

[0341] Figure 10 shows that both bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR induced dose-dependent cynomolgus monkey PBMC-induced killing of tumor cells expressing human ROR2, and that killing occurred at lower concentrations with bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR compared to bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR.

[0342] Figure 11 shows T cell activation in a cynomolgus monkey PMBC population in the presence of bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR or bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR and HeLa cells, as defined by the expression of activation markers CD69, CD25, and PD-1 on CD8+ T cells (determined by flow cytometry). In the presence of either bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR or bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR, approximately 80% of CD8+ T cells were activated and expressed CD69 and CD25 (at the highest antibody concentration), and approximately 40% of CD8+ T cells expressed PD-1. T cell activation induced by bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR occurred at lower concentrations than T cell activation induced by bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR. This indicates that both bsIgG1-huCD3-FEAL×ROR2-A-HC4LC3-FEAR and bsIgG1-huCD3-H101G-FEAL×ROR2-A-HC4LC3-FEAR can engage cynomolgus monkey T cells as effector cells and activate T cells.

[0343] Example 17 - ROR2 expression in various human cancer symptoms ROR2 mRNA levels were extracted from the Omicsoft TCGA database and visualized using Oncoland software (Qiagen, USA).

[0344] Figure 12 shows ROR2 mRNA expression levels ranked according to median expression in selected primary solid tumors. mRNA expression varied within each sign, with the highest median expression observed in sarcomas, uterine cancer, pancreatic cancer, breast cancer, and ovarian cancer, as well as lung squamous cell carcinoma.

[0345] ROR2 protein expression in fibrosarcoma, gastrointestinal stromal tumor (GIST), leiomyosarcoma, rhabdomyosarcoma, liposarcoma, ovarian adenocarcinoma (serous papillary), endometrioid carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, pancreatic cancer, clear cell carcinoma, transitional cell carcinoma, and colon adenocarcinoma was analyzed by immunohistochemistry (IHC) using Leica Bond RX with Leica Bond reagents in tissue microarrays (TMA; purchased from BioMax). Prior to staining, freshly cut TMA sections (5 μm) were deparaffinized and incubated with the targeted retrieval solution ER2. ROR2 IHC was performed using mouse anti-ROR2 antibody (clone ROR2 2535-2835, QED Bioscience, catalog number 34045) at a final concentration of 10 μg / mL. Next, the sections were washed and incubated with goat anti-mouse-IgG-HRP. HRP was visualized using the DAB refine substrate chromogen system. Hematoxylin was used to detect nucleated cells. Stained TMA sections were digitized at 20x magnification using an AxioScan slide scanner (Zeiss).

[0346] ROR2 staining intensity and the percentage of ROR2-positive cells in tumors were determined and quantified by a certified pathologist. Staining intensity was scored as negative (0), weak (1), moderate (2), or strong (3), and the percentage of cells was measured in 10% increments within the range of 0-100%. The histological score (H-score) was determined from the staining intensity and positive cell percentage according to the following formula. H-score = (0 × [percentage of cells with intensity 0] + 1 × [percentage of cells with intensity 1+] + 2 × [percentage of cells with intensity 2+] + 3 × [percentage of cells with intensity 3+])

[0347] Table 11 shows ROR2 protein expression (prevalence and H-score) determined by IHC analysis of BioMax TMA. ROR2 expression varied across different lesions. The highest prevalence and ROR2 H-score were found in sarcomas, GISTs, as well as ovarian cancer and endometrioid carcinoma.

[0348] (Table 11) ROR2 protein expression (incidence and H-score) determined by IHC analysis of BioMax TMA TIFF0007923755000024.tif195166

Claims

1. An antibody comprising at least one antigen-binding region capable of binding to human ROR2, comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9, respectively.

2. The antibody according to claim 1, which is humanized from an antibody comprising a VH region having the sequence shown in SEQ ID NO:2 and / or a VL region having the sequence shown in SEQ ID NO:

6.

3. a. The VH region shown in SEQ ID NO:10 (HC1); b. The VH region shown in SEQ ID NO:11 (HC2); c. The VH region shown in SEQ ID NO:12 (HC3); d. The VH region shown in SEQ ID NO:13 (HC4); e. The VH region shown in SEQ ID NO:14 (HC5); f. The VH region shown in SEQ ID NO:15 (HC6); g. The VH region shown in SEQ ID NO:16 (HC7), or h. VH region having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 10, 11, 12, 13, 14, 15, or 16 The antibody according to any one of claims 1 and 2, comprising a VH region having a sequence selected from the group including the group.

4. a. The VL region shown in SEQ ID NO:17 (LC1); b. The VL region shown in SEQ ID NO:18 (LC2); c. The VL region shown in SEQ ID NO:19 (LC3); d. The VL region shown in SEQ ID NO:20 (LC4); or e. VL regions having at least 90% sequence identity to any one of the sequences with SEQ ID NO: 17, 18, 19, or 20. The antibody according to any one of claims 1 to 3, comprising a VL region having a sequence selected from the group including the group.

5. a. VH region containing the sequence of SEQ ID NO. 10 and VL region containing the sequence of SEQ ID NO. 17; b. The VH region containing the sequence of SEQ ID NO. 10 and the VL region containing the sequence of SEQ ID NO. 18; c. VH region containing the sequence of SEQ ID NO. 10 and VL region containing the sequence of SEQ ID NO. 19; d. The VH region containing the sequence of SEQ ID NO. 10 and the VL region containing the sequence of SEQ ID NO. 20; e. The VH region containing the sequence of SEQ ID NO. 11 and the VL region containing the sequence of SEQ ID NO. 17; f. VH region containing the sequence of SEQ ID NO. 11 and VL region containing the sequence of SEQ ID NO. 18; g. The VH region containing the sequence of SEQ ID NO. 11 and the VL region containing the sequence of SEQ ID NO. 19; h. VH region containing the sequence of SEQ ID NO. 11 and VL region containing the sequence of SEQ ID NO. 20; i. The VH region containing the sequence of SEQ ID NO. 12 and the VL region containing the sequence of SEQ ID NO. 17; j. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 18; k. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 19; l. VH region containing the sequence of SEQ ID NO. 12 and VL region containing the sequence of SEQ ID NO. 20; m. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 17; n. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 18; o. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 19; p. VH region containing the sequence of SEQ ID NO. 13 and VL region containing the sequence of SEQ ID NO. 20; q. The VH region containing the sequence of SEQ ID NO. 14 and the VL region containing the sequence of SEQ ID NO. 17; r. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 18; s. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 19; t. VH region containing the sequence of SEQ ID NO. 14 and VL region containing the sequence of SEQ ID NO. 20; u. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 17; v. The VH region containing the sequence of SEQ ID NO. 15 and the VL region containing the sequence of SEQ ID NO. 18; w. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 19; x. VH region containing the sequence of SEQ ID NO. 15 and VL region containing the sequence of SEQ ID NO. 20; y. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 17; z. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 18; aa. VH region having the sequence of SEQ ID NO. 16 and VL region having the sequence of SEQ ID NO. 19; and bb. VH region containing the sequence of SEQ ID NO. 16 and VL region containing the sequence of SEQ ID NO. 20 An antibody according to any one of claims 1 to 4, comprising a VH region and a VL region having a sequence selected from the group including the group.

6. The antibody according to any one of claims 1 to 5, comprising a VH region and a VL region having sequences of SEQ ID No. 13 and 19.

7. The antibody according to any one of claims 1 to 6, wherein the heavy chain constant region is human IgG1.

8. The antibody according to any one of claims 1 to 7, wherein the light chain constant region is human kappa.

9. The antibody according to any one of claims 1 to 8, which is a full-length antibody such as a full-length IgG1 antibody.

10. The antibody according to any one of claims 1 to 9, wherein the human ROR2 is human ROR2 of SEQ ID NO.

1.

11. An antibody according to any one of claims 1 to 10, which can bind to the kringle domain of human ROR2.

12. The antibody according to any one of claims 1 to 11, which binds to an epitope or antibody-binding region on human ROR2 involving the 322nd amino acid residue of human ROR2, with a numbering indicating its position in SEQ ID NO:

1.

13. An antibody according to any one of claims 1 to 12, which can bind to the human ROR2 extracellular domain with a binding affinity corresponding to a KD value in the range of 10 nM to 1 nM.

14. An antibody comprising a first antigen-binding region capable of binding to human ROR2 according to any one of claims 1 to 13, and a second antigen-binding region capable of binding to a different target.

15. The antibody according to claim 14, wherein the second antigen-binding region can bind to human CD3ε (epsilon) as indicated by SEQ ID NO:

21.

16. The antigen-binding region that binds to human CD3ε, The heavy chain variable regions (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, 24, and 25, respectively. and Light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:27, GTN, and SEQ ID NO:28, respectively. The antibody according to claim 15, comprising:

17. The antigen-binding region that binds to human CD3ε, a. A heavy chain variable region (VH) containing the sequence of SEQ ID NO:22 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:22, and b. A light chain variable region (VL) containing the sequence of SEQ ID NO:26 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:

26. The antibody according to claim 16, comprising:

18. The antigen-binding region that binds to human CD3ε, a. A heavy chain variable region (VH) containing the sequence of SEQ ID NO:29 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:29, and b. A light chain variable region (VL) containing the sequence of SEQ ID NO:30 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with respect to the sequence of SEQ ID NO:

30. The antibody according to claim 16, comprising:

19. The antigen-binding region that binds to human CD3ε, a. Heavy chain variable region (VH) containing the sequence of SEQ ID NO:29 and b. Light chain variable region (VL) containing the sequence of SEQ ID NO:30 The antibody according to claim 16 or 18, comprising:

20. The antibody according to any one of claims 15 to 18, having a lower human CD3ε binding affinity than an antibody having an antigen-binding region including the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:

30.

21. The antibody according to any one of claims 15 to 19, wherein the antigen-binding region that binds to human CD3ε binds with an equilibrium dissociation constant KD in the range of 200 to 1000 nM.

22. The antibody according to any one of claims 15 to 19, wherein the antigen-binding region that binds to human CD3ε binds with an equilibrium dissociation constant KD in the range of 1 to 100 nM.

23. The antibody according to claim 15, wherein the antigen-binding region capable of binding to human CD3ε comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 23, 24, and 31, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 27, sequence GTN, and SEQ ID NO: 28, respectively.

24. The antibody according to claim 15 or 23, wherein the antigen-binding region capable of binding to human CD3ε comprises a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:32 and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:

30.

25. This is a bispecific antibody containing a first antigen-binding region that can bind to human ROR2 and a second antigen-binding region that can bind to human CD3. The first antigen-binding region is Heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 3, 4, and 5 respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having sequences shown in SEQ ID NO: 7, 8, and 9 respectively. including and The second antigen-binding region is A heavy chain variable (VH) region CDR1 having the sequence shown in SEQ ID NO:23, a heavy chain variable (VH) region CDR2 having the sequence shown in SEQ ID NO:24, and a heavy chain variable (VH) region CDR3 having the sequence shown in either SEQ ID NO:25 or 31, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:27, GTN, and SEQ ID NO:28, respectively. including, The antibody according to any one of claims 1 to 24.

26. It comprises a first antigen-binding region that can bind to human ROR2 and a second antigen-binding region that can bind to human CD3, The first antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:29 and a VL region containing the sequence shown in SEQ ID NO:

30. The antibody according to any one of claims 1 to 23 and 25.

27. It comprises a first antigen-binding region that can bind to human ROR2 and a second antigen-binding region that can bind to human CD3, The first antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:13 and a VL region containing the sequence shown in SEQ ID NO:19, and the second antigen-binding region includes a VH region containing the sequence shown in SEQ ID NO:32 and a VL region containing the sequence shown in SEQ ID NO:

30. The antibody according to any one of claims 1 to 18, 20, 21, and 23 to 25.

28. comprising a first heavy chain and a second heavy chain, In the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R, and in the second heavy chain, the amino acid at the position corresponding to F405 in the human IgG single chain is L, or In the second heavy chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R, and in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG single chain is L. The antibody according to any one of claims 1 to 27, wherein the amino acid positions in the constant region are numbered according to Eu numbering.

29. The primary and secondary heavy chains of the antibody have been modified to induce Fc-mediated effector function to a lower degree compared to the unmodified antibody. An antibody according to any one of claims 1 to 28, comprising:

30. It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residues corresponding to positions L234, L235, and D265 in the human IgG monohelic acid according to Eu numbering are F, E, and A, respectively. The antibody according to any one of claims 1 to 29.

31. It includes a primary heavy chain and a secondary heavy chain, In both the first and second heavy chain constant regions, the amino acid residues at positions L234, L235, and D265 in the human IgG monohelic acid according to Eu numbering are F, E, and A, respectively, and the first heavy chain constant region further includes a K409R substitution, and the second heavy chain constant region further includes an F405L substitution. The antibody according to any one of claims 1 to 30.

32. It is a bispecific antibody that can bind to human ROR2 and human CD3 epsilon. a. The first connecting arm that connects to ROR2 is i. VH region having amino acid sequence SEQ ID NO:13 ii. VL region having amino acid sequence SEQ ID NO:19 iii. A heavy chain constant region having the amino acid sequence of SEQ ID NO:34, and iv. The steady-state region of the human kappa light chain including and b. The second binding arm that binds to CD3 epsilon, i. VH region having amino acid sequence SEQ ID NO: 29 or 32 ii. VL region having amino acid sequence SEQ ID NO:30 iii. A heavy chain constant region having the amino acid sequence of SEQ ID NO:35, and iv. Steady-state region of human lambda light chain including, The antibody according to any one of claims 1 to 31.

33. a. It can bind to ROR2-expressing human tumor cells included in the HeLa, LCLC103-H, NCI-H1650, 786-O, NCI-H23, and ZR-75-1 cell groups, b. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of HeLa cells. c. When purified PBMCs or T cells are used as effector cells, they can mediate concentration-dependent cytotoxicity of 786-O, LCLC103-H, NCI-H23, NCI-H1650, or ZR-75-1 cells. d. In the presence of HeLa, 786-O, LCLC103-H, NCI-H23, NCI-H1650 tumor cells, T cells can be activated in vitro, and / or e. When HeLa and 786-O tumor cells are used as target cells, T cell cytokine production can be induced. The antibody according to any one of claims 1 to 32.

34. A composition comprising the antibody according to any one of claims 1 to 33.

35. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 33 and a pharmaceutically acceptable carrier.

36. An antibody according to any one of claims 1 to 33, for use as a pharmaceutical.

37. An antibody for use as a pharmaceutical, according to claim 36, for use in the treatment of a disease.

38. The antibody for pharmaceutical use according to claim 37, wherein the disease is cancer, and the cancer is characterized by the expression of ROR2 on the surface of cancer cells.

39. The antibody for use according to claim 38, wherein the cancer is selected from the group including sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine cancer, lung cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, and breast cancer.

40. a. A nucleic acid sequence encoding a heavy chain variable region sequence of an antigen-binding region that can bind to ROR2 according to any one of claims 1 to 3, 5, and 6, and b. A nucleic acid sequence encoding a corresponding light chain variable region sequence of an antigen-binding region that can bind to ROR2 according to any one of claims 1, 2, and 4-6. Nucleic acids, including

41. a. A nucleic acid sequence encoding the heavy chain sequence of an antibody containing an antigen-binding region that can bind to ROR2 as shown in SEQ ID NO:13, and b. A nucleic acid sequence encoding the light chain sequence of an antibody containing an antigen-binding region capable of binding to ROR2, as shown in SEQ ID NO:

19. One or more nucleic acids, including one or more nucleic acids.

42. A nucleic acid or one or more nucleic acids according to any one of claims 40 and 41, which is RNA or DNA.

43. A nucleic acid according to any one of claims 40 to 42, or one or more nucleic acids, for use in expression in mammalian cells.

44. a) A nucleic acid sequence encoding a heavy chain sequence of an antibody having an antigen-binding region that can bind to ROR2 according to any one of claims 40 to 43, and b) A nucleic acid sequence encoding the light chain sequence of an antibody having an antigen-binding region that can bind to ROR2 according to any one of claims 40 to 43. An expression vector containing [the specified element].

45. a. A nucleic acid sequence encoding a heavy chain sequence of an antibody having an antigen-binding region capable of binding to human CD3ε as described in any one of claims 16-20 and 23-24, and b. A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to human CD3ε as described in any one of claims 16-20 and 23-24. The expression vector according to claim 44, further comprising:

46. A cell comprising the nucleic acid according to any one of claims 40 to 43, or one or more nucleic acids, or the expression vector according to claim 44 or 45.

47. a. A step of providing an antibody capable of binding to ROR2, comprising an antigen-binding region capable of binding to ROR2 as described in any one of claims 1 to 33. b. A step of providing an antibody capable of binding to human CD3ε, comprising an antigen-binding region capable of binding to human CD3ε as described in any one of claims 15 to 33. c. A step of incubating an antibody capable of binding to ROR2 together with an antibody capable of binding to human CD3ε under conditions of sufficient reduction to allow cysteine ​​in the hinge region to undergo isomerization of the disulfide bond, and d. Steps to obtain antibodies that can bind to ROR2 and human CD3ε. A method for producing an antibody capable of binding to both ROR2 and human CD3ε according to any one of claims 15 to 33, comprising:

48. A kit for use as a companion diagnostic for identifying patients within a patient population who are responsive to treatment with an antibody according to any one of claims 1 to 33, the kit comprising an antibody according to any one of claims 1 to 33 and instructions for use of the kit.

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