Antibodies that bind to B7H4

Abstract

The present invention relates to antibodies that bind to B7H4, including bispecific antibodies that bind to B7H4 and CD3. The invention also provides pharmaceutical compositions comprising these antibodies, as well as uses of the antibodies for therapeutic and diagnostic procedures, particularly in cancer therapy.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antibodies that bind to B7H4, particularly bispecific antibodies that bind to B7H4 and CD3. The present invention also provides pharmaceutical compositions comprising the antibodies and uses of the antibodies for therapeutic and diagnostic procedures, particularly in cancer therapy. [Background technology]

[0002] Introduction B7H4 (B7-H4, V-set domain-containing T-cell activation inhibitor 1, or VTCN1) is a member of the B7 family of proteins, which contains cell surface protein ligands that bind to receptors on lymphocytes. The B7 family plays an important role in regulating immune responses. B7H4 negatively regulates T cell-mediated immune responses by inhibiting T cell activation, proliferation, cytokine production, and cytotoxic activity (Prasad et al., 2003, Immunity 18: 863-873). B7H4 is a type I transmembrane protein containing a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with IgV-like and IgC-like domains containing four conserved cysteine ​​residues and seven sites for N-linked glycosylation (Sica et al., 2003, Immunity 18: 849-861). To date, no receptor for B7H4 has been identified.

[0003] While B7H4 expression is very limited in normal mature tissues, it has been confirmed in tumor cells in many cancer tissues (Kaur and Janakiram, 2019, ESMO Open 4:e000554). In cancer, B7H4 expression correlates with advanced cancer stage, poor prognosis, and reduced overall patient survival.

[0004] Therefore, targeting B7H4 for cancer treatment has been proposed (Podojil and Miller, Immunological Reviews, 2017: 276; 40-51). Antibodies that bind to B7H4 are currently being developed for cancer therapy. For example, FPA150 is an afucosylated human antibody that attenuates B7H4-mediated suppression of T cell activation and exhibits antibody-dependent cellular cytotoxicity (ADCC) activity (Wainberg et al., 2019, Annals of Oncology 30, Suppl. 5, v489 (1198P)). FPA150 is currently in early clinical trials in advanced solid tumors as a monotherapy or in combination with pembrolizumab.

[0005] Attempts have also been made to target T cells to B7H4. A B7H4 / CD3 bispecific single-chain antibody, Fab scFv, was created based on the structures of the Fab fragment and single-chain variable fragment (scFv) of a mouse anti-human B7H4 antibody and a mouse anti-human CD3 antibody (Iizuka et al., 2019, Clin Cancer Res 25: 2925-2934 (Non-Patent Document 6)). Smith et al. described engineered T cells bearing a chimeric antigen receptor (CAR) specific for B7H4, which showed anti-tumor activity against B7H4-positive human ovarian tumor xenografts in mice, but also showed multi-organ lymphocyte infiltration and lethal toxicity (Smith et al. 2016, Molecular Therapy, Vol. 24 Iss. 11 pp 1987-99 (Non-Patent Document 7)).

[0006] Although some progress has been made, there is a need for the development of antibody-based cancer therapies that target B7H4 that are effective and / or safe for use in humans. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Prasad et al., 2003, Immunity 18: 863-873 [Non-Patent Document 2] Sica et al., 2003, Immunity 18: 849-861 [Non-Patent Document 3] Kaur and Janakiram, 2019, ESMO Open 4:e000554 [Non-Patent Document 4] Podojil and Miller, Immunological Reviews, 2017: 276; 40-51 [Non-Patent Document 5] Wainberg et al., 2019, Annals of Oncology 30, Suppl. 5, v489 (1198P) [Non-Patent Document 6] Iizuka et al., 2019, Clin Cancer Res 25: 2925-2934 [Non-Patent Document 7] Smith et al. 2016, Molecular Therapy, Vol.24 Iss. 11 pp 1987-99 [Summary of the Invention] <0OO0119>

[0008] An object of the present invention is to provide antibodies comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region that binds to CD3, such as human CD3ε (epsilon). The antigen-binding region of such antibodies comprises at least human framework regions, e.g., FR1, FR2, FR3, and FR4. Most preferably, all framework regions are human. Such antigen-binding regions are humanized and / or human. These antibodies are useful in treating conditions, such as cancer, in which specific targeting and T cell-mediated killing of B7H4-expressing cells is desirable. Preferably, such antibodies are suitable for use in humans, e.g., in medical therapy. Potentially suitable cancers for treatment are solid tumors. For example, the B7H4 expression and T cell-mediated killing in cancer cells described herein can range from relatively low B7H4 expression, e.g., in MCF-7 cells, to relatively high B7H4 expression, e.g., in SK-BR3 cells, as shown in Example 12. More preferably, such bispecific antibodies have substitutions in the constant region that render the Fc region inactive, if present.

[0009] In one embodiment, a bispecific antibody comprises an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to CD3, such as human CD3ε (epsilon), wherein the antigen-binding region capable of binding to human B7H4 comprises a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25, SEQ ID NO: 29, or SEQ ID NO: 31, and a variable light chain region comprising the CDR1, CDR2, and CDR3 of SEQ ID NO: 33, and the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN, and SEQ ID NO: and a light chain variable region (VL) comprising 24 CDR1, CDR2, and CDR3 sequences.

[0010] In another aspect, there are provided nucleic acids, such as DNA or RNA, that encode the antibodies defined herein, as well as methods of making the antibodies or components thereof defined herein.

[0011] In a further aspect, the antibody or nucleic acid according to the invention is for use in medical therapy. [The present invention 1001] An antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are humanized and / or derived from a human. [The present invention 1002] The antibody of the present invention, which is a bispecific antibody. [The present invention 1003] The antibody of the present invention 1001 or 1002, which is capable of binding to cancer cells and T cells. [The present invention 1004] The antibody of any one of claims 1001 to 1003, wherein the cancer cells express human B7H4. [The present invention 1005] The antibody of the present invention 1003 or 1004, wherein the cancer cells are from a solid tumor. [The present invention 1006] The antibody of any one of 1001 to 1005 of the present invention, which is capable of inducing T cell-mediated cell death. [The present invention 1007] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to CD3 is capable of binding to human CD3ε (epsilon), for example, human CD3ε (epsilon) shown in SEQ ID NO: 13. [The present invention 1008] The antigen-binding region that binds to CD3 is A heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 16 or SEQ ID NO: 17; and A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22 The antibody of any one of 1001 to 1007 of the present invention, comprising: [The present invention 1009] The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21, respectively; and A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively. The antibody of any one of 1001 to 1007 of the present invention, comprising: [The present invention 1010] The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 16 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 16; and A light chain variable region (VL) comprising 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 to the sequence of SEQ ID NO: 22. The antibody of any one of 1001 to 1007 of the present invention, comprising: [The present invention 1011] The dissociation equilibrium constant K D is within the range of 1 to 100 nM, for example, within the range of 5 to 100 nM, within the range of 10 to 100 nM, within the range of 1 to 80 nM, within the range of 1 to 60 nM, within the range of 1 to 40 nM, within the range of 1 to 20 nM, within the range of 5 to 80 nM, within the range of 5 to 60 nM, within the range of 5 to 40 nM, within the range of 5 to 20 nM, within the range of 10 to 80 nM, within the range of 10 to 60 nM, within the range of 10 to 40 nM, or for example, within the range of 10 to 20 nM. [The present invention 1012] Any of the antibodies of the present invention 1007 to 1010, which has a lower binding affinity for human CD3ε than an antibody having an antigen-binding region comprising the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 22, preferably the affinity is at least 5-fold, for example at least 10-fold, for example at least 20-fold, at least 30-fold, at least 40-fold, at least 45-fold, or for example at least 50-fold lower. [The present invention 1013] The antigen-binding region that binds to CD3 has an equilibrium dissociation constant K 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 900 nM, in the range of 400 to 700 nM, in the range of 500 to 900 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. D The antibody of any one of 1007 to 1010 of the present invention, [The present invention 1014] the antigen-binding region that binds to CD3 comprises a heavy chain variable (VH) region including a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; the heavy chain variable (VH) region has, when compared to a heavy chain variable (VH) region comprising the sequence set forth in SEQ ID NO: 16, an amino acid substitution at a position selected from the group consisting of T31, N57, H101, G105, S110, and Y114, wherein these positions are numbered based on the sequence of SEQ ID NO: 16; and the wild-type light chain variable (VL) region comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively; Any one of the antibodies 1011 to 1013 of the present invention. [The present invention 1015] 1014. An antibody of the present invention, wherein the antigen-binding region that binds to CD3 comprises a substitution in the heavy chain variable (VH) region selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V. [The present invention 1016] An antibody of any of inventions 1008 to 1010, wherein the CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding domain that binds to CD3 contain a total of at most 1, 2, 3, 4, or 5 amino acid substitutions when compared to the CDR1, CDR2, and CDR3 of the sequence of SEQ ID NO: 16, and the amino acid substitutions preferably include the amino acid substitutions defined in invention 1014 or invention 1015. [The present invention 1017] Any of the antibodies of the present invention, wherein the human B7H4 is human B7H4 of SEQ ID NO: 1. [The present invention 1018] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to the extracellular domain of human B7H4. [The present invention 1019] The antibody of the present invention 1017 or 1018, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to the IgC-like constant region of human B7H4. [The present invention 1020] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to B7H3-IgV / B7H4-IgC having the sequence of SEQ ID NO: 11. [The present invention 1021] An antibody of the present invention 1020, wherein the antigen-binding region capable of binding to human B7H4 is incapable of binding to B7H4-IgV / B7H3-IgC having the sequence of SEQ ID NO: 10. [The present invention 1022] An antigen-binding region capable of binding to human B7H4, a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 36, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 40; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 43, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 47; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 50, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 54; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 31, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 65, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 69; The antibody of any one of 1017 to 1020 of the present invention, comprising: [The present invention 1023] An antigen-binding region capable of binding to human B7H4, a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 41, DTS, and SEQ ID NO: 42, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 48, YTS, and SEQ ID NO: 49, respectively; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 55, GAS, and SEQ ID NO: 56, respectively; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 70, GAS, and SEQ ID NO: 71, respectively; The antibodies of the present invention 1017 to 1020, comprising: [The present invention 1024] An antigen-binding region capable of binding to human B7H4, a) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; c) the variable heavy chain (VH) region of SEQ ID NO: 36 and the variable light chain region of SEQ ID NO: 40; d) the variable heavy chain (VH) region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; e) the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; or f) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; g) the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 The antibody of any one of 1017 to 1020 of the present invention, comprising: [The present invention 1025] The antigen-binding region capable of binding to human B7H4 has a K of 5E-7M or less, for example, 1E-7M or less. D K values ​​corresponding to, for example, a K value in the range of 5E-7 to 2E-10 M, for example, a K value in the range of 2E-7 to 1E-10 M or 1E-7 to 5E-9 M. D Any of the antibodies of the present invention having a binding affinity corresponding to a value. [The present invention 1026] 1025 antibodies of the invention, whose binding affinity is determined by biolayer interferometry as optionally set forth in Example 3 herein. [The present invention 1027] The binding affinity is as follows: I) immobilizing an antibody in an amount of 1 μg / mL on an anti-human IgG Fc capture biosensor for 600 seconds; II) determining the association over a 300-second period and the dissociation over a 1000-second period of human recombinant His-tagged B7H4 protein (Sino Biological catalogue number 10738-H08H; a protein with a C-terminal polyhistidine tag expressed from a construct of a DNA sequence encoding human VTCN1 (Uniprot accession number Q7Z7D3) (Phe29-Ala258)) using a two-fold dilution series ranging from 1.56 nM to 100 nM; III) Relating data to buffer control (0 nM) Determined using biolayer interferometry, including The antibody of the present invention 1025 or 1026. [The present invention 1028] The antibody of any of claims 1025 to 1027, wherein the binding affinity is determined using an antibody as defined in any of the preceding claims of the invention that is a monospecific bivalent antibody, for example an antibody that is a full-length IgG1. [The present invention 1029] containing an antigenic region capable of binding to human B7H4, The antigen-binding region is An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 36 and a variable light chain region of SEQ ID NO: 40 can be cross-hindered, and the antigen-binding region is an antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 47; An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; and An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 Intersections cannot be obstructed, Any one of the antibodies 1001 to 1028 of the present invention. [The present invention 1030] containing an antigenic region capable of binding to human B7H4, The antigen-binding region is an antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 47; An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; and An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 can be cross-hindered, and the antigen-binding region is An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 36 and a variable light chain region of SEQ ID NO: 40 , which is unable to cross-block antibodies containing Any one of the antibodies 1001 to 1028 of the present invention. [The present invention 1031] The antibody of the present invention 1029 or 1030, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to B7H3-IgV / B7H4-IgC of SEQ ID NO: 11, and optionally is incapable of binding to B7H4-IgV / B7H3-IgC of SEQ ID NO: 10. [The present invention 1032] Any of the antibodies of the present invention, wherein each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each variable region comprises three CDR sequences, i.e., CDR1, CDR2, and CDR3, respectively, and four framework sequences, i.e., FR1, FR2, FR3, and FR4, respectively. [The present invention 1033] c) the antigen-binding region capable of binding to B7H4 is of human origin; and d) the antigen-binding region capable of binding to CD3 is humanized; Any of the antibodies of the present invention. [The present invention 1034] b) the antigen-binding region capable of binding to B7H4 is of human origin; and / or c) the antigen-binding region capable of binding to CD3 is humanized; Any one of the antibodies 1001 to 1032 of the present invention. [This invention 1035] Contains two heavy chain constant regions (CH) and two light chain constant regions (CL), Any of the antibodies of the present invention. [The present invention 1036] The antibody of the present invention, wherein the two heavy chain constant domains and the two light chain constant regions are of human origin. [This invention 1037] Any of the antibodies of the present invention which are full-length antibodies. [The present invention 1038] Any of the antibodies of the present invention which are of the IgG1 isotype. [This invention 1039] Any of the antibodies of the invention, wherein the antibody comprises a first heavy chain and a second heavy chain, each of the first heavy chain and the second heavy chain comprising at least a hinge region, a CH2 region, and a CH3 region, wherein the first heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and the second heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, wherein the substitutions in the first heavy chain and the second heavy chain are not at the same position, and these amino acid positions are numbered according to EU numbering. [The present invention 1040] 1039. The antibody of the invention, wherein the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in the first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain, or vice versa. [This invention 1041] the antibody comprises a first heavy chain and optionally a second heavy chain; and the antibody wherein the first heavy chain, and, if present, the second heavy chain, are modified such that the antibody induces Fc-mediated effector function to a lesser extent than the same unmodified antibody; Any of the antibodies of the present invention. [The present invention 1042] 1041. The antibody of the present invention, wherein the antibody comprises a first heavy chain and a second heavy chain, and in both the first heavy chain and the second heavy chain, the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain based on EU numbering are F and E, respectively. [This invention 1043] The antibody of the present invention 1041 or 1042, wherein the antibody comprises a first heavy chain and a second heavy chain, and in both the first heavy chain and the second heavy chain, the amino acid residue at a position corresponding to position D265 in a human IgG1 heavy chain based on EU numbering is A. [This invention 1044] Any of the aforementioned antibodies of the present invention, which comprises a kappa (κ) light chain. [This invention 1045] Any of the aforementioned antibodies of the present invention, which comprise a lambda (λ) light chain. [The present invention 1046] Any of the antibodies of the present invention, wherein the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain, for example, an antibody having a heavy chain and a lambda light chain comprising a binding region capable of binding to CD3, and a heavy chain and a kappa light chain comprising a binding region capable of binding to B7H4. [This invention 1047] an antigen-binding region capable of binding to human B7H4 is contained in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a kappa light chain constant region; and an antigen-binding region capable of binding to human CD3 is contained in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a lambda light chain constant region; Any of the antibodies of the present invention. [This invention 1048] The antibody of the present invention 1047, wherein one IgG1 heavy chain constant region is as defined in SEQ ID NO: 60 and the other is as defined in SEQ ID NO: 61, and the kappa light chain constant region is as defined in SEQ ID NO: 63, and the lambda light chain constant region is as defined in SEQ ID NO: 64. [This invention 1049] 1048. The antibody of the present invention, wherein the terminal lysine is deleted in the IgG1 heavy chain constant region defined in SEQ ID NO: 60 and SEQ ID NO: 61. [The present invention 1050] lacking or having reduced Fc-mediated effector function, and a) capable of binding to B7H4-expressing human tumor cells as described in Examples 9 and 10 herein; b) mediate concentration-dependent cytotoxicity in B7H4-expressing human tumor cells, e.g., when PBMCs or T cells are used as effector cells, as assayed as described in Examples 11 and 12 herein; c) mediating concentration-dependent cytotoxicity in one or more human B7H4-expressing tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650, e.g., when assayed as described in Examples 11 and 12 herein, using PBMCs or T cells as effector cells; d) T cells can be activated in vitro in the presence of B7H4-expressing human tumor cells, for example, when assayed as described in Example 13 herein; e) T cells can be activated in vitro in the presence of one or more B7H4-expressing human tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650, for example, when analyzed as described in Example 13 herein; f) capable of inducing cytotoxicity of B7H4-expressing human tumor cells, for example, when assayed as described in Examples 11 and 12 herein; and / or g) capable of inducing T cell-mediated cytotoxicity in one or more B7H4-expressing human tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650, when assayed, for example, as described in Examples 11 and 12 herein; Any of the antibodies of the present invention. [This invention 1051] The antibody has an IC50 in the range of 0.001 to 5 μg / ml, wherein the IC50 is determined to be in the following order: i) providing peripheral blood mononuclear cells (PBMCs) or purified T cells isolated from the buffy coat of a healthy human donor; ii) providing B7H4-expressing tumor cells; iii) mixing PBMCs or purified T cells with a human B7H4-expressing tumor cell line selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650 in a plurality of samples, wherein the ratio of the number of T cells derived from the PBMCs or purified T cells to the selected tumor cells is 8:1; iv) providing the antibody in a dilution series ranging from, for example, 0.0128 ng / mL to 10,000 ng / mL to the sample; and v) incubating the sample, for example at 37°C for 72 hours; followed by vi) assessing the viability of B7H4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample; and viii) determining IC50 as determined in an in vitro cytotoxicity assay comprising The antibody of the present invention 1047. [This invention 1052] The antibody of the present invention 1048, having an IC50 in the range of 0.001 to 0.03 μg / ml. [This invention 1053] The antibody of the present invention 1048, having an IC50 in the range of 0.05 to 5 μg / ml. [This invention 1054] The antigen-binding region that can bind to human B7H4 a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; e) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; or f) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33 An antibody comprising an antigen-binding region capable of binding to human B7H4, comprising: [This invention 1055] The antibody of the present invention 1054, which is a monospecific antibody. [The present invention 1056] The antibody of the present invention 1054 or 1055, which is a bivalent antibody. [This invention 1057] The antibody of the present invention 1056, which is a bispecific antibody comprising an additional antigen-binding region capable of binding to human CD3, preferably wherein the additional antigen-binding region capable of binding to CD3 is an antigen-binding region capable of binding to human CD3 of any of the antibodies of the present inventions 1007 to 1016. [This invention 1058] A composition comprising any one of the antibodies 1001 to 1057 of the present invention. [This invention 1059] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1058 and a pharmaceutically acceptable carrier. [The present invention 1060] An antibody according to any one of claims 1001 to 1057 of the present invention for use as a pharmaceutical. [This invention 1061] An antibody of the present invention for use as a medicament for use in the treatment of a disease. [This invention 1062] The antibody of the present invention for use as a pharmaceutical according to claim 1061, wherein the disease is cancer. [This invention 1063] The antibody of the present invention for use as a medicament according to claim 1062, wherein the cancer is characterized by the expression of B7H4 in cancer cells. [This invention 1064] An antibody for use as a medicament for use in accordance with the present invention 1063, wherein expression of B7H4 is determined in cancer cells obtained from a patient. [This invention 1065] An antibody for use as a pharmaceutical according to any one of claims 1062 to 1064 of the present invention, wherein the cancer is a solid tumor. [The present invention 1066] The antibody for use in any of the present inventions 1062 to 1065, wherein the cancer is selected from the group consisting of lung cancer, NSCLC (ADC or SQCC), gastric cancer, pancreatic cancer, bile duct cancer, bladder cancer, cervical cancer, head and neck cancer, breast cancer, ovarian cancer, and uterine cancer. [This invention 1067] A method for treating a disease, comprising administering to a subject in need thereof any of the antibodies of the present inventions 1001 to 1057, the composition of the present invention 1058, or the pharmaceutical composition of the present invention 1059. [The present invention 1068] The method of claim 1067, which is for treating cancer. [The present invention 1069] The method of claim 1068, wherein the cancer is selected from the group consisting of uterine carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), invasive breast cancer (BRCA), uterine endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), and cholangiocarcinoma (CHOL). [The present invention 1070] a) a nucleic acid sequence encoding the heavy chain variable region sequence of an antigen-binding region capable of binding to B7H4 of the present invention; and / or b) a nucleic acid sequence encoding the corresponding light chain variable region sequence of said antigen-binding region capable of binding to B7H4 of the invention 1054; A nucleic acid comprising: [This invention 1071] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to any one of B7H4 of the present inventions 1022 to 1024; b) a nucleic acid sequence encoding the corresponding light chain sequence of an antibody comprising an antigen-binding region capable of binding to B7H4 of any one of 1022 to 1024 of the present invention; c) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of 1008 to 1010 of the present invention; and d) A nucleic acid sequence encoding the corresponding light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of 1008 to 1010 of the present invention. One or more nucleic acids comprising: [This invention 1072] The nucleic acid or nucleic acids of the invention 1070 or 1071, which is RNA or DNA. [This invention 1073] 1070-1072. A nucleic acid or nucleic acids of any one of claims 1070-1072 for use in expression in a mammalian cell. [This invention 1074] A cell comprising any one of the nucleic acids of the present inventions 1070 to 1073 or one or more types of nucleic acids. [This invention 1075] The cell of the present invention 1074, which is of human origin, e.g., a human embryonic kidney (HEK) cell, such as an HEK / Expi cell, or of rodent origin, e.g., a Chinese hamster ovary cell, such as a CHO / N50 cell. [This invention 1076] The following stages: a) providing an antibody capable of binding to B7H4, wherein the antibody comprises an antigen-binding region capable of binding to B7H4 of any one of the antibodies 1001 to 1006 and 1017 to 1034 of the present invention; b) providing an antibody capable of binding to CD3, wherein the antibody comprises an antigen-binding region capable of binding to CD3 of any one of the antibodies 1001 to 1016 and 1032 to 1034 of the present invention; c) incubating the antibody capable of binding to B7H4 with the antibody capable of binding to CD3 under reducing conditions sufficient to cause disulfide bond isomerization at cysteines in the hinge region; and d) obtaining antibodies capable of binding to B7H4 and CD3; A method for producing an antibody capable of binding to both B7H4 and CD3 according to any one of claims 1001 to 1053 of the present invention, comprising: [This invention 1077] Steps a) and / or b) - providing a cell comprising an expression vector for producing one or more of said antibodies; and - causing said cells to produce said antibody or antibodies; and subsequently - obtaining one or more of said antibodies, thereby providing one or more of said antibodies 1076. A method for producing an antibody capable of binding to both B7H4 and CD3, comprising: [This invention 1078] For example, a kit for use as a companion diagnostic / for identifying patients within a patient population who have a tendency to respond to treatment with an antibody of any one of 1001 to 1057 of the present invention, the kit comprising an antibody of any one of 1001 to 1057 of the present invention and instructions for using the kit. Kit of parts such as: [Brief explanation of the drawings]

[0012] [Figure 1-1] Determination of the B7H4 domain involved in binding using B7H4-B7H3 chimeric molecules. The B7H4 domain specificity of B7H4 antibodies was determined using a panel of cells transfected to express human B7H4 (I), the human B7H4-B7H3 chimeric molecules B7H3-IgV / B7H4-IgC (II) or B7H4-IgV / B7H3-IgC (III), or human B7H3 (IV). Binding was determined by flow cytometry. A=bsIgG1-huCD3-FEAL×B7H4-C4-FEAR;B=bsIgG1-huCD3-FEAL×B7H4-C3-FEAR;C=bsIgG1-huCD3-FEAL×B7H4-C2-FEAR;D=bsIgG1-huCD3-FEAL×B7H4-C1-FEAR;E=IgG1-B7H3-BRCA84D. [Figure 1-2] See description of Figure 1-1. [Figure 2]Binding of B7H4 antibodies to B7H4, B7H3, or B7H4-B7H3 chimeric molecules. The binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C2-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C3-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR, and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR to HEK cells transiently transfected to express human B7H4 or the B7H4-B7H3 chimeric molecules B7H3-IgV / B7H4-IgC or B7H4-IgV / B7H3-IgC was assessed by flow cytometry. [Figure 3A] Binding of B7H4 antibody to B7H4 variants with alanine mutations in the ECD. Binding was expressed as fold change compared to the reference antibody. Fold change was defined as Log10(normalized gMFI [ala variant] / normalized gMFI [wt]). Residues with fold changes in binding smaller than the mean fold change - 1.5 × SD were considered "reduced binding variants." Residues with positive fold changes in binding are residues with reduced binding relative to the reference antibody. Numbers below the x-axis indicate amino acid positions. (A) Results for C1-N52S with C2 as the reference antibody. (B) Results for C2 with C1-N52S as the reference antibody. (C) Results for C3 with C2 as the reference antibody. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4]Binding of B7H4 antibody and CD3xB7H4 bispecific antibody to human B7H4 and cynomolgus monkey B7H4. Binding of IgG1-B7H4-C1-N52S-FEAR (A) and bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (B) to HEK-293F cells transiently transfected with human B7H4 or cynomolgus monkey B7H4 was determined by flow cytometry. Untransfected HEK-293F cells (C) were used as a negative control; for these, binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR is shown. [Figure 5] Binding of B7H4 antibody and CD3xB7H4 bispecific antibody to B7H4 from rabbit, rat, mouse, dog, and pig. Binding of IgG1-B7H4-C1-N52S-FEAR (A) and bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (B) to HEK-293F cells transiently transfected with B7H4 from rabbit, rat, mouse, dog, or pig was determined by flow cytometry. Untransfected HEK-293F cells (C) were used as a negative control; for these, binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR is shown. [Figure 6] Binding of B7H4 antibodies to HEK-293F cells transiently transfected with B7H4 from various species. Binding of IgG1-B7H4-C1-N52S-FEAR (A), IgG1-B7H4-C3-FEAR (B), IgG1-B7H4-C2-FEAR (C), IgG1-B7H4-C4-FEAR (D), and IgG1-B7H4-C5-FEAR (E) to HEK-293F cells transfected with B7H4 from human, cynomolgus monkey, mouse, rat, or pig, or to untransfected HEK-293F cells, was determined by flow cytometry. IgG1-b12 was used as a non-binding control antibody (not shown). [Figure 7]Binding of IgG1-B7H4-C1-N52S-FEAR (A) and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (B) to MCF-7 and MDA-MB-468 cells. Binding was determined by flow cytometry. IgG1-b12 (C) and bsIgG1-huCD3-H101G-FEAL×b12-FEAR (D) were used as non-binding control antibodies. [Figure 8] Binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A) to NIH-OVCAR-3, HCC1954, and HeLa cells. Binding was determined by flow cytometry. bsIgG1-huCD3-H101G-FEAL×b12-FEAR (B) was used as a non-binding control antibody. [Figure 9] Binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A) and bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (B) to SK-BR3 and MDA-MB-486 cells. Binding was determined by flow cytometry. bsIgG1-huCD3-FEAL×b12-FEAR (C) and bsIgG1-huCD3-H101G-FEAL×b12-FEAR (D) were used as non-binding control antibodies. [Figure 10]Binding of various B7H4 antibodies in homodimeric and bsAb forms to MDA-MB-486 and HCC1954 cells. IgG1-B7H4-C1-N52S-FEAR (A homodimer), IgG1-B7H4-C2-FEAR (B homodimer), IgG1-B7H4-C3-FEAR (C homodimer), IgG1-B7H4-C4-FEAR (D homodimer), IgG1-B7H4-C5-FEAR (E homodimer), bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A bsAb), bsIgG1-huCD3-FEAL×B7H4-C2-FEAR [MDA-MB-468] or bsIgG1-huCD3-H101G-FEAL×B7H4-C2-FEAR [HCC1954] (B Binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C3-FEAR (bsAb), bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR (bsAb), and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR (bsAb) was determined by flow cytometry. bsIgG1-huCD3-H101G-FEAL×b12-FEAR (bsAb) or IgG1-b12-K409R (bsAb) were used as non-binding control antibodies. [Figure 11] In vitro induction of T cell-mediated cytotoxicity against SK-BR3 cells by CD3×B7H4 bispecific antibody using purified T cells as effector cells at various effector-to-target (E:T) ratios. bsIgG1-huCD3-FEAL×b12-FEAR was used as a non-binding control antibody. A = bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR; B = bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR; C = bsIgG1-huCD3-FEAL×b12-FEAR. [Figure 12]In vitro induction of T cell-mediated cytotoxicity in various tumor cell lines in the presence of CD3×B7H4 bispecific antibodies with different CD3 arms. bsIgG1-huCD3-FEAL×b12-FEAR was used as a non-binding control antibody. A=bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR; B=bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR; C=bsIgG1-huCD3-FEAL×b12-FEAR, D=bsIgG1-huCD3-H101G-FEAL×b12-FEAR. [Figure 13] B7H4 expression levels and IC50 values ​​for T cell-mediated tumor cell killing. (A) Quantitative flow cytometry analysis of B7H4 expression levels in tumor cell lines. Individual measurements (dots), geometric means (bars), and standard deviations (error bars) are shown. sABC = specific antibody binding capacity. (B) IC50 values ​​for T cell-mediated tumor cell killing against various tumor cell lines in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (I) or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (II). Each dot represents an experiment performed with an individual T cell donor (4–6 donors per cell line), and the horizontal line indicates the median. Cell lines are ranked based on B7H4 expression level. [Figure 14A] T cell activation by B7H4 bispecific antibodies in cocultures of T cells and tumor cells. (A) T cell activation (percentage of CD69 on CD8+ cells) in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (I) or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (II) for various B7H4-positive tumor cell lines as determined by flow cytometry. (B) EC50 values ​​for T cell activation using T cells from three to five donors for each target cell line. Each point represents an experiment performed with an individual T cell donor, and the horizontal line indicates the geometric mean. [Figure 14B] See legend to Figure 14A. [Figure 15]IFNγ in the supernatants of T cell and tumor cell cocultures at EC50, EC90, and EC99 concentrations of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A) and bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (B). Determined by multiplex U-plex assay using T cells from three to four donors. Individual measurements (dots), geometric means (horizontal lines), and standard deviations (error bars) are shown. [Figure 16] IL-6 and MCP-1 levels in plasma of cynomolgus monkeys treated with a single IV infusion of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A) or bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (B). [Figure 17-1] Mean plasma concentration-time profiles after a single IV infusion of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (A) or bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (B). [Figure 17-2] See description of Figure 17-1. [Figure 18]B7H4 mRNA expression levels in various primary solid tumors. B7H4 mRNA levels were extracted from the Omicsoft TCGA database and visualized using Oncoland software. Indications are ranked based on median B7H4 mRNA expression. THYM = thymoma, UVM = uveal melanoma, PCPG = pheochromocytoma and paraganglioma, ACC = adrenocortical carcinoma, MESO = mesothelioma, SKCM = skin cutaneous melanoma, READ = rectal adenocarcinoma, COAD = colon adenocarcinoma, GMB = glioblastoma multiforme, SARC = sarcoma, LIHC = liver hepatocellular carcinoma, LGG = brain low-grade glioma, KIRC = kidney renal clear cell carcinoma, TGCT = testicular germ cell tumor, KICH = chromophobe renal cell carcinoma, STAD = gastric adenocarcinoma, THCA = Thyroid cancer, HNSC=head and neck squamous cell carcinoma, PRAD=prostate adenocarcinoma, LUAD=lung adenocarcinoma, ESCA=esophageal carcinoma, CESC=cervical squamous cell carcinoma and adenocarcinoma, KIRP=papillary renal cell carcinoma of the kidney, UCS=uterine carcinosarcoma, BLCA=bladder urothelial carcinoma, PAAD=pancreatic adenocarcinoma, LUSC=lung squamous cell carcinoma, BRCA=invasive breast cancer, UCEC=uterine endometrial carcinoma, OV=ovarian serous cystadenocarcinoma, and CHOL=cholangiocarcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0013] Table 1: Amino acid and nucleic acid sequences TIFF0007749575000001.tif125152TIFF0007749575000002.tif235152TIFF0007749575000003.tif225152TIFF00077495750 00004.tif226152TIFF0007749575000005.tif227152TIFF0007749575000006.tif221152TIFF0007749575000007.tif250152

[0014] The CDR regions in the above tables (CDR1, CDR2, and CDR3, as well as the underlined sequences in the VH and VL sequences) are annotated according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)). The references to K405L and K409R used in the above tables are based on the EU index numbering (as 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)).

[0015] Detailed Description definition The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological and / or tumor-specific conditions, with a significant half-life, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days, or any other suitable functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the antibody binding to the antigen and / or a period sufficient for the antibody to be internalized). An antibody includes a binding region (or, as used herein, a binding domain, both of which have the same meaning) capable of interacting with an antigen, or a binding region including both the heavy and light chain variable regions of an immunoglobulin molecule. The antibody can comprise an antibody (Ab) constant region that can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation.

[0016] In the present invention, the term "antibody" includes monoclonal antibodies (mAbs), antibody-like polypeptides, chimeric antibodies, humanized antibodies, and "antibody fragments" or "fragments thereof" (antigen-binding fragments) that retain the ability to specifically bind to an antigen, which are provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant DNA technology. The term "antibody" also includes bispecific antibodies and / or antibodies with further modifications, for example, antibody-drug conjugates thereof.

[0017] Antibodies defined by the present invention can have any isotype, unless otherwise limited by the disclosure herein.

[0018] It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antibody" include: (i) a Fab' or Fab fragment, i.e., a monovalent fragment consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL) domain, and a heavy chain constant region domain 1 (CH1) domain, or a monovalent antibody as described in WO2007 / 059782; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of a VH domain and a CH1 domain; (iv) a Fv fragment consisting essentially of the VL and VH domains of one arm of an antibody; (v) a domain antibody consisting essentially of a VH domain (Holt et al.; Trends Biotechnol. 2003 Nov; 21 (11):484-90) (Ward et al., Nature 341 , 544-546 (1989));(vi) camelid antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, the VL and VH regions can be paired to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv). See, e.g., Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24 and Bird et al., Science 242Using recombinant techniques, these can be linked by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (see, e.g., J. Am. Chem. Soc., 423-426 (1988)). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Generally, such fragments are included within the meaning of antibody, but these fragments, collectively and each independently, are unique features of the present invention and exhibit different biological properties and utilities. These and other useful antibody fragments in the present invention are discussed further herein.

[0019] Antibodies can be produced in and recovered from a variety of in vitro or ex vivo expression or production systems, such as recombinantly engineered host cells, hybridomas, or systems that use cell extracts to facilitate the in vitro transcription and / or translation of nucleic acid sequences encoding the antibodies. It should be understood that a population of multiple different antibodies, i.e., antibodies as defined in the present invention, can be provided by producing each antibody separately in the aforementioned production system and then mixing the antibodies, or by producing several types of antibodies in the same production system.

[0020] As used herein, the term "immunoglobulin heavy chain" or "immunoglobulin heavy chain" is intended to refer to one of the heavy chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, namely CH1, CH2, and CH3. As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: one pair of low-molecular-weight light (L) chains and one pair of heavy (H) chains, with all four chains optionally interconnected by disulfide bonds. The structural characteristics of immunoglobulins have been well characterized (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989). Within the structure of an immunoglobulin, two heavy chains are interconnected by disulfide bonds in the so-called "hinge region." Like heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain: CL. Furthermore, the VH and VL regions can be further subdivided into regions of hypervariability, also called complementarity-determining regions (CDRs) (or hypervariable regions in which the sequence and / or shape of the structured loops may be hypervariable), interrupted by highly conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0021] As used herein, the terms "half molecule," "Fab arm," and "arm" refer to one heavy-light chain pair. When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was produced by recombining half molecules derived from each of the first and second antibodies by any known method to produce the resulting bispecific antibody. In this context, "recombining" is not intended to be limited by any particular recombination method and thus includes all of the methods for producing bispecific antibodies described herein below, including, for example, recombining by half molecule exchange, as well as recombining at the nucleic acid level and / or co-expressing two half molecules in the same cell.

[0022] As used herein, the term "antigen-binding region" or "binding region" refers to the region of an antibody that can bind to an antigen. An antigen can be any molecule, such as a polypeptide. An antigen can be presented, for example, on a cell, a bacterium, or a virus particle. The terms "antigen" and "target" may be used interchangeably in the present invention, unless the context indicates otherwise. The terms "antigen-binding region" and "antigen-binding site" may be used interchangeably in the present invention, unless the context indicates otherwise.

[0023] The terms "blocking binding" or "blocking antibody binding" or "cross-blocking binding" or "cross-blocking binding" refer to a situation in which one antibody bound to a specific antigen blocks the binding of a second antibody to the same antigen, and vice versa. In the absence of the other antibody, each antibody has the ability to bind to the antigen, as determined by a significant binding response, whereas in the presence of the other antibody, one of the antibodies lacks a binding response. The ability of one antibody to block the binding of another antibody can be determined by biolayer interferometry in a conventional sandwich epitope binning assay format, as described, for example, in Example 5 of the present application and by Abdiche et al. (Abdiche YN, Malashock DS, Pinkerton A, Pons J. Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal Biochem. 2009; 386(2): 172-180). Briefly, sandwich epitope binning assay examines the binding of an antibody in solution to its specific antigen that has been captured through immobilized antibody.In the present invention, if an antibody can bind to an antigen in the presence of a second antibody, the antibody will not interfere with the binding of the second antibody, and vice versa.The terms " interfere with binding " and " interfere with the binding of antibody " and " cross-blocking binding " and " cross-blocking binding " can be used interchangeably in the present invention, unless the context is inconsistent.An antibody that is described as interfering with the binding of another antibody can also be described as competing with other antibodies to obtain target binding.

[0024] "K D The term "(M)" as used herein refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, k d k a It is obtained by dividing by K D can also be referred to as "binding affinity."

[0025] "k d" (sec -1 The term k ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is known as k off Also called the off-value or off-rate.

[0026] "k a " (M -1 ×sec -1 The term k ) as used herein refers to the binding rate constant of a particular antibody-antigen interaction. This value is known as k on Also called value or on-rate.

[0027] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, typically as determined by biolayer interferometry using the antibody as the ligand and the antigen as the analyte. -6 M or less, e.g., 5E -7 M or less, 1E -7 M or less, e.g. 5E -8 M or less, e.g. 1E -8 M or less, e.g. 5E -9 M or less, or for example, 1E -9 M or less K D and a K that is at least 10-fold, e.g., at least 100-fold, e.g., at least 1,000-fold, e.g., at least 10,000-fold, e.g., at least 100,000-fold lower than the affinity of binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D It binds to a given antigen with an affinity equivalent to

[0028] As used herein, the term "B7H4" refers to the protein designated B7H4, which is also known as B7-H4, V-set domain-containing T-cell activation inhibitor 1, or VTCN1. B7H4 is a member of the B7 family of proteins, which includes cell surface protein ligands that bind to receptors on lymphocytes. B7H4 is a type I transmembrane protein containing a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with IgV-like and IgC-like domains, containing four conserved cysteine ​​residues and seven sites for N-linked glycosylation. (Sica et al., 2003, Immunity 18: 849-861). B7H4 proteins are known from various species, including human (Homo sapiens) B7H4 (Uniprot accession number Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7H4 transcript 1 (Uniprot accession number A0A2K5U6P5), dog (Canis familiaris) B7H4 (Uniprot accession number F1P8R9), rabbit (Lepus rufocanus) B7H4 (Uniprot accession number G1TQE8), rat (Rattus norvegicus) B7H4 (Uniprot accession number Q501W4), mouse (Mus musculus) B7H4 (Uniprot accession number Q7TSP5), and pig (Sus scrofa) B7H4 (Uniprot accession number F1SAY4). Naturally occurring variants of the listed B7H4 sequences may exist.

[0029] As used herein, the term "CD3" refers to the human cluster of differentiation 3 protein, which is part of the T cell coreceptor protein complex and is composed of four separate chains. CD3 is found in a variety of species, and therefore the term "CD3" may not be limited to human CD3 unless the context indicates otherwise. In mammals, the complex comprises a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot number P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot number Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot number P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot number Q95LI8), two CD3ε (epsilon) chains (human CD3ε: UniProtKB / Swiss-Prot number P07766, the sequence of which is incorporated herein as SEQ ID NO: 13, in which amino acid residues 1-22 correspond to the signal peptide and amino acid residues 23-207 correspond to the mature CD3ε polypeptide; cynomolgus monkey CD3ε These include the CD3ε (human CD3ζ UniProtKB / Swiss-Prot number Q95LI5; or rhesus monkey CD3ε UniProtKB / Swiss-Prot number G7NCB9), and the CD3ζ (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot number P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot number Q09TK0). These chains bind to a molecule known as the T cell receptor (TCR), generating an activation signal in T lymphocytes. Together, the TCR and CD3 molecules comprise the TCR complex.

[0030] 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 identified by epitope binning using biolayer interferometry, by alanine scanning, or by domain shuffling assays (using an antigen construct in which a region of the antigen has been exchanged with that of another species to determine whether the antibody still binds to the antigen). The amino acids in the antibody binding region that are involved in the interaction with the antibody can be identified by hydrogen / deuterium exchange mass spectrometry and / or crystal structure analysis of the antibody bound to the antigen.

[0031] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface collections of molecules such as amino acids, sugar side chains, or a combination thereof, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, e.g., amino acid residues that are substantially obstructed or covered by an antibody when it binds to an antigen (in other words, these amino acid residues are included in or closely adjacent to the binding zone of a specific antibody).

[0032] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," or "mAb" refer to a preparation of antibody molecules of a single molecular composition, typically exhibiting a single binding specificity and affinity for a particular epitope. Typically, monoclonal antibodies are produced using identical cells that are clones of only one parent cell, such as hybridomas or stable cell lines. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, with variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas containing B cells fused to immortalized cells, and the B cells can be obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, whose genomes contain human heavy and light chain transgenes. Human monoclonal antibodies can be derived from human B cells or plasma cells. Monoclonal antibodies can also be produced from recombinantly modified host cells or systems that use cell extracts to facilitate the in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0033] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)), encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or a lambda (λ) light chain.

[0034] As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent antibody or a variant antibody) comprising one pair of heavy and light chains or two different pairs of heavy and light chains, each pair comprising the heavy and light chain constant and variable domains normally found in the heavy-light chain pair of a wild-type antibody of that isotype. In a full-length variant antibody, the heavy and light chain constant and variable domains may contain amino acid substitutions that alter and / or improve the functional properties of the antibody, particularly compared to the full-length parent or wild-type antibody. Full-length antibodies according to the present invention can be produced by a method comprising (i) cloning CDR sequences into one or more suitable vectors containing the complete heavy and light chain sequences, and (ii) expressing the resulting suitable vectors carrying the heavy and light chain sequences in a suitable expression system. It is within the knowledge of one skilled in the art to produce full-length antibodies starting from either CDR sequences or complete variable region sequences. Thus, one skilled in the art will know how to generate full-length antibodies according to the present invention.

[0035] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology to the human variable domain. This can be achieved by grafting the non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto the cognate human acceptor framework regions (FRs) (see generally WO92 / 22653 and EP0629240). To fully restore the binding affinity and specificity of the parent antibody, it may be necessary to replace the human framework regions with framework residues from the parent antibody (i.e., non-human antibody) (backmutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, framework regions of primarily human origin, optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a constant region that is entirely human. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with preferred characteristics, such as particular useful affinity and biochemical properties, for example, to avoid deamidation, to provide an "inert Fc region", and / or to include modifications to improve manufacturability.

[0036] The term "human antibody," as used herein, is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and constant domains derived from human immunoglobulin constant domains. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). "Human antibodies" can incorporate VH and VL sequences generated from human germline immunoglobulin sequences, such as in humans, transgenic animals as described in the Examples herein, or HIS mice. Such VH and VL sequences are considered, for example, to be human VH and VL sequences fused to constant domains derived from human immunoglobulin constant domains.

[0037] Thus, a "human antibody" may be an engineered antibody. A "human antibody" may have been subjected to further manipulations, including, for example, modifications to avoid deamidation, to provide an "inert Fc region," to enable the generation of bispecific antibodies, and / or to improve manufacturability. Human antibodies may also be produced in non-human cells, such as CHO cells. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0038] As used herein, the term "Fc region" refers to a region comprising, from the N-terminus to the C-terminus, at least the hinge, CH2, and CH3 regions of the two heavy chains of an antibody. The Fc region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0039] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the EU numbering scheme 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 of any of the other subtypes described herein.

[0040] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the EU numbering scheme set forth in Kabat (ibid.). However, the CH1 region may also be of any of the other subtypes described herein.

[0041] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering scheme set forth in Kabat (ibid.). However, the CH2 region may also be of any of the other subtypes described herein.

[0042] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering scheme described in Kabat (ibid.). However, the CH3 region may also be of any of the other subtypes described herein.

[0043] The term "Fc-mediated effector function," as used herein, is intended to mean a function that results from the binding of a polypeptide or antibody to its target or antigen on a cell membrane, where the Fc-mediated effector function can be 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 cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-γ receptor (FcgR) binding, (vi) antibody-dependent FcγR-mediated antigen cross-linking, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement-dependent cellular cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) binding of an opsonized antibody to a complement receptor mediated by the antibody, (xi) opsonization, and (xii) any combination of (i)-(xi).

[0044] As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is at least incapable of binding to any FcγR, of inducing Fc-mediated FcγR cross-linking, or of inducing cross-linking of target antigens via FcγRs mediated by the two Fc regions of an individual antibody, or of binding to C1q. An example of this is the FEA substitution within the constant domain described herein. The inactivity of an antibody Fc region can be tested using an antibody in a monospecific or bispecific format.

[0045] The term "full length," when used in reference to an antibody, indicates that the antibody is not a fragment, but includes all of the domains corresponding to a particular isotype as normally found in nature for that isotype, e.g., the VH domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, VL domain, and CL domain in the case of an IgG1 antibody.

[0046] The term "monovalent antibody" as used herein refers to an antibody molecule that can interact with an antigen using only one antigen-binding domain (e.g., one Fab arm). In the context of bispecific antibodies, "monovalent antibody binding" refers to the binding of a bispecific antibody to one antigen using only one antigen-binding domain (e.g., one Fab arm).

[0047] The term "monospecific antibody" in the present invention means an antibody that has binding specificity for only one antigen, one epitope. The antibody may be a monospecific, monovalent antibody (i.e., having only one antigen-binding region) or a monospecific, bivalent antibody (e.g., having two identical antigen-binding regions).

[0048] The term "bispecific antibody" refers to an antibody having two antigen-binding domains that bind to different epitopes, such as two non-identical pairs of VH and VL regions, two non-identical Fab arms, or two Fab arms with non-identical CDR regions. In the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may be present on the same or different antigens or targets. When epitopes are present on different antigens, these antigens may be present on the surface of the same cell or different cells, cell types, or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, a bispecific antibody may be able to cross-link multiple antigens, such as two different cells.

[0049] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions that bind to the same two epitopes on the same antigen or to two different epitopes on the same or different antigens. Thus, a bivalent antibody can be a monospecific antibody or a bispecific antibody.

[0050] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) functional group and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the present invention, amino acids can be classified based on their structure and chemical characteristics. Thus, the classes of amino acids can be represented in one or both of the following tables:

[0051] Table 2. Major classifications based on the structure and general chemical characteristics of the R group. TIFF0007749575000008.tif52128

[0052] Table 3. Alternative physical and functional classification of amino acid residues TIFF0007749575000009.tif88128

[0053] Substitution of one amino acid for another can be classified as conservative substitution or non-conservative substitution. In the present invention, "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, for example, the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above: for example, leucine can be substituted with isoleucine, since both leucine and isoleucine are aliphatic branched hydrophobic substances. Similarly, aspartic acid can be substituted with glutamic acid, since both aspartic acid and glutamic acid are small negatively charged residues.

[0054] In the present invention, substitution in an antibody is represented as the original amino acid-position-substituted amino acid. In accordance with the well-recognized nomenclature for amino acids, three-letter or one-letter symbols, including the code "Xaa" or "X", are used to represent any amino acid residue. Thus, Xaa or X typically represents any of the 20 naturally occurring 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.

[0055] Thus, the symbols "K409R" or "Lys409Arg" mean that the antibody contains a substitution of lysine with arginine at amino acid position 409. Substitution of an amino acid at a given position with any other amino acid is referred to as the original amino acid position, or, for example, "K409." In cases where the original and / or replacement amino acids are modifications that may include multiple, but not all, amino acids, the multiple amino acids may be separated by "," or " / ." For example, a substitution of lysine at position 409 with arginine, alanine, or phenylalanine is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409 to R, A, or F." Such designations may be used synonymously in the present invention and may have the same meaning and purpose.

[0056] Furthermore, the term "substitution" encompasses substitution with any one of the natural amino acids or the other 19 natural amino acids, or with other amino acids, such as unnatural amino acids. For example, substitution of the amino acid K at position 409 includes 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. Alternatively, this is equivalent to the designation 409X, where X represents 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. By analogy, the same applies to each and every position mentioned herein, and any one of such substitutions specifically included herein.

[0057] Antibodies according to the present invention may also include deletions of amino acid residues. Such deletions may be designated as "del," including, for example, K409del. Thus, in such embodiments, the lysine at position 409 has been deleted from the amino acid sequence.

[0058] The term "host cell," as used herein, is intended to mean a cell into which a nucleic acid, such as an expression vector, has been introduced. It should be understood that such a term is not intended to refer only to the particular subject cell, but may also include the progeny of such a cell. Because some modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocyte cells, as well as prokaryotic cells such as Escherichia coli (E. coli), and other eukaryotic hosts such as plant cells and fungi.

[0059] The term "transfectoma," as used herein, includes recombinant eukaryotic host cells, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells, that express an antibody or target antigen.

[0060] For purposes of the present invention, sequence identity between two amino acid sequences is preferably determined over the entire length of the referenced sequence using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle, labeled "longest identity" (obtained using the -nobrief option), is used as the percent identity and is calculated as follows: (Identical residues × 100) / (total length of alignment − total number of gaps in alignment)

[0061] Retention of similar residues can also or alternatively be determined using a similarity score, which is determined using a BLAST program (e.g., BLAST 2.2.8 available from NCBI using standard settings BLOSUM62, open gap = 11, and extension gap = 1). Typically, suitable variants exhibit at least about 45% similarity to the parent or reference sequence, e.g., at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or even higher (e.g., about 99%).

[0062] As used herein, the term "internalized" or "internalization" refers to the biological process by which a molecule, such as an antibody according to the present invention, is enveloped by the cell membrane and introduced into the interior of the cell. Internalization may also be referred to as "endocytosis."

[0063] Bispecific antibodies targeting CD3×B7H4 In a first aspect of the present invention, an antibody is provided that comprises an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the antigen-binding region is a human variable region and / or a humanized variable region. For example, one antigen-binding region may comprise a human heavy chain variable region and a human light chain variable region, and the other antigen-binding region may comprise a humanized heavy chain variable region and a humanized light chain variable region. Alternatively, both antigen-binding regions may comprise a human heavy chain variable region and a human light chain variable region, or both antigen-binding regions may comprise a humanized heavy chain variable region and a humanized light chain variable region. Accordingly, an antibody is provided that comprises an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise human framework regions. An antibody according to the invention described herein that comprises an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3 may also be referred to herein as, for example, a B7H4xCD3 antibody.

[0064] Such antibody is preferably a bispecific antibody.In a further embodiment, such antibody as described above can bind to cancer cells and T cells, for example, as described in the Examples.Cancer cells that can be selected are cancer cells that express human B7H4 and / or cancer cells derived from solid tumors.Such antibody is preferably capable of inducing T cell-mediated cell death of cancer cells.

[0065] Being able to bind is understood to include, as shown in the Examples, that the antibody binds to its target in a binding assay, as shown, for example, by typical binding curves such as those shown in Figures 3 and 4 herein, or by determining binding affinity using, for example, bio-layer interferometry, as shown in Examples 3 and 4. An antigen-binding region that cannot bind to a designated target will, for example, have an undetectable binding affinity for that target, e.g., a response of less than 0.05 nm at the highest concentration used in a typical bio-layer interferometry assay such as that shown in Example 3. In any event, one of skill in the art will be familiar with how to determine whether an antigen-binding region is able to bind to its target.

[0066] Bispecific morphology The present invention provides bispecific CD3xB7H4 antibodies that efficiently promote T cell-mediated killing of B7H4-expressing tumor cells. A specific antigen-binding region can be selected from the range of antibodies or antigen-binding regions provided by the present invention depending on the functional properties desired for a particular application. Many different forms and uses of bispecific antibodies are known in the art and are reviewed in Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97. Bispecific antibodies according to the present invention may not be limited to any particular bispecific form or method of making them.

[0067] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody with two arms containing different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an added peptide linker; and (iii) a dual-variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains tandemly linked by a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM ) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) chemically linked bispecific (Fab') fragments; (v) Tandabs, which are fusions of two single-chain diabodies, resulting in tetravalent bispecific antibodies with two binding sites for each target antigen; (vi) Flexibodies, which are combinations of scFvs and diabodies, resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when added to Fab, can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which, for example, contain two scFvs fused to both ends of human Fab arms; and (ix) diabodies.

[0068] In one embodiment, the bispecific antibody of the invention is a diabody, crossbody, or bispecific antibody obtained by directed Fab arm exchange (as described in WO2011131746 (Genmab)).

[0069] Examples of different classes of bispecific antibodies include: (i) IgG-like molecules with complementary CH3 domains that force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, in which the two sides of the molecule each comprise an Fab fragment or a portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or stabilized diabody is fused to the constant domain of a heavy chain, an Fc region, or a portion thereof; and (v) recombinant IgG-like dual targeting molecules, in which the two sides of the molecule each comprise an Fab fragment or a portion of an Fab fragment of at least two different antibodies. and (vi) ScFv-based 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 carrier molecule fused to a heavy chain constant domain, Fc region, or portion thereof.

[0070] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called knob-into-hole molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically coupled molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body molecules and PIG body molecules (Pharmabcine, WO2010134666, WO2014081202), strand-exchange engineered domain body (SEEDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254), and DuoBody® molecules (Genmab A / S, WO2011131746).

[0071] Examples of recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al. 2009. Science 323, 1610-1614.), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506.)

[0072] Examples of IgG fusion molecules include dual variable domain (DVD)-Ig molecules (Abbott, US 7,612,181), dual-domain double-head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), as well as BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US 007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246), and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0073] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), dual affinity retargeting technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538), and dual (ScFv)2-Fab molecules (National Research Center of Antibody Medicine (China)).

[0074] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al. J. Immunol. 1998 Feb 15;160(4):1677-86), dual-action or bis-Fab molecules (Genentech, Bostrom, et al. 2009. Science 323, 1610-1614), dock-and-lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J. Immunol. 2000 Dec 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0075] Examples of ScFv-based antibodies, diabody-based antibodies, and domain antibodies include bispecific T cell-triggering (BiTE) molecules (Micromet, WO2005061547), tandem diabody molecules (TandAb) (Affimed Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), dual affinity retargeting technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual-targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual-targeting heavy chain-only domain antibodies.

[0076] The bispecific antibodies of the present invention can be of any isotype. Exemplary isotypes include, but are not limited to, any of the human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably, the bispecific antibodies can be selected to be of the human IgG1 isotype, as shown in the Examples. Any human light chain constant region, i.e., kappa or lambda, can be used. In one embodiment, both heavy chains of the antibodies of the present invention are of the IgG1 isotype, e.g., IgG1, κ. In one embodiment, the two heavy chains of the bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. Preferably, the bispecific antibodies can be selected to be of the human IgG1 isotype, as shown in the Examples.

[0077] Optionally, and preferably, the heavy chain and its Fc sequence of a selected isotype may be modified in the hinge and / or CH3 region as described herein to allow for the creation of bispecific antibodies and to introduce inactivation.

[0078] In one aspect, the bispecific antibody of the present invention comprises an Fc region comprising a first heavy chain having a first Fc sequence comprising a first CH3 region, and a second heavy chain having a second Fc sequence comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the respective homodimeric interactions between the first CH3 region and the second CH3 region. Further details regarding these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0079] As further described herein, stable bispecific CD3xB7H4 antibodies can be obtained in high yields based on one B7H4 antibody and one CD3 antibody, each composed of two identical heavy chains and two identical light chains, each containing only a few fairly conservative (asymmetric) mutations in their CH3 regions. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain one or more amino acid substitutions at positions that are not identical.

[0080] Antigen-binding region capable of binding to CD3 As described above, the present invention provides antibodies according to the present invention, which comprise an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3. The present invention further provides antibodies according to the present invention, which comprise an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region capable of binding to CD3 is capable of binding to human CD3ε(epsilon), for example, human CD3ε(epsilon) designated as SEQ ID NO: 13. Such antigen-binding regions are capable of binding to human CD3ε(epsilon) displayed on T cells, such as primary human T cells.

[0081] The antibody according to the present invention comprises an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 16 or SEQ ID NO: 17, and optionally A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22 The antibody may be an antibody comprising:

[0082] CDR1 region, CDR2 region and CDR3 region can be identified from the variable heavy chain region and the variable light chain region by using methods known in the art.The CDR regions derived from the variable heavy chain region and the variable light chain region can be annotated according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).Therefore, an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region binding to CD3 is: a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21, respectively; and optionally, A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively. Also disclosed is the antibody, comprising:

[0083] An antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 16, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 16; and optionally A light chain variable region (VL) comprising 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 to the sequence of SEQ ID NO: 22. Also disclosed is the antibody, comprising:

[0084] Such antigen-binding regions capable of binding to human CD3 are generally described in WO2015001085 and WO2017009442. Additional antigen-binding regions capable of binding to human CD3 are disclosed and described in WO2015001085 and WO2017009442, which are incorporated herein by reference, and can further be contemplated and serve as building blocks for generating antibodies according to the present invention.

[0085] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human CD3 and human CD3, which is in the range of 1 to 1000 nM. D And can be combined.

[0086] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human CD3 and human CD3 that is in the range of 1 to 100 nM, for example, 5 to 100 nM, 10 to 100 nM, 1 to 80 nM, 1 to 60 nM, 1 to 40 nM, 1 to 20 nM, 5 to 80 nM, 5 to 60 nM, 5 to 40 nM, 5 to 20 nM, 10 to 80 nM, 10 to 60 nM, 10 to 40 nM, or 10 to 20 nM. D Exemplary suitable antigen-binding regions include the heavy chain variable region (VH) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22. Such variable regions were generally described in WO2015001085.

[0087] In another aspect of the invention, the antibody has a lower binding affinity for human CD3ε than an antibody having an antigen-binding region comprising the VH sequence as set forth in SEQ ID NO: 16 and the VL sequence as set forth in SEQ ID NO: 22, preferably the affinity is at least 5-fold lower, such as at least 10-fold lower, such as at least 20-fold lower, at least 30-fold lower, at least 40-fold lower, at least 45-fold lower, or such as at least 50-fold lower.

[0088] In another aspect of the present invention, the antibody has an equilibrium dissociation constant K between the antigen-binding region that binds to human CD3 and the human CD3 antigen that is in the range of 200 to 1000 nM, for example, 300 to 1000 nM, 400 to 1000 nM, 500 to 1000 nM, 300 to 900 nM, 400 to 900 nM, 400 to 700 nM, 500 to 900 nM, 500 to 800 nM, 500 to 700 nM, 600 to 1000 nM, 600 to 900 nM, 600 to 800 nM, or for example, 600 to 700 nM. DExemplary suitable antigen-binding regions include the heavy chain variable region (VH) of SEQ ID NO: 16 or SEQ ID NO: 17 and the light chain variable region (VL) of SEQ ID NO: 22. Such variable regions were generally described in WO2017009442.

[0089] Binding affinity can be determined by bio-layer interferometry, as optionally described herein in Example 4. Thus, antibodies according to the invention, which have binding affinity for human CD3 as defined herein, may have their binding affinity determined using bio-layer interferometry, which comprises the following steps: I) immobilizing an antibody in an amount of 1 μg / mL on an anti-human IgG Fc capture biosensor for 600 seconds; II) determining the association over a period of 1000 seconds and the dissociation over a period of 2000 seconds of human recombinant soluble CD3ε (CD3E27-GSKa) (mature protein of SEQ ID NO: 13) using a 3-fold dilution series ranging from 1.40 nM to 1000 nM; III) Relating the data to a buffer control (0 nM).

[0090] Additionally, binding affinity may be determined using an antibody, such as a monospecific, bivalent antibody, for example, an antibody that is a full-length IgG1.

[0091] Thus, in a further embodiment, the antibody according to the invention comprises: the antigen-binding region that binds to CD3 comprises a heavy chain variable (VH) region as defined herein, comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein the heavy chain variable (VH) region has amino acid substitutions at positions selected from the group consisting of T31, N57, H101, G105, S110, and Y114, when compared to a heavy chain variable (VH) region comprising the sequence set forth in SEQ ID NO: 16, wherein these positions are numbered based on the sequence of SEQ ID NO: 16; and the wild-type light chain variable (VL) region comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively; It is an antibody.

[0092] Specifically, the antibody according to the present invention is an antibody whose antigen-binding region that binds to CD3 comprises a substitution in the heavy chain variable (VH) region as defined herein selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V.

[0093] Furthermore, the antibody according to the present invention is an antibody in which the antigen-binding region that binds to CD3 comprises a heavy chain variable region as defined herein, having an M or P at amino acid position 31, or an E at amino acid position 57, or a G or N at amino acid position 101, or a P at amino acid position 105, or an A or G at amino acid position 110, or an M, R, or V at amino acid position 114, which positions correspond to the amino acid position numbering of the heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 16.

[0094] Furthermore, the antibody according to the present invention is an antibody in which the CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 as defined herein contain a total of at most 1, 2, 3, 4, or 5 amino acid substitutions when compared to the CDR1, CDR2, and CDR3 of the sequence of SEQ ID NO: 16, and the amino acid substitutions preferably include the amino acid substitutions defined above.

[0095] Antigen-binding region capable of binding to B7H4 Specifically, the present invention provides an antibody according to the present invention, comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the human B7H4 is human B7H4 of SEQ ID NO: 1. Preferably, the antibody according to the present invention comprises an antigen-binding region capable of binding to human CD3ε (epsilon) designated in SEQ ID NO: 13 and an antigen-binding region capable of binding to human B7H4 of SEQ ID NO: 1.

[0096] Specifically, the antibody according to the present invention is an antibody in which the antigen-binding region capable of binding to human B7H4 is capable of binding to the extracellular domain of human B7H4. Preferably, B7H4 is expressed on cells, more preferably on human cells.

[0097] In a further embodiment, the antibody according to the present invention is an antibody in which the antigen-binding region capable of binding to human B7H4 is capable of binding to the IgC-like constant region of human B7H4. In another further embodiment, the antibody according to the present invention is an antibody in which the antigen-binding region capable of binding to human B7H4 is capable of binding to B7H3-IgV / B7H4-IgC. B7H3-IgV / B7H4-IgC refers to a fusion of human B7H3 and human B7H4 in which the B7H3 IgV-like domain is fused to the B7H4 IgC-like domain, and corresponds to SEQ ID NO: 11. B7H3-IgV / B7H4-IgC is expressed by cells as described herein in Example 7. In yet another further embodiment, the antibody according to the present invention is an antibody in which the antigen-binding region capable of binding to human B7H4 is incapable of binding to B7H4-IgV / B7H3-IgC. B7H4-IgV / B7H3-IgC refers to a fusion of human B7H3 and human B7H4 in which the B7H4 IgV-like domain is fused to the B7H3 IgC-like domain, which corresponds to SEQ ID NO: 10. B7H4-IgV / B7H3-IgC is expressed by cells as described herein in Example 7.

[0098] Suitable antigen-binding regions capable of binding to human B7H4 contemplated according to the present invention as described herein include: a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 36 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 40; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 43 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 47; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 50 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 54; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 31 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; g) A variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 65 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 69.

[0099] CDR1, CDR2 and CDR3 regions can be identified from the variable heavy chain region and the variable light chain region by methods known in the art.The CDR regions derived from the variable heavy chain region and the variable light chain region can be annotated according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).Therefore, as described herein, suitable antigen-binding regions that can bind to human B7H4 contemplated according to the present invention include: a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 41, DTS, and SEQ ID NO: 42, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 48, YTS, and SEQ ID NO: 49, respectively; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 55, GAS, and SEQ ID NO: 56, respectively; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; g) A variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 70, GAS, and SEQ ID NO: 71, respectively.

[0100] Yet another suitable antigen-binding region capable of binding to human B7H4 contemplated according to the present invention as described herein includes: a) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; c) the variable heavy chain (VH) region of SEQ ID NO: 36 and the variable light chain region of SEQ ID NO: 40; d) the variable heavy chain (VH) region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; e) the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; or f) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; g) The variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69.

[0101] Optionally, the antigen-binding region that binds to B7H4 comprises a heavy chain variable region and a light chain variable region (VH) that have at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the following sequences: a) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; c) the variable heavy chain (VH) region of SEQ ID NO: 36 and the variable light chain region of SEQ ID NO: 40; d) the variable heavy chain (VH) region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; e) the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; or f) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; g) The variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69.

[0102] The antibody according to the invention has a K of 5E-7M or less, for example 1E-7M or less. D The antigen-binding region may have a binding affinity for human B7H4 that corresponds to a K value, e.g., a K value within the range of 5E-7 to 2E-10 M, e.g., 2E-7 to 1E-10 M or 1E-7 to 5E-9 M. D Equivalent to the value.

[0103] Binding affinity can be determined by bio-layer interferometry, as optionally described herein in Example 3. Thus, antibodies according to the invention, which have binding affinity for human B7H4 as defined herein, can have their binding affinity determined using bio-layer interferometry, which comprises the following steps: I) immobilizing an antibody in an amount of 1 μg / mL on an anti-human IgG Fc capture biosensor for 600 seconds; II) determining the association over a 300-second period and the dissociation over a 1000-second period of human recombinant His-tagged B7H4 protein (Sino Biological catalogue number 10738-H08H; a protein with a C-terminal polyhistidine tag expressed from a construct of a DNA sequence encoding human VTCN1 (Uniprot accession number Q7Z7D3) (Phe29-Ala258)) using a two-fold dilution series ranging from 1.56 nM to 100 nM; III) Relating the data to a buffer control (0 nM).

[0104] Additionally, binding affinity may be determined using an antibody, such as a monospecific, bivalent antibody, for example, an antibody that is a full-length IgG1.

[0105] In a further aspect, there is provided an antibody according to the invention comprising an antigenic region capable of binding to human B7H4, wherein the antigen-binding region is An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 36 and a variable light chain region of SEQ ID NO: 40 can be cross-hindered, and The antigen-binding region comprises: an antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 47; An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; and An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 cannot be cross-hindered.

[0106] In yet another embodiment, the antibody according to the invention comprises an antigenic region capable of binding to human B7H4, The antigen-binding region is an antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 47; An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; and An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 can be cross-hindered, and The antigen-binding region comprises: An antibody comprising the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 36 and a variable light chain region of SEQ ID NO: 40 cannot cross-block antibodies containing

[0107] Specifically, "cross-blocking," or the ability of an antibody according to the invention to block the binding of another antibody to B7H4, is defined as the ability of a first antibody bound to B7H4 to block the binding of a second antibody to B7H4 bound to the first antibody. Cross-blocking can be determined using the assay described in Example 5. Such cross-blocking can also be determined, for example, by a procedure comprising the following steps: i) providing a set of samples, each sample comprising an antibody that binds to B7H4; ii) immobilizing a first antibody in an amount of 20 μg / mL from the set of samples on an amine-reactive second generation biosensor (AR2G) for 600 seconds; iii) adding human B7H4 (100 nM human recombinant His-tagged B7H4 protein (Sino Biological catalogue number 10738-H08H; a protein with a polyhistidine tag at its C-terminus, expressed from a construct of a DNA sequence encoding human VTCN1 (Uniprot accession number Q7Z7D3) (Phe29 to Ala258)) to the ARG2 biosensor with the immobilized antibody; iv) determining binding for a 10 μg / mL amount of second antibody from said set of samples for 300 seconds;

[0108] If the second antibody cannot bind, the first antibody is considered to cross-block the second antibody.Those skilled in the art will be familiar with suitable techniques for determining the ability of an antibody to cross-block the binding of another antibody to a target, and the present application discloses suitable procedures for determining binding blockage and displacement.In a further embodiment, the cross-blocking described herein is determined as described in Example 5.

[0109] In a further embodiment, an antibody according to the invention has an antigen-binding region capable of binding to human B7H4 with the aforementioned cross-blocking characteristics, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to B7H3-IgV / B7H4-IgC (SEQ ID NO: 11) and, optionally, is incapable of binding to B7H4-IgV / B7H3-IgC (SEQ ID NO: 10).

[0110] Combination of CD3 and B7H4 antigen-binding regions The present disclosure further provides an antibody according to the invention comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 16 and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22; and The antigen-binding region capable of binding to B7H4 is a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 36 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 40; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 43 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 47; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 50 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 54; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 31 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 65 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 69; Includes.

[0111] The present disclosure further provides an antibody according to the invention, which may be an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3; wherein the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 17 and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22; and The antigen-binding region capable of binding to B7H4 is a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 36 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 40; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 43 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 47; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 50 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 54; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 31 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 65 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 69; Includes.

[0112] The present disclosure also provides an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, Here, the antigen-binding region capable of binding to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively; and The antigen-binding region capable of binding to B7H4 is a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 41, DTS, and SEQ ID NO: 42, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 48, YTS, and SEQ ID NO: 49, respectively; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 55, GAS, and SEQ ID NO: 56, respectively; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 70, GAS, and SEQ ID NO: 71, respectively; Includes.

[0113] The present disclosure further provides an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, Here, the antigen-binding region capable of binding to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21, respectively; and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively; and The antigen-binding region capable of binding to B7H4 is a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 41, DTS, and SEQ ID NO: 42, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 48, YTS, and SEQ ID NO: 49, respectively; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 55, GAS, and SEQ ID NO: 56, respectively; or f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; g) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 70, GAS, and SEQ ID NO: 71, respectively; Includes.

[0114] Further disclosed is an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region that binds to CD3 comprises: a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 16 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 22; and The antigen-binding region capable of binding to B7H4 is a) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; c) the variable heavy chain (VH) region of SEQ ID NO: 36 and the variable light chain region of SEQ ID NO: 40; d) the variable heavy chain (VH) region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; e) the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; or f) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; g) the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 and a light chain variable region (VH) having the following antigen-binding region that binds to B7H4:

[0115] Also disclosed is an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 22; and The antigen-binding region capable of binding to B7H4 is a) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; c) the variable heavy chain (VH) region of SEQ ID NO: 36 and the variable light chain region of SEQ ID NO: 40; d) the variable heavy chain (VH) region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; e) the variable heavy chain (VH) region of SEQ ID NO: 50 and the variable light chain region of SEQ ID NO: 54; or f) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; g) the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 The antibody comprises an antigen-binding heavy chain variable region and a light chain variable region (VH) having the following structure:

[0116] In a further embodiment, in such a bispecific antibody, the antigen-binding regions capable of binding to human B7H4 are comprised in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a kappa light chain constant region; and the antigen-binding regions capable of binding to human CD3 are comprised in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a lambda light chain constant region. More preferably, in such a bispecific antibody, one IgG1 heavy chain constant region is as defined in SEQ ID NO: 60, and the other is as defined in SEQ ID NO: 61, the kappa light chain constant region is as defined in SEQ ID NO: 63, and the lambda light chain constant region is as defined in SEQ ID NO: 64. It will be understood that, optionally, the terminal lysines of the IgG1 heavy chain constant regions defined in SEQ ID NO: 60 and SEQ ID NO: 61 can be deleted.

[0117] As is well known to those skilled in the art, each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each variable region comprises three CDR sequences, i.e., CDR1, CDR2, and CDR3, respectively, and may comprise four framework sequences, i.e., FR1, FR2, FR3, and FR4, respectively. Each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each variable region comprises three CDR sequences, i.e., CDR1, CDR2, and CDR3, respectively, and may comprise four human framework sequences, i.e., FR1, FR2, FR3, and FR4, respectively. Preferably, this structure is also found in the antibodies of the present invention. Furthermore, the antibodies of the present invention may comprise two heavy chain constant regions (CH) and two light chain constant regions (CL). Examples of constant regions are generally provided in SEQ ID NOs: 57-64.

[0118] In a specific embodiment, the antibody according to the present invention comprises a first heavy chain and a second heavy chain, e.g., a first heavy chain and a second heavy chain, each comprising at least a hinge region, a CH2 region, and a CH3 region. Stable heterodimeric antibodies can be obtained in high yields based on two homodimeric starting proteins containing only a few asymmetric mutations in the CH3 region, for example, by so-called Fab arm exchange as provided in WO 2008 / 119353 and WO 2011 / 131746. Thus, in some embodiments of the present invention, an antibody comprises a first heavy chain having at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and a second heavy chain having at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, wherein the substitutions for the first and second heavy chains are not at the same position, and these amino acid positions are numbered according to EU numbering. For example, constant domains having such substitutions are generally provided in SEQ ID NO: 58 and SEQ ID NO: 62, and can be compared to SEQ ID NO: 57, which does not have such substitutions.

[0119] As used herein, the term "amino acid corresponding to a position" refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Unless otherwise stated or contradicted by context, amino acids in constant region sequences are numbered herein according to the EU index numbering system (as described in Kabat, EA et al., 1991, Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689). Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment when using standard sequence alignment programs such as ALIGN, ClustalW, or the like, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. Methods for aligning sequences or segments in a sequence and thereby determining the corresponding positions in the sequence for the amino acid positions according to the present invention are considered to be well known in the art.

[0120] In certain embodiments, the invention provides antibodies in which the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in the first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain, or vice versa.

[0121] In some embodiments, the antibody according to the present invention comprises, in addition to the antigen-binding region, an Fc region having two heavy chain Fc sequences. The first Fc sequence and the second Fc sequence may each be of any isotype, including any human isotype, for example, IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotype, or a mixed isotype. Preferably, the Fc region is of human IgG1, IgG2, IgG3, or IgG4 isotype, or a mixed isotype, for example, human IgG1 isotype. In some embodiments, the antibody according to the present invention is preferably a full-length antibody, and most preferably of the IgG1 type.

[0122] The antibodies of the present invention may contain modifications in the Fc region that render them inactive or non-activating. Thus, in the antibodies disclosed herein, one or both heavy chains can be modified so that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody containing unmodified first and second heavy chains. Fc-mediated effector function can be assessed by determining Fc-mediated CD69 expression in T cells (i.e., CD69 expression as a result of Fcγ receptor-dependent CD3 cross-linking via a CD3 antibody) by binding to Fcγ receptors, by binding to C1q, or by inducing Fc-mediated FcγR cross-linking. Specifically, the heavy chain constant sequence can 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 the Fc-mediated CD69 expression is determined in a functional assay using PBMCs, for example, as described in Example 3 of WO2015001085. Modification of the heavy and light chain constant sequences can also result in reduced C1q binding to the antibody. This reduction can be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the unmodified antibody, and C1q binding can be determined by ELISA. Additionally, the Fc region can be modified such that the antibody results in at least a 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% reduction in Fc-mediated T cell proliferation compared to the unmodified antibody, where T cell proliferation is determined in a functional assay using PBMCs.

[0123] A wide variety of non-activated antibody forms have been developed in which amino acid substitutions and combinations thereof have been introduced into the constant heavy chain region of IgG1 isotype antibodies to eliminate Fc-mediated effector functions (e.g., Chiu et al., Antibodies 2019 Dec; 8(4): 55; Liu et al., Antibodies, 2020 Nov 17; 9(4): 64; 29(10): 457-66; Shields et al., J Biol Chem,. 2001 Mar 2; 276(9): 6591-604).

[0124] For example, examples of amino acid positions that can be modified in an IgG1 isotype antibody include positions L234 and L235. Thus, an antibody according to the invention may comprise a first heavy chain and a second heavy chain in which the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively. It will be understood that in addition to modifying amino acid positions L234 and L235, further positions may be modified.

[0125] Furthermore, the D265A amino acid substitution can reduce binding to all Fcγ receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Thus, an antibody according to the present invention may comprise a first heavy chain and a second 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 first and second heavy chains. A further aspect of the present invention provides an antibody in which the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain in at least one, for example both, of the first and second heavy chains are F, E, and A, respectively. In the present application, antibodies having the combination of the three amino acid substitutions L234F, L235E, and D265A, and additionally the K409R or F405L mutations disclosed herein above, may be referred to with the suffix "FEAR" or "FEAL," respectively.

[0126] The amino acid sequence of a wild-type IgG1 heavy chain constant region is identified herein as SEQ ID NO: 57. Consistent with the above-disclosed embodiments, an antibody of the invention may comprise an IgG1 heavy chain constant region with an F405L substitution and may have the amino acid sequence set forth in SEQ ID NO: 58 and / or an IgG1 heavy chain constant region with a K409R substitution and may have the amino acid sequence set forth in SEQ ID NO: 62.

[0127] The amino acid sequence of an IgG1 heavy chain constant region having L234F, L235E, and D265A substitutions is identified herein as SEQ ID NO: 59. The amino acid sequence of an IgG1 heavy chain constant region having L234F, L235E, D265A, and F405L substitutions is identified herein as SEQ ID NO: 60. The amino acid sequence of an IgG1 heavy chain constant region having L234F, L235E, D265A, and K409R substitutions is identified herein as SEQ ID NO: 61.

[0128] The constant region sequences listed in SEQ ID NOs: 57-62 contain a terminal lysine (K), and such sequences were used in the Examples section of this specification. The origin of this lysine is the native sequence found in humans, from which these Fc regions are derived. During recombinant antibody production in cell culture, this terminal lysine can be proteolytically cleaved by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For antibody manufacturing purposes, the DNA encoding this terminal lysine can be removed from the sequence to produce a lysine-free antibody. For example, when using antibodies produced in a CHO-based production system, there is typically a high degree of processing of the terminal lysine, so antibodies produced from nucleic acid sequences that either do or do not encode the terminal lysine are substantially identical in terms of sequence and function (Dick, LW et al. Biotechnol. Bioeng. 2008;100:1132-1143). It is therefore understood that antibodies according to the invention can be made that do not encode or have a terminal lysine as recited herein, and therefore, for manufacturing purposes, antibodies can be made that do not have a terminal lysine.

[0129] The present invention provides a) the antigen-binding region capable of binding to B7H4 is of human origin; and b) the antigen-binding region capable of binding to CD3 is humanized; Antibodies are also provided.

[0130] The present invention also provides a) the antigen-binding region capable of binding to B7H4 is of human origin; and / or The antigen-binding region capable of binding to CD3 is humanized. Antibodies are also provided.

[0131] In some embodiments of the invention, the antibody comprises a kappa (κ) light chain. In certain embodiments of the invention, the sequences relate to bispecific antibodies, and the kappa light chain comprises the CDR1, CDR2, and CDR3 sequences of the B7H4 antibody light chain disclosed above.

[0132] In a further embodiment of the invention, the antibody of any one of the preceding claims comprises a lambda (λ) light chain. In a particular embodiment of the invention relating to bispecific antibodies, the lambda light chain comprises the CDR1, CDR2, and CDR3 sequences of a CD3 antibody light chain disclosed above, in particular the CDR1, CDR2, and CDR3 sequences of a CD3 antibody with reduced affinity for CD3 disclosed above. The amino acid sequence of the kappa light chain constant region is included herein as SEQ ID NO: 63, and the amino acid sequence of the lambda light chain constant region is included herein as SEQ ID NO: 64.

[0133] In certain embodiments, the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain, e.g., an antibody having a heavy chain and a lambda light chain that includes a binding region capable of binding to CD3, and a heavy chain and a kappa light chain that includes a binding region capable of binding to B7H4.

[0134] Thus, in a further embodiment, in the bispecific antibody defined herein, the antigen-binding regions capable of binding to human B7H4 are comprised in the heavy and light chains, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a kappa light chain constant region; and the antigen-binding regions capable of binding to human CD3 are comprised in the heavy and light chains, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a lambda light chain constant region. More preferably, in said bispecific antibody, one IgG1 heavy chain constant region is as defined in SEQ ID NO: 60, and the other is as defined in SEQ ID NO: 61, the kappa light chain constant region is as defined in SEQ ID NO: 63, and the lambda light chain constant region is as defined in SEQ ID NO: 64. It is understood that the IgG1 heavy chain constant regions defined in SEQ ID NO: 60 and SEQ ID NO: 61 may lack terminal lysines.

[0135] Binding, cytotoxicity, and T cell activation Antibodies described herein, e.g., bispecific antibodies, capable of binding to human CD3 and human B7H4, can advantageously target T cells to human B7H4-expressing cancer cells, thereby inducing T cell-mediated cancer cell killing. As shown in the Examples section, having reduced or inactive Fc functionality in such antibodies allows for the administration of safe, effective, and potent antibodies to human patients, while still being effective against a wide range of cancers with varying levels of B7H4 expression.

[0136] As mentioned above, preferably, the antibody according to the present invention lacks or has reduced Fc-mediated effector function, and further, the antibody a) capable of binding to B7H4-expressing human tumor cells, as described in Examples 9 and 10 herein; b) can mediate concentration-dependent cytotoxicity against B7H4-expressing human tumor cells, e.g., when purified PBMCs or T cells are used as effector cells, as assayed as described in Examples 11 and 12 herein; c) can mediate concentration-dependent cytotoxicity of one or more human B7H4-expressing tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650 when assayed as described in Examples 11 and 12 herein, e.g., using purified PBMCs or T cells as effector cells; d) T cells can be activated in vitro in the presence of B7H4-expressing human tumor cells, for example, when assayed as described in Example 13 herein; e) T cells can be activated in vitro in the presence of one or more B7H4-expressing human tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650, for example, when analyzed as described in Example 13 herein; f) capable of inducing cytotoxicity of B7H4-expressing human tumor cells, for example, when assayed as described in Examples 11 and 12 herein; and / or g) capable of inducing T cell-mediated cytotoxicity in one or more B7H4-expressing human tumor cell lines selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650, when assayed, for example, as described in Examples 11 and 12 herein.

[0137] Furthermore, antibodies according to the present invention may lack or have reduced Fc-mediated effector function and may further induce T cell-mediated cytotoxicity, wherein cytotoxicity is assessed in an in vitro IC50 assay comprising the following steps: i) providing peripheral blood mononuclear cells (PBMCs) or purified T cells isolated from the buffy coat of a healthy human donor; ii) providing a B7H4-expressing tumor cell, e.g., a human B7H4-expressing tumor cell line, selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650; iii) mixing PBMCs or purified T cells with a plurality of samples of B7H4-expressing tumor cells, wherein the ratio of the number of T cells derived from the PBMCs or the purified T cells to the selected tumor cells is 8:1; iv) providing the sample with a dilution series of antibodies, for example, ranging from 0.0128 ng / mL to 10,000 ng / mL, for selected human B7H4-expressing tumor cells; and v) incubating the sample obtained in step iv) for example at 37°C for 72 hours; and subsequently vi) assessing the viability of B7H4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample; and viii) Determining IC50.

[0138] Instead of isolated peripheral blood mononuclear cells (PBMCs), purified T cells may be provided in step i).

[0139] Thus, the antibody may have an IC50 in the range of 0.001 to 2 μg / ml, where the IC50 is determined in an in vitro cytotoxicity assay comprising the following steps: i) providing peripheral blood mononuclear cells (PBMCs) isolated from the buffy coat of a healthy human donor; ii) providing a B7H4-expressing tumor cell, e.g., a human B7H4-expressing tumor cell line, selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, and HCC1954; iii) mixing PBMCs with multiple samples of B7H4-expressing tumor cells, wherein the ratio of the number of T cells derived from the PBMCs to the selected tumor cells is 8:1; iv) providing the sample with a dilution series of antibodies, for example, ranging from 0.0128 ng / mL to 10,000 ng / mL, for selected human B7H4-expressing tumor cells; and v) incubating the sample obtained in step iv) for example at 37°C for 72 hours; and subsequently vi) assessing the viability of B7H4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample; and viii) Determining IC50.

[0140] Thus, the antibody may have an IC50 in the range of 0.001 to 5 μg / ml, where the IC50 is determined in an in vitro cytotoxicity assay comprising the following steps: i) providing peripheral blood mononuclear cells (PBMCs) or purified T cells isolated from the buffy coat of a healthy human donor; ii) providing a B7H4-expressing tumor cell, e.g., a human B7H4-expressing tumor cell line, selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650; iii) mixing PBMCs or purified T cells with a plurality of samples of B7H4-expressing tumor cells, wherein the ratio of the number of T cells derived from the PBMCs or the purified T cells to the selected tumor cells is 8:1; iv) providing the sample with a dilution series of antibodies, for example, ranging from 0.0128 ng / mL to 10,000 ng / mL, for selected human B7H4-expressing tumor cells; and v) incubating the sample obtained in step iv) for example at 37°C for 72 hours; and subsequently vi) assessing the viability of B7H4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample; and viii) Determining IC50.

[0141] In one embodiment, the antibody according to the present invention may have an IC50 in the range of 0.001 to 5 μg / ml. In one embodiment, the antibody according to the present invention may have an IC50 in the range of 0.001 to 2 μg / ml. In another embodiment, the antibody according to the present invention may have an IC50 in the range of 0.001 to 0.03 μg / ml. In yet another embodiment, the IC50 may be in the range of 0.05 to 2 μg / ml. In yet another further embodiment, the IC50 may be in the range of 0.05 to 5 μg / ml. The IC50 can be determined using a method such as that described in Example 12.

[0142] In a further embodiment, the ability of an antibody according to the invention to mediate T cell activation is determined in an in vitro assay comprising the following steps: i) providing peripheral blood mononuclear cells (PBMCs) isolated from the buffy coat of a healthy human donor; ii) providing B7H4-expressing tumor cells; iii) mixing PBMCs and B7H4-expressing tumor cells in a plurality of samples, wherein the ratio of the number of PBMCs to tumor cells is 8:1; iv) applying a dilution series of antibody, for example ranging from 0.0128 ng / mL to 10,000 ng / mL, to the sample; and v) incubating the sample, for example, at 37° C. for 72 hours; and vi) Subsequently detecting cytokines.

[0143] For example, an exemplary cytokine that may be detected is IFN-γ, e.g., as described in Example 13. Preferably, the B7H4-expressing tumor cell is a human B7H4-expressing tumor, e.g., a primary tumor or a tumor cell line selected from the group consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, and HCC1954.

[0144] B7H4 antibody In another embodiment, an antibody is provided comprising an antigen-binding region capable of binding to human B7H4, wherein the antigen-binding region capable of binding to human B7H4 comprises: a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 31 and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; e) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; f) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; g) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; or h) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; i) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; j) having a heavy chain (VH) variable region and a light chain (VH) variable region that have at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33, respectively.

[0145] Such antibodies do not necessarily contain an antigen-binding region that binds to CD3. Such antibodies may be useful, for example, in kits and assays for detecting B7H4. Such antibodies may also be useful in cancer treatment. Therefore, such antibodies may be monospecific antibodies that bind to B7H4. Such antibodies may be bivalent antibodies.

[0146] Preferably, such antibodies comprise a heavy chain constant region that is a human IgG1 constant region, for example, a heavy chain constant region listed in SEQ ID NOs: 57 to 62. A preferred light chain constant region is a kappa light chain, for example, a light chain constant region listed in SEQ ID NO: 63.

[0147] In one embodiment, the antibodies provided herein can bind to an epitope or antibody binding region on human B7H4 that includes one or more of amino acid residues S151, V157, D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242, and I245; the numbering of each amino acid residue refers to its position in SEQ ID NO: 1. In a further embodiment, the antibodies provided herein can bind to an epitope or antibody binding region on human B7H4 that includes one or more of amino acid residues V157, D158, Y159, E164, L166; the numbering of each amino acid residue refers to its position in SEQ ID NO: 1.

[0148] In another embodiment, the antibodies provided herein can bind to an epitope or antibody binding region on human B7H4 comprising amino acid residues S151, V157, D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242, and I245; the numbering of each amino acid residue refers to its position in SEQ ID NO: 1. In a further embodiment, the antibodies provided herein can bind to an epitope or antibody binding region on human B7H4 comprising amino acid residues V157, D158, Y159, E164, L166; the numbering of each amino acid residue refers to its position in SEQ ID NO: 1.

[0149] Based on the results provided in Example 7 herein, and without wishing to be bound by theory, it is hypothesized that any one or more of these amino acid residues (i.e., S151, V157, D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242, and I245) are directly involved in antibody binding, such as through non-covalent interactions with amino acid residues within the CDR sequences of the antibody.

[0150] The amino acid residues encompassed by the epitope or antibody binding region, and optionally one or more additional amino acid residues indirectly involved in binding, can be identified by alanine scanning of human B7H4 having the amino acid sequence shown in SEQ ID NO: 1 or the extracellular domain sequence of SEQ ID NO: 1. Alanine scanning can specifically be performed as described or essentially as described in Example 7 herein.

[0151] Furthermore, alanine scanning can be performed by a procedure comprising the following steps: i) expressing a mutant human B7H4 polypeptide in which amino acid residues other than cysteine ​​and alanine in the extracellular domain of human B7H4 are individually substituted with alanine, and the corresponding wild-type B7H4 polypeptide, in human embryonic kidney cells, e.g., HEK293 cells, so as to provide a sample containing 40 to 60,000 cells, e.g., 50,000 cells, for each of the mutant B7H4 and the wild-type B7H4; ii) incubating the cells in each sample with 20 μl of antibody, where the antibody consists of one heavy chain and one light chain and is labeled with a label appropriate for flow cytometry analysis, such as, for example, mNeogreen label, for 1 hour at room temperature; followed by washing with FACS buffer (e.g., phosphate-buffered saline [PBS; Lonza, catalog number BE17-517] + 0.1% [w / v] BSA [Roche, catalog number 10735086001] + 0.02% [w / v] sodium azide [NaN3; EMELCA Bioscience, catalog number 41920044-3]); and resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, determining the average amount of bound antibody per cell as the geometric mean fluorescence intensity (gMFI) of the viable single-cell population in that sample, and normalizing the data for each test antibody to the binding intensity of a non-cross-blocking B7H4-specific reference antibody using the formula: TIFF0007749575000010.tif9128In the formula, "aa position" refers to the position mutated to alanine, To represent the decrease or increase in antibody binding, a fold change or Z-score is calculated based on the following formula: TIFF0007749575000011.tif10128Here, amino acid positions where substitution of the amino acid with alanine results in neither a decrease nor an increase in binding by a particular antibody were given a result of "0", an increase in binding resulted in ">0", a decrease in binding resulted in "<0", and only B7H4 amino acid residues where the fold change in binding was less than the mean fold change -1.5 x SD (SD is the standard deviation of the fold change calculated from four independent experiments for a particular test antibody) were considered "reduced binding mutants", and data were excluded from analysis if the gMFI of the reference antibody for a particular B7H4 mutant was less than the mean gMFI -2.5 x (SD of the mean gMFI of the control Ab).

[0152] Furthermore, such antibodies may also be bispecific antibodies, comprising an antigen-binding region capable of binding to B7H4 as well as another antigen-binding region. Such another antigen-binding region may be an antigen-binding region capable of binding to human CD3. Such an antigen-binding region capable of binding to human CD3 may be the antigen-binding region capable of binding to CD3 described and disclosed herein.

[0153] In a further embodiment, in such a bispecific antibody, the antigen-binding regions capable of binding to human B7H4 are comprised in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a kappa light chain constant region; and the antigen-binding regions capable of binding to human CD3 are comprised in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a lambda light chain constant region. More preferably, in such a bispecific antibody, one IgG1 heavy chain constant region is as defined in SEQ ID NO: 60, and the other is as defined in SEQ ID NO: 61, the kappa light chain constant region is as defined in SEQ ID NO: 63, and the lambda light chain constant region is as defined in SEQ ID NO: 64. It will be understood that, optionally, the terminal lysines of the IgG1 heavy chain constant regions defined in SEQ ID NO: 60 and SEQ ID NO: 61 can be deleted.

[0154] A highly preferred bispecific antibody according to the invention is as described and used in the Examples section and is called BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR.

[0155] Thus, in a preferred embodiment, a bispecific antibody capable of binding to human CD3 and human B7H4 is provided, comprising: - a first heavy chain and a first light chain comprising a binding region capable of binding to human CD3, wherein the first heavy chain comprises a heavy chain variable region defined by SEQ ID NO: 17 and a human IgG1 heavy chain constant region as defined herein, and the first light chain comprises a light chain variable region defined by SEQ ID NO: 22 and a human lambda light chain constant region; and - a second heavy chain and a second light chain comprising a binding region capable of binding to human B7H4, wherein the second heavy chain comprises a heavy chain variable region defined by SEQ ID NO: 29 and a human IgG1 heavy chain constant region defined herein, and the second light chain comprises a light chain variable region defined by SEQ ID NO: 33 and a human kappa light chain constant region.

[0156] It is understood that the human IgG1 heavy chain constant region as defined herein may include substitutions as defined herein (e.g., FEAR / FEAL), etc. It is also understood that the human IgG1 heavy chain constant region may lack the terminal lysine (K).

[0157] In a further preferred embodiment, a bispecific antibody capable of binding to human CD3 and human B7H4 is provided, comprising: - a first heavy chain and a first light chain comprising a binding region capable of binding to human CD3, wherein the first heavy chain comprises a heavy chain variable region defined by SEQ ID NO: 17 and a heavy chain constant region defined by SEQ ID NO: 60, and the first light chain comprises a light chain variable region defined by SEQ ID NO: 22 and a light chain constant region defined by SEQ ID NO: 64; and - a second heavy chain and a second light chain comprising a binding region capable of binding to human B7H4, wherein the second heavy chain comprises a heavy chain variable region defined by SEQ ID NO: 29 and a heavy chain constant region defined by SEQ ID NO: 61, and the second light chain comprises a light chain variable region defined by SEQ ID NO: 33 and a light chain constant region defined by SEQ ID NO: 63.

[0158] Similarly, it is understood that the human IgG1 heavy chain constant region may lack the terminal lysine (K).

[0159] In yet another further preferred embodiment, a bispecific antibody capable of binding to human CD3 and human B7H4 is provided, comprising: - a first heavy chain and a first light chain comprising a binding region capable of binding to human CD3, wherein the first heavy chain consists of a heavy chain variable region defined by SEQ ID NO: 17 and a heavy chain constant region defined by SEQ ID NO: 60, and the first light chain consists of a light chain variable region defined by SEQ ID NO: 22 and a light chain constant region defined by SEQ ID NO: 64; and - a second heavy chain and a second light chain comprising an antigen-binding region capable of binding to human B7H4, wherein the second heavy chain consists of a heavy chain variable region defined by SEQ ID NO: 29 and a heavy chain constant region defined by SEQ ID NO: 61, and the second light chain consists of a light chain variable region defined by SEQ ID NO: 33 and a light chain constant region defined by SEQ ID NO: 63.

[0160] In another further preferred embodiment, a bispecific antibody capable of binding to human CD3 and human B7H4 is provided, comprising: - a first heavy chain and a first light chain comprising a binding region capable of binding to human CD3, wherein the first heavy chain consists of a heavy chain variable region defined by SEQ ID NO: 17 and a heavy chain constant region defined by SEQ ID NO: 60, wherein the terminal lysine (K) is deleted, and the first light chain consists of a light chain variable region defined by SEQ ID NO: 22 and a light chain constant region defined by SEQ ID NO: 64; and - a second heavy chain and a second light chain comprising a binding region capable of binding to human B7H4, wherein the second heavy chain consists of a heavy chain variable region defined by SEQ ID NO: 29 and a heavy chain constant region defined by SEQ ID NO: 61, wherein the heavy chain lacks a terminal lysine (K), and the second light chain consists of a light chain variable region defined by SEQ ID NO: 33 and a light chain constant region defined by SEQ ID NO: 63.

[0161] Methods for preparing bispecific antibodies Conventional methods, such as hybrid hybridoma technology and chemical ligation (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare the bispecific antibodies of the invention. Co-expression of two antibodies consisting of different heavy and light chains in a host cell produces a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or similar methods.

[0162] Strategies that promote the formation of functional bispecific products when various antibody constructs are coexpressed can also be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to species-restricted preferential heavy / light chain pairing. Another strategy that promotes heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which a protuberance is introduced into the surface of a first heavy chain polypeptide and a corresponding depression is introduced into a second heavy chain polypeptide, so that the protuberance fits into the depression at the interface between the two heavy chains, promoting heterodimer formation and preventing homodimer formation. The "protuberance" is created by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain. By replacing large amino acid side chains with smaller ones, a compensatory "dimple" of the same or similar size as the protuberance is created at the interface of the second polypeptide (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies for promoting heterodimer formation when different antibody domains are co-expressed in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are substituted with charged amino acids, resulting in electrostatically unfavorable homodimer formation and favorable heterodimerization. WO2007110205 (Merck) describes yet another strategy that exploits the differences in the CH3 domains of IgA and IgG to promote heterodimerization.

[0163] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (swapping of light chains attached to heavy chains) between two monospecific IgG4 antibodies or IgG4-like antibodies upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences from each other.

[0164] Preferred methods for preparing the bispecific CD3xB7H4 antibodies of the present invention include those described in WO2011131746 and WO13060867 (Genmab), which comprise the following steps: a) providing a first antibody comprising an Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region comprising a second CH3 region; wherein said first antibody is a CD3 antibody and said second antibody is a B7H4 antibody, or vice versa; the sequences of the first CH3 region and the second CH3 region are different from each other, such that the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than each homodimer interaction between the first CH3 region and the second CH3 region; c) incubating the first antibody with the second antibody under reducing conditions; and d) Obtaining a bispecific CD3xB7H4 antibody.

[0165] In one embodiment, the first antibody is incubated with the second antibody under reducing conditions sufficient to cause disulfide bond isomerization at the cysteines in the hinge region, wherein the heterodimeric interaction between the first and second antibodies in the resulting heterodimeric antibody is such that no Fab arm exchange occurs at 0.5 mM GSH after 24 hours at 37°C.

[0166] Without being limited by theory, in step c), heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteines can then form inter-heavy chain disulfide bonds with cysteine ​​residues of another parent antibody molecule (originally with different specificity). In one embodiment of this method, the reducing conditions of step c) comprise the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to non-reducing or less reducing by, for example, removing the reducing agent by desalting, etc.

[0167] For this method, any of the CD3 and B7H4 antibodies described herein can be used. In certain embodiments, the CD3 and B7H4 antibodies can each be selected to obtain a bispecific CD3xB7H4 antibody described herein.

[0168] In one embodiment of this method, the first antibody and / or the second antibody is a full-length antibody.

[0169] The Fc regions of the first and second antibodies may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of these antibodies is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains an IgG1 Fc region and an IgG4 Fc region and may therefore have interesting intermediate properties with respect to activation of effector function.

[0170] In a further embodiment, one of the antibody starting proteins is engineered not to bind to Protein A, thus allowing the heterodimeric protein to be separated from the homodimeric starting protein by passing the product over a Protein A column.

[0171] As mentioned above, the sequences of the first CH3 region and the second CH3 region of the starting antibody homodimer are different, and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region. More details about these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), the entire contents of which are incorporated herein by reference.

[0172] Specifically, stable bispecific CD3xB7H4 antibodies can be obtained in high yield using the above-described method of the present invention based on two homodimeric starting antibodies that bind to CD3 and B7H4, respectively, and that contain only a few, fairly conservative, asymmetric mutations in their CH3 regions, meaning that the sequences of the first and second CH3 regions contain amino acid substitutions at positions that are not identical.

[0173] The bispecific antibodies of the present invention can also be obtained by co-expressing constructs encoding the first and second polypeptides in a single cell.

[0174] Thus, in a further aspect, the present invention relates to a method for producing a bispecific antibody, said method comprising the steps of: a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc region comprising a first CH3 region and a first antigen-binding region of a first antibody heavy chain; b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region comprising a second CH3 region and a second antigen-binding region of a second antibody heavy chain; wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than each homodimeric interaction between the first CH3 region and the second CH3 region, and wherein the first homodimeric protein has an amino acid other than Lys, Leu, and Met at position 409, and the second homodimeric protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407; Optionally, the first nucleic acid construct and the second nucleic acid construct encode the light chain sequences of a first antibody and a second antibody. c) co-expressing the first nucleic acid construct and the second nucleic acid construct in a host cell; and d) Obtaining the heterodimeric protein from the cell culture.

[0175] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells producing the bispecific antibodies of the present invention.

[0176] Suitable expression vectors containing promoters, enhancers, etc., and suitable host cells for producing antibodies are well known in the art. Examples of host cells include yeast cells, bacterial cells, and mammalian cells, such as CHO cells or HEK cells.

[0177] In embodiments, there is provided a method for producing an antibody according to the invention capable of binding to both B7H4 and CD3, the method comprising the steps of: a) providing an antibody capable of binding to B7H4, wherein the antibody comprises an antigen-binding region capable of binding to B7H4 as defined herein; b) providing an antibody capable of binding to CD3, wherein the antibody comprises an antigen-binding region capable of binding to CD3 as defined herein; c) incubating the antibody capable of binding to B7H4 with the antibody capable of binding to CD3 under reducing conditions sufficient to cause disulfide bond isomerization at cysteines in the hinge region; and d) Obtaining antibodies capable of binding to B7H4 and CD3.

[0178] In such methods, the step of providing an antibody capable of binding to B7H4 and / or CD3 may include the steps of: - providing a cell comprising an expression vector for producing one or more of said antibodies; and - causing said cells to produce said antibody or antibodies; and subsequently - Obtaining one or more of said antibodies, thereby providing one or more of said antibodies.

[0179] The present invention further provides: a) a nucleic acid sequence encoding a heavy chain sequence of an antigen-binding region capable of binding to B7H4 as defined herein, and / or b) A nucleic acid sequence encoding the corresponding light chain sequence of said antigen-binding region capable of binding to B7H4.

[0180] Additionally, the present invention provides one or more nucleic acids comprising: a) a nucleic acid sequence encoding a heavy chain sequence of an antigen-binding region capable of binding to B7H4 as defined herein; b) a nucleic acid sequence encoding the corresponding light chain sequence of said antigen-binding region capable of binding to B7H4; c) a nucleic acid sequence encoding a heavy chain sequence of an antigen-binding region capable of binding to CD3 as defined herein; and d) A nucleic acid sequence encoding the corresponding light chain sequence of said antigen-binding region capable of binding to CD3.

[0181] The nucleic acid or one or more nucleic acids defined herein can be RNA or DNA.The nucleic acid or one or more nucleic acids defined herein can be used for expression in mammalian cells.Therefore, the present invention further provides one or more cells that contain nucleic acid or one or more nucleic acids defined herein.

[0182] The nucleic acid of the present invention may be an expression vector, which may be any suitable vector (a nucleic acid sequence comprising an appropriate set of expression control elements), including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the B7H4 or CD3 antibody is expressed in, for example, a naked DNA or RNA vector comprising a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compact nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119, the minimal size nucleic acid vector "Midge" (e.g., as described in Schakowski et al., Mol Ther 3, 793 800 (2001)), or in a precipitated nucleic acid vector construct, such as a CaP04 precipitated construct (e.g., as described in WO200046147; Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0183] In one embodiment, the vector is suitable for expressing B7H4 and / or CD3 antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison WI).

[0184] Additionally or alternatively, the expression vector may be a vector suitable for expression in yeast system. Any vector suitable for expression in yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0185] The nucleic acid and / or expression vector may also contain a nucleic acid sequence encoding a secretion / localization sequence capable of directing a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides. The nucleic acid and / or expression vector may contain any suitable elements that facilitate expression of the nucleic acid, i.e., transcription and / or translation, such that components of the (bispecific) antibody are expressed. The nucleic acid and / or vector may be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer, as well as the RSV promoter, SV40 promoter, SL33 promoter, MMTV promoter, and HIV LTR promoter), an effective poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0186] In one embodiment, an expression vector encoding a B7H4 antibody and / or a CD3 antibody may be placed in a cell and / or delivered to a cell. Thus, in a further aspect, the present invention relates to a host cell comprising a nucleic acid or vector as defined herein. The cell may be of human origin, for example, a human embryonic kidney (HEK) cell such as HEK / Expi cell, or of rodent origin, for example, a Chinese hamster ovary cell such as CHO / N50 cell.

[0187] Composition and (medical) uses Furthermore, the present invention also provides compositions comprising the antibodies defined herein. Preferably, such compositions are pharmaceutical compositions, i.e., the antibodies are contained in a pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention may comprise a bispecific antibody of the present invention that targets both B7H4 and CD3. Pharmaceutical compositions may also comprise an antibody that targets B7H4. Pharmaceutical compositions may also comprise a combination of antibodies, including an antibody that targets B7H4 and / or a bispecific antibody according to the present invention.

[0188] Pharmaceutical compositions may be formulated according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention may include, for example, diluents, excipients, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or non-protein amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0189] The antibody, composition, or pharmaceutical composition according to the present invention is preferably for use as a pharmaceutical. The antibody, composition, or pharmaceutical composition according to the present invention is preferably for use in the treatment of a disease. The bispecific antibody of the present invention can be used for several purposes. In particular, the bispecific antibody of the present invention can be used for the treatment of various forms of cancer, including metastatic cancer and refractory cancer. Preferably, the cancer may be of the solid tumor type.

[0190] In particular, bispecific antibodies according to the invention may be useful in therapeutic situations where it is desirable to specifically target B7H4-expressing cells and kill them by T cell mediation.

[0191] In one embodiment, the invention provides a method for treating cancer in a subject comprising administering a therapeutically effective amount of a bispecific B7H4xCD3 antibody of the invention. In a further embodiment, the invention provides a method for treating a disorder associated with B7H4-expressing cells in a subject comprising administering a therapeutically effective amount of a bispecific antibody of the invention.

[0192] In another embodiment, the invention provides a method for treating cancer in a subject, comprising administering a therapeutically effective amount of an antibody of the invention capable of binding to human B7H4. In a further embodiment, the invention provides a method for treating a disorder associated with B7H4-expressing cells in a subject, comprising administering a therapeutically effective amount of a monospecific antibody of the invention capable of binding to human B7H4.

[0193] As mentioned above, a suitable disease that can be contemplated in the methods and uses according to the present invention is cancer. Most preferably, the cancer is characterized by the expression of B7H4. B7H4 expression in cancer can be easily determined by using methods known in the art, such as PCR, immunostaining, or FACS analysis, i.e., by detecting the expression of B7H4 transcripts and / or B7H4 protein. The antibodies described herein that can bind to human B7H4 can be used, for example, in immunostaining and / or FACS analysis.

[0194] Cancers that may express B7H4 include breast cancer, uterine / endometrial cancer, uterine carcinosarcoma cancer, ovarian cancer, cervical cancer, non-small cell lung cancer (squamous cell carcinoma and adenocarcinoma), head and neck squamous cell carcinoma, bladder cancer, esophageal cancer, bile duct cancer, pancreatic cancer, gastric cancer, kidney cancer, and prostate cancer.

[0195] Cancers that may express B7H4 include gastric cancer, bile duct cancer, bladder cancer, non-small cell lung cancer (especially squamous cell NSCLC), pancreatic cancer, cervical cancer, head and neck cancer, breast cancer (including triple-negative breast cancer), ovarian cancer, and uterine cancer. Potentially preferred cancer types are cancers selected from uterine carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), invasive breast cancer (BRCA), endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), and cholangiocarcinoma (CHOL).

[0196] In a further embodiment, patients diagnosed with cancer may be evaluated for B7H4 expression in cancer cells, and if B7H4 is detected, it may range from low to high levels, and such patients may be selected for treatment with an antibody according to the present invention. Patients diagnosed with gastric cancer, bile duct cancer, bladder cancer, non-small cell lung cancer (particularly squamous cell NSCLC), pancreatic cancer, cervical cancer, head and neck cancer, breast cancer (including triple-negative breast cancer), ovarian cancer, or uterine cancer may be subjected to such testing. In a further embodiment, patients diagnosed with uterine carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), invasive breast cancer (BRCA), endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), or bile duct carcinoma (CHOL) may be subjected to such testing. However, including such an evaluation when selecting patients for treatment may not necessarily be a requirement.

[0197] kit The present invention further provides a kit-of-parts comprising an antibody as disclosed above, e.g. a kit for use as a companion diagnostic / to identify patients within a patient population who have a propensity to respond to treatment with an antibody as defined herein above or an immunoconjugate or antibody-drug conjugate (ADC) as defined herein above, or for predicting the efficacy or anti-tumor activity of said antibody or immunoconjugate or ADC when used in the treatment of a patient, said kit comprising an antibody as disclosed above and instructions for using said kit.

[0198] A kit of parts, for example a kit for use as a companion diagnostic / for identifying patients within a patient population who have a propensity to respond to treatment with an antibody according to any one of claims 1 to 55, comprising an antibody according to any one of claims 1 to 55 and instructions for using the kit.

[0199] Thus, in one aspect, the present invention relates to diagnostic compositions comprising a bispecific CD3xB7H4 antibody as defined herein or a B7H4 antibody as defined herein, and uses thereof.

[0200] In another aspect, the present invention relates to a kit for detecting crosslinking of CD3-expressing cells and B7H4-expressing cells in a sample derived from a patient, the kit comprising: i) the bispecific antibody of any one of the embodiments disclosed herein; and ii) Instructions for using the kit.

[0201] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a bispecific CD3xB7H4 antibody and one or more reagents for detecting crosslinking of B7H4-expressing cells and CD3-expressing cells. The reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies, or reagents for enzymatic reactions that result in a visualized product.

[0202] In a further aspect, the present invention relates to a method for detecting whether cross-linking of CD3-expressing cells and B7H4-expressing cells occurs in a sample derived from a patient upon administration of a bispecific antibody according to any one of the embodiments disclosed herein, the method comprising the steps of: (i) contacting a sample with the bispecific antibody of any one of the embodiments disclosed herein under conditions that allow complex formation between the bispecific antibody and CD3-expressing cells and B7H4-expressing cells; and (ii) analyzing whether a complex is formed;

[0203] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0204] Example 1: Production and screening of B7H4 antibodies Expression of B7H4 constructs Constructs encoding various full-length B7H4 variants were generated: human (Homo sapiens) B7H4 (Uniprot accession number Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7H4 transcript 1 (Uniprot accession number A0A2K5U6P5), dog (Canis familiaris) B7H4 (Uniprot accession number F1P8R9), rabbit (Lepus rufocanus) B7H4 (Uniprot accession number G1TQE8), rat (Rattus norvegicus) B7H4 (Uniprot accession number Q501W4), mouse (Mus musculus) B7H4 (Uniprot accession number Q7TSP5), and pig (Sus scrofa) B7H4 (Uniprot accession number F1SAY4) (see Table 1).

[0205] Additionally, a construct was generated for the extracellular domain (ECD of human B7H4 (amino acids 25-259 of Uniprot accession number Q7Z7D3)) fused to a human IgG1 Fc domain with a C-terminal His tag and C tag (B7H4ECD-FcHisC) (SEQ ID NO: 12). In SEQ ID NO: 1, amino acid residues 1-24 are the signal peptide; therefore, the mature B7H4ECD-FcHisC protein corresponds to amino acid residues 25-259 of SEQ ID NO: 1.

[0206] The constructs contained appropriate restriction sites and optimal Kozak (GCCGCCACC) sequences for cloning (Kozak, M., Gene 1999;234(2):187-208). The full-length B7H4 construct and the B7H4 ECD construct were cloned into pSB, a mammalian expression vector containing Sleeping Beauty inverted terminal repeats flanking an expression cassette consisting of a CMV promoter and an HSV-TK poly(A) signal.

[0207] Generation of HEK-293F cell lines transiently expressing full-length B7H4 variants Freestyle™ 293-F (a HEK-293 subclone [HEK-293F] adapted to suspension growth and chemically defined Freestyle medium) cells were obtained from Invitrogen (catalog no. R790-07) and transfected with the above constructs using 293fectin (Invitrogen, catalog no. 12347-019) according to the manufacturer's instructions.

[0208] Purification of His-tagged B7H4 B7H4ECD-FcHisC was expressed using the Expi293F expression platform (Thermo Fisher Scientific, Waltham, MA, USA, catalogue no. A14527) essentially as described by the manufacturer.

[0209] The His tag allows for purification using immobilized metal affinity chromatography (Ni-NTA). His-tagged proteins bind strongly to the column material, whereas other proteins present in the culture supernatant either do not bind or bind more weakly than the His-tagged proteins and are eluted in the flow-through fraction. The column was then washed to remove weakly bound proteins. 2+ Strongly bound His-tagged proteins were eluted with an imidazole-containing buffer, which competes with His binding to the ATP. The eluent was removed by buffer exchange using a desalting column.

[0210] immunization OmniRat® animals (transgenic rats expressing a diverse antibody repertoire with fully human idiotypes; Ligand Pharmaceuticals Inc., San Diego, USA) were immunized by subcutaneous injection (twice weekly for 7 weeks) into the heel joints of both hind paws with 50 μg of B7H4ECD-FcHisC in PBS mixed with an equal volume of adjuvant (Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA, catalog no. S6322) or CFA, complete Freund's adjuvant (first injection) and IFA, incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA, catalog no. F5881 / F5506) (subsequent injections), followed by a final subcutaneous injection of antigen in PBS without adjuvant.

[0211] Antibody production Three days after the final booster immunization, lymph node cells from immunized animals were fused with mouse myeloma SP2.0 cells according to standard procedures. RNA from hybridomas producing B7H4-specific antibodies was extracted, and 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. The VH and VL coding regions were amplified by PCR and directly cloned in-frame into p33G1f, p33kappa, and p33lambda expression vectors (pcDNA3.3-based vectors containing codon-optimized human IgG1m(f) constant domains, human kappa constant domains, and human lambda constant domains, respectively) by ligation-independent cloning (Aslanidis, C. and PJ de Jong, Nucleic Acids Res 1990;18(20):6069-74). The variable domains from these expression vectors were sequenced, and the CDRs were annotated according to the IMGT definition (Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)). Clones with the correct open reading frame (ORF) were expressed and tested for antigen binding. After performing antigen-specific screening assays, the sequences of the heavy and light chain variable regions were gene synthesized and cloned into expression vectors containing a human IgG1 heavy chain with the following amino acid mutations: L234F, L235E, D265A, and K409R (FEAR) (where amino acid position numbers are according to EU numbering (corresponding to SEQ ID NO: 60)), and into expression vectors containing a human kappa or lambda light chain. For some of the antibodies, variants were generated with point mutations in the variable domain to remove cysteine ​​residues that could potentially result in unwanted disulfide bridge formation, or to replace asparagine with serine or germline residues to remove potential N-linked glycosylation sites.For example, variants were made from the C1 heavy and light chain variable region sequences with an N52S substitution, which corresponds to a substitution in CDR2 (see Table 1, SEQ ID NO: 25 and SEQ ID NO: 29). Another variant can have an N52Q substitution (SEQ ID NO: 31).

[0212] Antigen-specific screening assays The presence of B7H4 antibodies in the sera of immunized animals or in the culture supernatants of hybridomas and transfectomas was determined by a homogeneous binding assay. Samples were analyzed for antibody binding to HEK-293F cells expressing human B7H4, cynomolgus monkey B7H4, or mouse B7H4, transiently transfected with constructs designed to express full-length B7H4 variants, or HEK-293F wild-type cells (negative control). Samples were added to these cells to allow antibody binding to B7H4. Antibody binding was then detected using appropriate fluorescent conjugates (AffiniPure Goat Anti-Rat IgG (H+L) Alexa Fluor® 647; Jackson ImmunoResearch, Cat. No. 112-605-143; AffiniPure Goat Anti-Human IgG Fc gamma-Alexa Fluor® 647; Jackson ImmunoResearch, Cat. No. 109-605-098). Depending on the antibody framework, cells (2.5 × 10 5Cells (0.2 μg / ml) were mixed with goat anti-human AffiniPure goat anti-human IgG Fc gamma-Alexa Fluor® 647 (0.2 μg / ml; Jackson ImmunoResearch Laboratories, 109-605-098) or AffiniPure goat anti-rat IgG (H+L) Alexa Fluor® 647 (0.2 μg / ml; Jackson ImmunoResearch, 112-605-143). Serial dilutions (ranging from 0.003 to 3 μg / ml in 2-fold dilution steps) of test and control antibodies were prepared, and 2 μl of antibody dilution was added to 5 μl of cell / conjugate mixture in a 1536-well plate (Greiner, catalog no. 789866). The plates were incubated at room temperature for 9 hours, after which the fluorescence intensity was determined using an ImageXpress Velos laser scanning cytometer (Molecular Devices, LLC, Sunnyvale, CA, USA), and the total fluorescence was used as the readout. Samples were considered positive if the count was greater than 50 and the count multiplied by the fluorescence intensity was at least three times greater than the negative control.

[0213] Results of B7H4 antibody panel production Heavy and light chain variable region sequences were successfully obtained from 176 of the 193 hybridomas generated. Of the 351 heavy / light chain combinations tested, 98 showed binding in the antigen screening assay using HEK-293F cells transfected with human B7H4 described above. Thirty-five antibodies were selected: 26 with the original sequence and 9 variants with point mutations introduced into the variable domains. The antibodies were generated as monovalent binding antibodies (as CD3 bispecifics) and bivalent binding antibodies (as IgG1 molecules) and tested for tumor cell binding as described below. Of the antibodies from the generated panel, only antibody B7H4-C1 and its variant B7H4-C1-N52S provided antibodies that bound to tumor cells, as described below. Their corresponding VH and VL antibody variable domain coding sequences are listed in Table 1.

[0214] Additional B7H4 antibodies In the examples, additional antibodies specific for B7H4 were used, comprising variable domains previously described in the following documents: those described in WO2014159835 (referenced therein as SEQ ID NO: 38 and SEQ ID NO: 35), which correspond to B7H4-C2 herein, and the relevant sequences of these variable domains are set out in Table 1 herein, including SEQ ID NO: 43 and SEQ ID NO: 47; those described in WO2014159835 (referenced therein as SEQ ID NO: 56 and SEQ ID NO: 55), which correspond to B7H4-C3 herein, and the relevant sequences of these variable domains are set out in Table 1 herein, including SEQ ID NO: 36 and SEQ ID NO: 40; and those described in WO2009073533 (referenced therein as SEQ ID NO: 2 and SEQ ID NO: and those described in US20190085080A1, which correspond to B7H4-C5 herein, the relevant sequences of which are set forth in Table 1 herein, including SEQ ID NO: 65 and SEQ ID NO: 69. These corresponding VH and VL antibody variable domain coding sequences were synthesized and cloned into pcDNA3.3-based vectors with codon-optimized human IgG1m(f) constant domains and human kappa or human lambda constant domains or variants thereof to generate monospecific and bispecific antibodies. When referring to the antibody IgG1-B7H4-CX-FEAL, this refers to an antibody having a B7H4-CX variable region, being of the IgG1 isotype, and having the amino acid substitutions L234F, L235E, D265A, and F409R in the constant region of the heavy chain.

[0215] IgG1-b12 antibody Antibody b12, an HIV-1 gp120-specific antibody (Barbas, C. F. J. Mol. Biol. 1993 Apr. 5; 230(3):812-23), was used in some examples as a negative control IgG1 or as a non-binding control Fab arm of a control bispecific. A codon-optimized antibody coding sequence for this control antibody was synthesized and cloned into a pcDNA3.3-based vector containing codon-optimized human IgG1m(f) constant domains and human kappa constant domains or variants thereof. The sequences of the variable heavy (VH) and variable light (VL) chains are included herein as SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0216] Example 2 Humanized CD3 antibody for generating CD3xB7H4 bispecific antibody The production of humanized antibody IgG1-huCD3-H1L1 (whose variable heavy and light region sequences are set forth herein in SEQ ID NO: 16 and SEQ ID NO: 22) is described in Example 1 of WO2015 / 001085. IgG1-huCD3-H1L1 is referred to herein as "IgG1-huCD3." Antibody IgG1-huCD3-H1L1-FEAL is a variant thereof with three amino acid substitutions in the Fc domain (L234F, L235E, D265A) in addition to an amino acid substitution (F405L) that allows for the generation of bispecific antibodies by directed Fab arm exchange, as described herein below. Such mutations have been shown to not affect target binding of the antibody into which they are introduced (see, e.g., US 2015 / 0337049 and Engelberts et al., 2020, EBioMedicine 52: 102625).

[0217] The production of humanized antibody IgG1-huCD3-H1L1-H101G (whose variable heavy and light chain region sequences are set forth herein in SEQ ID NO: 17 and SEQ ID NO: 22) is described in Example 2 of WO 2017 / 009442. IgG1-huCD3-H1L1-H101G is referred to as "IgG1-huCD3-H101G." This variant contains the substitution H101G in the variable heavy chain region sequence (compare SEQ ID NO: 16 and SEQ ID NO: 17) and has the same light chain as IgG1-huCD3-H1L1. Antibody IgG1-huCD3-H101G-FEAL is this variant with amino acid substitutions L234F, L235E, D265A, and F405L.

[0218] Example 3 Determination of B7H4 Binding Affinity Using Biolayer Interferometry The target binding affinity of the B7H4 antibody was determined by label-free biolayer interferometry (BLI) on an Octet HTX instrument (ForteBio). Experiments were performed at 30°C with shaking at 1,000 RPM. First, the affinities of IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C4-FEAR for human and mouse B7H4 were determined using BLI. Anti-human IgG Fc capture (AHC) biosensors (ForteBio, catalog no. 18-5060) were primed by exposure to 10 mM glycine (Sigma-Aldrich, catalog no. 15527) buffer, pH 1.7, for 5 seconds, followed by neutralization in sample diluent (ForteBio, catalog no. 18-1048) for 5 seconds. Both steps were repeated twice. Next, antibody (1 μg / mL in sample diluent) was added to the AHC sensor for 600 seconds. After a baseline measurement (100 seconds) in sample diluent, the binding (300 seconds) and dissociation (1,000 seconds) of human B7H4 (Sino Biological, catalog number 10738-H08H-100) or mouse B7H4 (R&D Systems, catalog number 2154-B7-050) were determined by two-fold dilution steps in sample diluent using concentrations ranging from 1.56 to 100 nM (0.04 to 2.68 μg / mL) and 5.9 to 375 nM (0.16 to 10 μg / mL) of human B7H4 and mouse B7H4, respectively. The theoretical molecular weights of human B7H4 and mouse B7H4 (as ECD-His-tagged molecules) based on the amino acid sequences (26.8 kDa and 26.6 kDa, respectively) were used for calculations. 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. The antibody was then added back to the sensor for the next cycle of kinetic measurements.

[0219] Data were acquired using data acquisition software v9.0.0.49d (ForteBio) and analyzed using data analysis software v9.0.0.12 (ForteBio). Data traces were corrected for each antibody by subtracting a reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and step-by-step correction alignment and Savitzky-Golay filtering for dissociation were applied. Data traces with responses below 0.05 nm were excluded from analysis. Data were fitted with a 1:1 Global Full-fit model, with the time windows of interest for association and dissociation times set to 300 and 200 seconds, respectively.

[0220] In a second experiment, the affinity of IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR, and IgG1-B7H4-C5-FEAR for human B7H4 and mouse B7H4 was determined using BLI. This experiment was performed as previously described, with some minor exceptions. The preparatory steps were repeated five times. The binding (200 s) and dissociation (1,000 s) times of human B7H4 or mouse B7H4 were determined using a concentration range of 0.78–800 nM in two-fold dilution steps in diluent. Data were acquired using data acquisition software v12.0.1.8 (ForteBio) and analyzed using data analysis software v12.0.1.2 (ForteBio). Data were fitted with a 1:1 Global Full-fit model using a time window of interest of 200 seconds for association times and 200 seconds for dissociation times, except for IgG1-B7H4-C2-FEAR, for which a dissociation time of 1,000 seconds was used. Dissociation times were calculated using the R 2 Selection was based on values, visual inspection of the curves, and a signal decay of at least 5% during the dissociation phase. Data traces generated using antigen concentrations greater than 100 nM were excluded from analyses involving antibodies with affinities below 50 nM.

[0221] Furthermore, the affinity for cynomolgus monkey B7H4 was also determined by BLI. In a first experiment, the affinity of bsIgG1-huCD3-FEAL×B7H4-C1-FEAR, bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR, bsIgG1-huCD3-FEAL×B7H4-C2-FEAR, bsIgG1-huCD3-FEAL×B7H4-C3-FEAR, and bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR for cynomolgus monkey B7H4 was determined. Amine-reactive second-generation (AR2G) biosensors (ForteBio, catalog no. 18-5092) were activated by reacting with 20 mM EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (ForteBio, catalog no. 18-1033) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (ForteBio, catalog no. 18-1067) for 300 s. To the activated sensor, 10 μg / mL of recombinant hIgG1 Fc-tagged cynomolgus monkey B7H4 (Creative BioMart, Catalog No. VTCN1-1517R) dissolved in 10 mM sodium acetate, pH 4.0 (ForteBio, Catalog No. 18-1068) was added for 600 seconds, and the reaction was stopped with 1 M ethanolamine, pH 8.5 (ForteBio, Catalog No. 18-1071) for 300 seconds. After a baseline measurement in sample diluent (300 seconds; ForteBio, Catalog No. 18-1048), the association (100 seconds) and dissociation (1,000 seconds) times for functionally monovalent B7H4 binding by the CD3×B7H4 bispecific antibody (shown in Table 8) were determined using a concentration range of 0.23–15 μg / mL (1.56–100 nM) in two-fold dilution steps in sample diluent. The molecular weight of these antibodies, 150 kDa, was used for the calculations. For each antibody, a reference sensor incubated with sample diluent instead of the antibody was used.

[0222] Data were acquired using data acquisition software v9.0.0.49d (ForteBio) and analyzed using data analysis software v9.0.0.12 (ForteBio). Data traces were corrected for each antibody by subtracting a reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and step-by-step alignment and Savitzky-Golay filtering for dissociation were applied. Data traces with responses below 0.05 nm were excluded from analysis. Data were fitted with a 1:1 Global Full-fit model, with the time windows of interest for association and dissociation times set to 100 and 200 seconds, respectively.

[0223] In a second experiment to determine the affinity of the B7H4 antibody for cynomolgus monkey B7H4, the affinities of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR were determined. This experiment was performed as previously described, with a few minor exceptions. After a 600-second baseline determination in sample diluent, the association (200 seconds) and dissociation (1,000 seconds) times for functionally monovalent B7H4 binding by the CD3×B7H4 bispecific antibody (shown in Table 9) were determined using a concentration range of approximately 0.1 to 116 μg / mL (0.78 to 800 nM) in two-fold dilution steps in sample diluent. The intrinsic molecular weight of each antibody (approximately 145 kDa) was used for calculations. Data were acquired using Data Acquisition Software v12 (ForteBio) and analyzed with Data Analysis Software v12 (ForteBio). Data traces with responses less than 0.03 nm were excluded from the analysis. Data were fitted with a 1:1 Global Full Fit model using a time window of interest of 200 seconds for association and dissociation times. Dissociation times were calculated using R 2Selection was based on the R value, visual inspection of the curve, and a signal decay of at least 5% during the dissociation phase. Date traces made with antibody concentrations above 200 nM were excluded from the analysis, which focused on antibodies with affinities below 50 nM. All results determined had an R of at least 0.98. 2 showed.

[0224] "K D " (M) refers to the equilibrium dissociation constant of the antibody-antigen interaction, and k d k a It is obtained by dividing by "k d " (sec -1 ) refers to the dissociation rate constant of the antibody-antigen interaction. d is k off It is sometimes called the k value or off rate. a " (M -1 ×sec -1 ) refers to the binding rate constant of the antibody-antigen interaction. a is k on It is sometimes called the value or on-rate.

[0225] Tables 4 and 5 show the results of the first and second experiments, which show the binding rate constants k for the antibodies shown in the tables to human B7H4. a (1 / Ms), dissociation rate constant k d (1 / s), and the equilibrium dissociation constant K D (M) was determined by biolayer interferometry.

[0226] Table 4: Binding affinities of antibodies to the human B7H4 extracellular domain as determined by label-free biolayer interferometry. ND = not determined. TIFF0007749575000012.tif65128

[0227] Table 5. Binding affinities of antibodies to the human B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF0007749575000013.tif65128 1 The results shown are the average of three experiments.

[0228] Tables 6 and 7 show the results of two experiments in which the kJ of the antibodies shown in the tables against mouse B7H4 was a (1 / Ms), k d (1 / s), and K D (M) was determined by biolayer interferometry.

[0229] Table 6: Binding affinity of antibodies to mouse B7H4 extracellular domain as determined by label-free biolayer interferometry. ND = not determined. - = no binding (response less than 0.05 nM at highest concentration used). TIFF0007749575000014.tif65128

[0230] Table 7: Binding affinity of antibodies to mouse B7H4 extracellular domain as determined by label-free biolayer interferometry. - = no binding (response less than 0.05 nM at highest concentration used). TIFF0007749575000015.tif65128

[0231] Tables 8 and 9 show the results of two experiments in which the kJ of the antibodies shown in the table against cynomolgus monkey B7H4 was a (1 / Ms), k d (1 / s), and K D (M) was determined by biolayer interferometry.

[0232] Table 8. Binding affinity of functionally monovalent antibodies to the cynomolgus monkey B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF0007749575000016.tif69153

[0233] Table 9. Binding affinities of functionally monovalent antibodies to the cynomolgus monkey B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF0007749575000017.tif37164 a The results shown are the average of three experiments. b R is strict quality control 2 The threshold of 0.98 was not met.

[0234] Example 4 Determination of CD3 Binding Affinity Using Biolayer Interferometry The binding affinities of IgG1-huCD3-FEAL and IgG1-huCD3-H101G-FEAL were determined as described in Example 7 of WO2017 / 009442.

[0235] Briefly, the binding affinity of selected CD3 antibodies in the IgG1-huCD3-FEAL format to recombinant soluble CD3ε (CD3E27-GSKa) (mature protein of SEQ ID NO: 13) was determined using biolayer interferometry in a ForteBio Octet HTX (ForteBio). hIgG (1 μg / mL) was added to an anti-human Fc capture biosensor (ForteBio, catalog no. 18-5060) for 600 seconds. After a baseline measurement (200 seconds), the association (1000 seconds) and dissociation (2000 seconds) of CD3E27-GSKa were determined using a CD3E27-GSKa concentration range of 27.11 μg / mL to 0.04 μg / mL (1000 nM to 1.4 nM) in 3-fold dilution steps (sample diluent, ForteBio, catalog no. 18-5028). The theoretical molecular weight of CD3E27-GSKa, based on the amino acid sequence, i.e., 27.11 kDa, was used for 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 using a 1:1 model and a global full fit with an association time of 1000 s and a dissociation time of 100 s. Data traces were corrected by subtracting a reference curve (determined by loading the antibody onto the biosensor and using sample diluent only). The Y-axis was fitted to the last 10 s of baseline, and step-to-step correction and Savitzky-Golay filtering were applied. Data traces with responses below 0.05 nm were excluded from the analysis.

[0236] Table 10 shows the binding rate constant k for recombinant CD3ε determined by biolayer interferometry. a (1 / Ms), dissociation rate constant k d (1 / s), and the equilibrium dissociation constant K D (M) shows the IgG1-huCD3-FEAL antibody. D : 683nM) D : 15 nM), showing binding affinity to recombinant CD3ε.

[0237] Table 10. Binding affinities of monospecific bivalent CD3 antibodies to recombinant CD3ε determined by label-free biolayer interferometry. TIFF0007749575000018.tif37128

[0238] Example 5 Cross-blocking of B7H4 antibody determined by biolayer interferometry Conventional sandwich-type antibody cross-blocking analysis (epitope binning) was performed using BLI on an Octet HTX instrument (ForteBio). The first cross-blocking experiment using IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C4-FEAR was performed at 30°C with shaking at 1,000 RPM.

[0239] Amine-reactive second-generation (AR2G) biosensors (ForteBio, catalog no. 18-5092) were activated for 300 seconds with a solution of 20 mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) (Sigma-Aldrich, catalog no. 03449) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (Sigma-Aldrich, catalog no. 56485). The activated AR2G sensors were then loaded with 20 μg / mL of primary antibody in 10 mM sodium acetate, pH 6.0 (ForteBio, catalog no. 18-1070) for 600 seconds, and the reaction was quenched with 1 M ethanolamine, pH 8.5 (ForteBio, catalog no. 18-1071) for 300 seconds. After a baseline measurement in sample diluent (50 seconds; ForteBio, catalog no. 18-1048), human B7H4 (100 nM or 2.68 μg / mL diluted in sample diluent; Sino Biological, catalog no. 10738-H08H) was added to the AR2G biosensor containing the immobilized antibody for 300 seconds. The theoretical molecular weight of human B7H4 (26.8 kDa) based on the amino acid sequence was used for calculations. Binding of the second antibody (10 μg / mL in sample diluent) was determined (300 seconds). The sensor was regenerated by exposure to 10 mM glycine (Riedel-de Haen, catalog no. 15527) buffer, pH 2.5, for 5 seconds, followed by neutralization in sample diluent for 5 seconds. Both steps were repeated twice. The sensor containing the immobilized first antibody was then used again, starting from the baseline step.

[0240] Data were acquired using Data Acquisition Software v9.0.0.49d (ForteBio) and analyzed with Data Analysis HT Software v10.0.17 (ForteBio). To correct for dissociation of B7H4 from the immobilized primary antibody, data traces were corrected by subtracting a reference curve (sample diluent instead of the secondary antibody). The Y-axis was aligned to the start of the binding phase, and Savitzky-Golay filtering was applied. The corrected binding responses of the secondary antibodies were presented in matrix format. Responses above 0.05 nM were typically considered to be non-cross-interfering antibodies, whereas responses below 0.05 nM were considered to be interfering antibody pairs.

[0241] The cross-blocking experiment was repeated to include IgG1-B7H4-C5-FEAR and performed as described above with minor modifications. Experiments were performed at 22°C with shaking at 1,000 RPM. Data were acquired using Data Acquisition Software v12.0.1.8 (ForteBio) and analyzed with Data Analysis HT Software v12.0.1.55 (ForteBio). Responses above 0.1 nM were typically considered to represent non-cross-blocking antibodies, while responses below 0.1 nM were considered to represent blocking antibody pairs.

[0242] A first set of cross-blocking experiments was performed with the antibodies IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR, and IgG1-B7H4-C2-FEAR. The results are summarized in Table 11. A second set of cross-blocking experiments was performed to include IgG1-B7H4-C5-FEAR. The results are summarized in Table 12. The first column shows the immobilized antibody; the first row shows the antibody in solution (referred to above as "second antibody"). The corrected binding response of the antibody in solution is shown. Antibody cross-blocking is shown in dark gray; non-blocking antibody combinations are unmarked (clear background). This shows that IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C5-FEAR cross-block with each other, but do not cross-block with IgG1-B7H4-C4-FEAR and IgG1-B7H4-C2-FEAR, and vice versa.

[0243] Table 11: First antibody cross-blocking experiment using biolayer interferometry. The first column shows immobilized antibodies, and the first row shows antibodies in solution. Corrected binding responses of antibodies in solution are shown. Antibody cross-interference is shown in dark gray; non-interfering antibody combinations are unmarked (clear background). TIFF0007749575000019.tif78133

[0244] Table 12: Second antibody cross-blocking experiment using biolayer interferometry. The first column shows immobilized antibodies, and the first row shows antibodies in solution. Corrected binding responses of antibodies in solution are shown. Antibody cross-interference is shown in dark gray; non-interfering antibody combinations are unmarked (clear background). TIFF0007749575000020.tif85148

[0245] Example 6 Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific antibodies were generated in vitro using DuoBody® platform technology, i.e., 2-MEA-induced Fab arm exchange, as described in WO2011147986, WO2011131746, and WO2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS 2013, 110: 5145-50; Gramer et al., MAbs 2013, 5: 962-973). To enable the production of bispecific antibodies by this method, the following IgG1 molecules were generated with specific point mutations in the CH3 domain: an F405L mutation in one parent IgG1 antibody (i.e., the CD3 antibody in this application) and a K409R mutation in the other parent IgG1 antibody (i.e., the B7H4 antibody or an HIV-1 gp120-specific control antibody in this application). In addition to these mutations, the parent IgG1 antibody also contained the substitutions L234F, L235E, D265A (FEA).

[0246] To generate bispecific antibodies, equal masses of two parent antibodies were mixed in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4 2- , 1.8mM H2PO4 - , 163.9mM Na + , 140.3 mM Cl - The mixture was mixed in PBS buffer (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 reoxidation of the interchain disulfide bonds and formation of intact bispecific antibodies, 2-MEA was removed by dialysis into PBS buffer using a Slide-A-Lyzer carriage with a 10 kDa molecular weight cutoff (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0247] The following antibodies were used in the examples: B7H4 antibody IgG1-B7H4-C1-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 33). IgG1-B7H4-C1-N52S-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 29 and SEQ ID NO: 33). IgG1-B7H4-C2-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 43 and SEQ ID NO: 47). IgG1-B7H4-C3-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 36 and SEQ ID NO: 40). IgG1-B7H4-C4-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 50 and SEQ ID NO: 54). IgG1-B7H4-C5-FEAR (having the VH and VL sequences set forth in SEQ ID NO: 65 and SEQ ID NO: 69). 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 contained the amino acid substitutions L234F, L235E, D265A, and K409R, and the light chain constant region was of the kappa type (SEQ ID NO: 61 and SEQ ID NO: 63, respectively).

[0248] CD3 antibody IgG1-huCD3-FEAL (having the VH and VL sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 22). IgG1-huCD3-H101G-FEAL (having the VH and VL sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 22). The annotation IgG1 indicates that a full-length antibody of IgG1 isotype was produced, and the annotation FEAL indicates that the heavy chain constant region contained the amino acid substitutions L234F, L235E, D265A, and F405L, and the light chain constant region was of the lambda type (SEQ ID NO: 60 and SEQ ID NO: 64, respectively).

[0249] Control antibody IgG1-b12-K409R (having the VH and VL sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15). The annotation IgG1 indicates that a full-length antibody of the IgG1 isotype was produced, and the annotation K409R indicates that the heavy chain constant region contained the amino acid substitution K409R and the light chain constant region was of the kappa type (SEQ ID NO: 62 and SEQ ID NO: 63, respectively).

[0250] bispecific antibody The aforementioned CD3 and B7H4 antibodies were combined to create a bispecific antibody with one antigen-binding region capable of binding to human CD3 and another antigen-binding region capable of binding to B7H4, providing a bispecific antibody of isotype IgG1, which is annotated as bsIgG1. bsIgG1-huCD3-FEAL×B7H4-C1-FEAR bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR bsIgG1-huCD3-FEAL×B7H4-C2-FEAR bsIgG1-huCD3-FEAL×B7H4-C3-FEAR bsIgG1-huCD3-FEAL×B7H4-C4-FEAR bsIgG1-huCD3-H101G-FEAL×B7H4-C2-FEAR bsIgG1-huCD3-H101G-FEAL×B7H4-C3-FEAR bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR bsIgG1-huCD3-FEAL×b12-FEAR (for b12 arms with VH and VL sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15) bsIgG1-huCD3-H101G-FEAL×b12-FEAR

[0251] Example 7 Determination of B7H4 domains involved in binding and functional epitopes using B7H4-B7H3 chimeric molecules and a B7H4 alanine scanning library Domain mapping using B7H4-B7H3 chimeric molecules by end-point analysis The B7H4 domain specificity of the B7H4 antibody was determined using a panel of cells transfected to express human B7H4, human B7H3 (structurally similar proteins with sufficient amino acid sequence variance in the extracellular domain), or two different human B7H4-B7H3 chimeric molecules. Expression constructs encoding human B7H4, human B7H3 (Uniprot accession number Q5ZPR3-1; SEQ ID NO: 9), a chimeric molecule containing the IgV domain of B7H3 and the IgC domain of B7H4 (B7H3-IgV / B7H4-IgC; SEQ ID NO: 11), or a chimeric molecule containing the IgV domain of B7H4 and the IgC domain of B7H3 (B7H4-IgV / B7H3-IgC; SEQ ID NO: 10) were prepared. HEK cells were transiently transfected to express these constructs.

[0252] Cells (3 × 10 ) were cultured in a polystyrene 96-well round-bottom plate (Greiner bio-one, catalog no. 650101) containing serial dilutions of antibodies (ranging from 0.0046 to 10 μg / mL in 3-fold dilutions) in 50 μL of PBS / 0.1% BSA / 0.02% azide (FACS buffer). 4The cells (100 cells / well) were incubated at 4°C for 30 minutes. After washing twice in FACS buffer, the cells were incubated with a secondary antibody at 4°C for 30 minutes. The secondary antibody used was R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (1:500 in staining buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, catalog number 109-116-098). The cells were then washed twice in FACS buffer, resuspended in 20 μL of FACS buffer, and analyzed using an iQue screener (Intellicyt Corporation, USA). Binding of 10 μg / mL of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR, bsIgG1-huCD3-FEAL×B7H4-C4-FEAR, bsIgG1-huCD3-FEAL×B7H4-C3-FEAR, and bsIgG1-huCD3-FEAL×B7H4-C2-FEAR was determined as the mean fluorescence intensity (MFI) percent of binding of 10 μg / mL of the following agents: Binding of IgG1-B7H3-BRCA84D (a B7H3-specific IgG1 antibody produced as described above, having the CDR sequences described for antibody BRCA84D in WO2011109400) to B7H3-expressing cells; Binding of bsIgG1-huCD3-FEAL×B7H4-C4-FEAR to B7H3-IgV / B7H4-IgC-expressing cells Binding of bsIgG1-huCD3-FEAL×B7H4-C2-FEAR to B7H4-IgV / B7H3-IgC-expressing cells · and bsIgG1-huCD3-FEAL×B7H4-C3-FEAR binding to B7H4-expressing cells.

[0253] Figure 1 shows that the IgC domain of B7H4 is involved in the binding of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-FEAL×B7H4-C4-FEAR, that both the IgC and IgV domains of B7H4 are involved in the binding of bsIgG1-huCD3-FEAL×B7H4-C3-FEAR, and that at least the IgV domain of B7H4 is involved in the binding of bsIgG1-huCD3-FEAL×B7H4-C2-FEAR. Regarding the C2 antibody, from which the variable domains used to create bsIgG1-huCD3-FEAL×B7H4-C2-FEAR were derived, it has been described that the C2 antibody binds to the IgV domain; the data in Figure 1 indicate that the IgC domain is also involved in binding (WO2014159835 and Leong et al 2015, Mol. Pharmaceutics 12, 1717-1729).

[0254] Domain mapping using B7H4-B7H3 chimeric molecules with complete dose-response curve analysis Further experiments were performed to investigate the B7H4 domain specificity of the B7H4 antibody in more detail by analyzing full dose-response curves. These experiments also determined the domain specificity of bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR. bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C2-N52S-FEAR, and bsIgG1-huCD3-H101G-FEAL×B7H4-C2-N52S-FEAR were injected into HEK cells transiently transfected to express human B7H4 or the B7H4-B7H3 chimeric molecules B7H3-IgV / B7H4-IgC or B7H4-IgV / B7H3-IgC. The binding of serial dilutions (0.014–30 μg / mL in 3-fold dilution steps) of bsIgG1-huCD3-H101G-FEAL×B7H4-C3-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR, and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR was determined as previously described. Figure 2 shows the dose-response curves, indicating that the IgC domain of B7H4 is responsible for the binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, consistent with the findings of the alanine scanning library experiments. Furthermore, the IgV domain was involved in the binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C2-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR, and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR, whereas both the IgC and IgV domains appeared to be involved in the binding of bsIgG1-huCD3-H101GFEAL×B7H4-C3-FEAR.

[0255] Determining the contribution of B7H4 amino acid residues to the binding of B7H4 antibodies using a B7H4 alanine scanning library Library Design A single-residue alanine library of human B7H4 (Uniprot Q7Z7D3-1) was synthesized (GeneArt) by individually mutating all amino acid residues in the extracellular domain of human B7H4 to alanine, except for positions containing alanine or cysteine. Cysteines were not mutated to minimize potential disruption of the antigen's structure. This library was cloned into the pMAC expression vector, which contains a CMV / TK-polyA expression cassette, an Amp resistance gene, and a pBR322 origin of replication.

[0256] Library construction and screening Antibodies C1-N52S, C2, and C3 were produced as recombinant monovalent antibodies using the mNeonGreen tag as described in WO2007059782. Wild-type B7H4 and alanine mutants were individually expressed in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). One day after transfection, cells were harvested. Approximately 50,000 cells were incubated with 20 μL of mNeoGreen-labeled antibody of interest. The cells were incubated for 1 hour at room temperature. Subsequently, 150 μL of FACS buffer was added, and the cells were washed twice with FACS buffer. The cells were resuspended in 30 μL of fresh FACS buffer and analyzed by flow cytometry using an iQue screener (Intellicyt Corporation, USA).

[0257] The entire experiment was performed twice in duplicate.

[0258] Data analysis For each sample, the average antibody binding per cell was determined as the geometric mean fluorescence intensity (gMFI) of the ungated cell population. gMFI is affected by the affinity of the antibody for the B7H4 variant and the expression level of the B7H4 variant per cell. Because individual alanine mutations can affect the surface expression level of mutant B7H4, to correct for differences in expression across each B7H4 variant, data were normalized to the binding intensity of a non-cross-blocking B7H4-specific reference antibody using the following formula: TIFF0007749575000021.tif10128 where C2 is used as the reference antibody for C1-N52S and C3, C1-N52S is used as the reference antibody for C2, and "aa position" refers to either a specific ala mutant of B7H4 or wild-type (wt) B7H4.

[0259] To express the decrease or increase in antibody binding on a linear scale of fold change, the following calculation was used: TIFF0007749575000022.tif10128

[0260] The increased binding is in most cases due to decreased binding of the reference antibody to the individual ala variants.

[0261] In these calculations, amino acid positions that result in neither a decrease nor an increase in binding by a particular antibody when the amino acid is substituted with alanine are assigned a result of "0," an increase in binding results in ">0," and a decrease in binding results in "<0." To correct for sample variability, only B7H4 amino acid residues with a fold change in binding less than the mean fold change - 1.5 x SD (SD is the standard deviation of the fold change calculated from four independent experiments with a particular test antibody) were considered "reduced binding mutants."

[0262] The gMFI of the reference antibody for a particular B7H4 variant is calculated as the mean gMFI - 2.5 × (mean gMFI 対照Ab If the difference was smaller than the standard deviation (SD), the data were excluded from the analysis (assuming that the expression levels were not sufficient for these B7H4 variants).

[0263] Figure 3 shows the fold change in binding of the B7H4 antibody to B7H4 variants with ala mutations in the ECD, annotating amino acid residues for which the fold change in binding was less than the mean fold change - 1.5 x SD. Fold changes are shown as Z scores in Figure 3. These results demonstrate the following: that the binding of antibody C1-N52S is dependent on at least the following amino acids in the IgC domain of human B7H4: S151, V157, D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242, and I245; that the binding of antibody C2 is dependent on at least the amino acids R98, G99, R116, K118, N119, and D124, which are present in the IgV of human B7H4; and that the binding of antibody C3 is dependent on at least the amino acids N156, E164, V217, and R248, which are present in the IgC domain of human B7H4; and Antibodies C1-N52S, C2, and C3 recognize different functional epitopes on B7H4.

[0264] Example 8 Binding of B7H4 Monospecific Antibody and CD3xB7H4 Bispecific Antibody to B7H4 from Various Species First, we analyzed the binding of bispecific CD3xB7H4 and monospecific B7H4 antibodies to HEK-293F cells transiently transfected with human B7H4 or cynomolgus monkey (Macaca fascicularis) B7H4 by flow cytometry. Untransfected HEK-293F cells were used as a negative control; these cells also did not express CD3.

[0265] Cells (3 × 10 ) were cultured in a polystyrene 96-well round-bottom plate (Greiner bio-one, catalog no. 650180) containing serial dilutions of antibody (ranging from 0.000458 to 30 μg / mL in 4-fold dilution steps) in 100 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer). 4 Cells (1000 cells / well) were incubated at 4°C for 30 minutes. Experiments were performed in technical duplicates. After washing twice in staining buffer, cells were incubated in 50 μL of secondary antibody at 4°C for 30 minutes. The secondary antibody used was R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (1:500 in FACS buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, catalog number 109-116-098). Cells were washed twice in staining 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 by nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0266] Figure 4 shows that both IgG1-B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR bound to cells expressing human B7H4 or cynomolgus B7H4.

[0267] Next, binding to HEK-293F cells transiently transfected with B7H4 derived from dog, rabbit, rat, mouse, or pig was determined as described above. Figure 5 shows that IgG1-B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR bound to B7H4 derived from dog, rabbit, rat, and mouse to varying degrees; for each, the apparent affinity (EC50) of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR was lower than that of IgG1-B7H4-C1-N52S-FEAR. bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR failed to bind to porcine B7H4, whereas IgG1-B7H4-C1-N52S-FEAR showed weak binding only at the highest antibody concentration tested.

[0268] The EC50s for binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and IgG1-B7H4-C1-N52S-FEAR to human B7H4 and cynomolgus B7H4 were in a similar range.

[0269] Similar studies were performed to compare the binding of IgG1-B7H4-C1-052S-FEAR, IgG1-B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and IgG1-B7H4-C5-FEAR to B7H4 from various species (human, cynomolgus monkey, mouse, rat, rabbit, dog, and pig). Figure 6 shows that binding to HEK cells transfected with human B7H4 or cynomolgus monkey B7H4 was similar among the antibodies tested. Similar results were obtained using cells expressing rabbit B7H4 and canine B7H4. However, the binding of IgG1-B7H4-C1-N52S-FEAR to mouse B7H4 appeared to be weaker than that of IgG1-B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and IgG1-B7H4-C5-FEAR, which is consistent with the results of Example 3. The binding of IgG1-B7H4-C1-N52S-FEAR and IgG1-B7H4-C3-FEAR to rat B7H4 appeared to be weaker than that of IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and IgG1-B7H4-C5-FEAR. Furthermore, IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and IgG1-B7H4-C5-FEAR bound to porcine B7H4, whereas binding of IgG1-B7H4-C1-052S-FEAR was very weak and only occurred at the highest antibody concentration tested. Binding of IgG1-B7H4-C3-FEAR to porcine B7H4 was undetectable.

[0270] Example 9 Binding of B7H4 Monospecific Antibody and CD3xB7H4 Bispecific Antibody to B7H4-Expressing Human Tumor Cell Lines IgG1-B7H4-C1-N52S-FEAR and / or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR to the B7H4-expressing human tumor cell lines MCF-7 (breast adenocarcinoma; ATCC catalog no. HTB-22), MDA-MB-468 (breast adenocarcinoma; ATCC, catalog no. HTB-132), and SK-BR3 (breast adenocarcinoma; ATCC catalog no. HTB-30). The binding of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and / or bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR to the B7H4-expressing human tumor cell lines NIH-OVCAR-3 (ovarian adenocarcinoma; ATCC, catalog no. HTB-161) or HCC1954 (breast ductal carcinoma; ATCC, catalog no. CRL-2338) was determined. Furthermore, MDA-MB-468 cells and HCC1954 cells were treated with IgG1-B7H4-C1-N52S-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, bsIgG1-huCD3-FEAL×B7H4-C2-FEAR, or bsIgG1-huCD3-H101G-FEAL×B7H4-C2-FEA. Binding of R, IgG1-B7H4-C3-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR, IgG1-B7H4-C5-FEAR, and / or bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR was also determined. Solid tumor cell lines typically do not express CD3. As a negative control, the tumor cell line HeLa (cervical adenocarcinoma; ATCC, catalog number CCL-2), which does not show detectable B7H4 expression, was used. Binding was analyzed by flow cytometry as described above.

[0271] Figure 7 shows that IgG1-B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR showed similar dose-dependent binding to MCF-7 and MDA-MB-468 cells, with similar maximum binding levels.

[0272] FIG. 8 shows dose-dependent binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR to NIH-OVCAR-3 and HCC1954 cells and no detectable binding to the non-B7H4 expressing cell line HeLa.

[0273] The binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR to B7H4-expressing tumor cells was compared using MDA-MB-486 and SK-BR3 cells. Figure 9 shows that bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR showed similar dose-dependent binding to these cells and similar maximum binding levels.

[0274] Figure 10 shows the dose-dependent binding of homodimeric or bispecific C1-N52S, C2, C3, C4, and C5 B7H4 antibodies to MDA-MB-468 and HCC1954 cells. The C4- and C5-based antibodies showed the most efficient binding, the C1-N52S- and C2-based antibodies showed intermediate binding efficiency, and the C3-based antibody showed the lowest binding efficiency. Maximum binding was comparable among the C1-N52S-, C2-, C4-, and C5-based antibodies, but was at a low level for the C3-based antibody.

[0275] Example 10 Binding of B7H4 Antibody to Primary Tumor Cells Primary tumor cells from an ovarian cancer patient were obtained from Discovery Life Sciences (Huntsville, AL, USA; patient ID 110045042). Binding of IgG1-B7H4-C1-N52S-FEAR to tumor cells was assessed by flow cytometry: cells were plated at 2 × 10 in a 96-well round-bottom polystyrene plate (Greinerbio-one, catalog number 650180). 4 Cells / well were plated, centrifuged, and incubated with 50 μl of fixable viability stain FVS-BV510 (BD Biosciences, Cat. No. 564406) diluted 1:1000 in PBS for 30 minutes at 4°C. After washing in staining buffer, cells were incubated with FITC-labeled IgG1-B7H4-C1-N52S-FEAR and a panel of CD3-specific antibodies (EF450-labeled; eBioscience, catalog no. 48-0037-42), CD45-specific antibodies (BV786-labeled; Biolegend, catalog no. 304048), CD14-specific antibodies (PE-Cy7-labeled; BD Biosciences, catalog no. 557742), CD86-specific antibodies (PerCP-Cy5.5-labeled; Biolegend, catalog no. 305420), CD163-specific antibodies (APC-Cy7-labeled; Biolegend, catalog no. 333622), and EpCAM-specific antibodies (AF700-labeled; R&D Systems, catalog no. FAB9601N) for 30 minutes at 4°C. After washing, cells were resuspended in staining buffer and analyzed using a FACS Fortessa (BD Biosciences). Live cells were identified by gating on single cells based on scatter FSC / SSC and excluding FVS-BV510-positive cells. Tumor cells were identified as EpCAM-positive cells.

[0276] Flow cytometry analysis showed that IgG1-B7H4-N52S-FEAR bound to viable EpCAM-positive tumor cells but not to monocytes or T cells in suspensions of dissociated tumor cells from ovarian cancer samples.

[0277] Example 11 In vitro induction of T cell-mediated cytotoxicity by CD3xB7H4 bispecific antibody using purified T cells as effector cells at various effector-to-target ratios. To determine the efficiency of T cell-mediated tumor cell killing in the presence of the bispecific antibodies bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, in vitro cytotoxicity assays were performed using B7H4-positive tumor cell lines as target cells and purified T cells as effector cells at various effector-to-target cell (E:T) ratios.

[0278] T cells were obtained from buffy coats of healthy human donors (Sanquin, Amsterdam, The Netherlands) and isolated using RosetteSep™ Human T Cell Enrichment Cocktail (Stemcell Technologies, France, Catalog No. 15061) according to the manufacturer's instructions. SK-BR3 cells (16,000 cells / well) were seeded into flat-bottom 96-well plates (Greiner-bio-one, The Netherlands, Catalog No. 655180) and allowed to adhere for 4 hours at 37°C. T cells were added to tumor cells at effector-to-target (E:T) ratios of 2:1, 4:1, or 8:1. Serial dilutions of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR were added (final concentrations ranging from 10,000 to 0.0128 ng / mL; 5-fold dilutions), and the plates were incubated at 37°C for 72 hours. The plates were washed three times with PBS, and the cells were incubated with 150 μl / well of 10% alamarBlue® solution (Invitrogen, catalog no. DAL1100) for 4 hours at 37°C. As a positive control for cytotoxicity, cells were incubated with 16 μg / mL phenylarsine oxide (PAO; Sigma-Aldrich, catalog no. P3075; dissolved in dimethyl sulfoxide [DMSO; Sigma-Aldrich, catalog no. D2438]). AlamarBlue fluorescence, an indicator of the metabolic activity of tumor cell cultures and therefore viable tumor cells, was determined at 615 nm (OD615) in an EnVision plate reader (PerkinElmer). The absorbance of tumor cell samples treated with PAO was set to 0% viability, and the absorbance of untreated tumor cell samples was set to 100% viability. The "% Viable Cells" was calculated as follows: Viability (%) = ([absorbance of sample - absorbance of PAO-treated target cells] / [absorbance of untreated target cells - absorbance of PAO-treated target cells]) x 100

[0279] Dose-response curves were constructed by nonlinear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA) to determine IC50 values.

[0280] Figure 11 shows that T cell-mediated cytotoxicity was observed at all E:T ratios, with maximal tumor cell killing (<10% surviving tumor cells) observed at an E:T ratio of 8:1.

[0281] Example 12 In vitro induction of cytotoxicity in various tumor cell lines by CD3xB7H4 bispecific antibodies and correlation with B7H4 expression levels T cell-mediated killing by the bispecific antibodies bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR against various B7H4-expressing tumor cell lines was determined in an in vitro cytotoxicity assay described previously. The following cell lines were used: MCF-7, MDA-MB-486, SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650. From each incubation, 150 μL of T cell-containing supernatant was transferred to a U-bottom 96-well culture plate (CellStar, catalog no. 650180) prior to washing and alamarBlue incubation (to determine T cell activation and cytokine release, as described below).

[0282] B7H4 expression was quantified in these tumor cell lines by quantitative flow cytometry (human IgG calibrator, BioCytex) according to the manufacturer's instructions, using bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR to detect B7H4.

[0283] Figure 12 shows that both bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR induced dose-dependent T cell-mediated cytotoxicity in MCF-7, MDA-MB-486, SK-BR3, NIH-OVCAR-3, and HCC1954 cells in vitro. Maximal cytotoxic activity (<10% surviving tumor cells) was achieved for both bsAb variants, but this occurred at lower concentrations for bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR compared to bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (Table 13).

[0284] No significant relationship between tumor cell lysis and B7H4 expression levels was observed (Figure 13A) for either bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (Figure 13B). Figure 13B shows the IC50 of T cell-mediated killing for each cell line using T cells from four to six donors in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR. Cell lines are ranked from lowest to highest B7H4 expression levels, demonstrating that T cell-mediated killing can occur across a wide range of B7H4 expression levels.

[0285] Table 13 summarizes the results from a panel of five cell lines and four donors.

[0286] Table 13. In vitro induction of cytotoxicity in various tumor cell lines by CD3xB7H4 bispecific antibodies. TIFF0007749575000023.tif58128bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR also induced T cell-mediated cytotoxicity against the tested NCI-H1650 NSCLC cell line in a dose-dependent manner.

[0287] Example 13 In vitro induction of T cell activation and cytokine production by CD3xB7H4 bispecific antibodies in the presence of B7H4-positive tumor cells U-bottom 96-well culture plates containing supernatants collected during the in vitro T cell-mediated cytotoxicity experiments described in Example 12 were centrifuged (300 × g) for 3 minutes at 4°C, after which 75 μL of supernatant was transferred to a new plate for cytokine production determination, and T cells were saved for assessment of T cell activation (described below). Cytokine production was analyzed by a multiplex U-plex assay (MeSo Scale Discovery, USA, catalog number K15049K) according to the manufacturer's instructions.

[0288] T cells were stained for T cell markers CD3 (1:200; eBioscience, clone OKT3, conjugated to eFluor450), CD4 (1:50; eBioscience, clone OKT4, conjugated to APC-eFluor780), CD8 (1:100; Biolegend, clone RPA-T8, conjugated to AF700), and T cell activation markers CD69 (1:50; BD Biosciences, clone AB2439, conjugated to APC), CD25 (1:50; eBioscience, clone BC96, conjugated to PE-Cy7), and CD279 / PD1 (1:50; Biolegend, clone EH12.2H7, conjugated to BV605). A single-stained sample (5 μL; Invitrogen, catalog number 01-2222-42) with Ultracomp beads was included and used for flow cytometer compensation. After a 30-minute incubation at 4°C, the plate was washed three times with PBS / 0.1% BSA / 0.02% azide (staining buffer). Cells were resuspended in 120 μL of staining buffer and analyzed using a FACS Fortessa (BD Biosciences). Data were processed using FlowJo (BD Biosciences).

[0289] Dose-response curves, EC50, EC90, and EC99 values ​​were calculated by nonlinear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0290] Figure 14A shows T cell activation against B7H4-positive tumor cell lines in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, as revealed by expression of the activation marker CD69 on the surface of CD8+ T cells (determined by flow cytometry). Figure 14B shows the EC50 of T cell activation using T cells from 3-4 donors against each tumor cell line.

[0291] Overall, a subset of CD8+ T cells (approximately 20–50% at the highest antibody concentration) became activated in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR. T cell activation induced by bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR generally occurred at higher concentrations than that induced by bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (Figure 14A). For both bispecific antibodies, the EC50 for T cell activation varied depending on the target cell line and donor used (Figure 14B).

[0292] Cytokine production in the supernatants of tumor cell-T cell cultures was assessed by Mesoscale Discovery U-plex multiplex ELISA. Among 10 cytokines analyzed across a cell line panel using T cells from four donors, significant increases in cytokine levels were primarily observed for IFN-γ and IL-8 (>2000 pg / ml). IL-4, IL-6, and IL-13 were regulated to much lower levels (<500 pg / ml), while IL-1β, IL-2, IL-10, IL-12p70, and TNFα levels were typically below 50 pg / ml. We present data on IFN-γ because its changes were robust and consistent and because it is one of the central cytokines elevated in the serum of patients with cytokine release syndrome.

[0293] Figure 15 shows IFN-γ levels in supernatants of T cell-tumor cell cocultures using T cells from at least three analyzed donors per cell line at antibody concentrations that induced T cell-mediated cytotoxicity in 50%, 90%, and 99% of tumor cells in the presence of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (EC50, EC90, and EC99, respectively). Cytokine production levels varied depending on the donor and target tumor cell line. Nevertheless, at antibody concentrations that induced similar percentages of tumor cell killing, cytokine production levels were typically lower after exposure of T cell-tumor cell cocultures to bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR compared to levels after exposure of the cocultures to bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR. Thus, at similar levels of tumor cell killing, incubation with bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR resulted in less cytokine production than bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR.

[0294] Example 14 Non-clinical safety study of CD3xB7H4 bispecific antibodies in cynomolgus monkeys The nonclinical safety profiles of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR in nonhuman primates (cynomolgus monkeys, Macaca fascicularis, from Mauritius) were evaluated at Citoxlab (France). Based on the species specificity of the CD3 arms of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, and further due to the similar binding of the B7H4 arms to human and cynomolgus B7H4, as well as additional pharmacological findings, cynomolgus monkeys were considered the only appropriate species for nonclinical safety studies. These studies were carried out in accordance with animal health regulations (Council Directive 2010 / 63 / EU of 22 September 2010 on the protection of animals used for scientific purposes and French Decree No. 2013-118 of 1 February 2013).

[0295] The goal of these studies was to characterize the potential toxicity and toxicokinetics of the CD3xB7H4 bispecific antibody. Only the results of the toxicokinetics and plasma cytokine level determinations are described here.

[0296] In two separate studies, animals were treated with a single intravenous (IV) infusion of 0.1 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR or bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR (one female animal per dose). The day of infusion was designated study day 1. Blood samples were collected twice pre-dose and at 0.5, 2, 4, 12, 24, and 48 hours post-dose to assess toxicokinetic profiles and plasma cytokine levels, and additionally at 168, 336, and 504 hours post-dose for toxicokinetics.

[0297] Cytokine levels Plasma samples were analyzed for cytokine levels (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF, IL-12p70, IL-15, and CCL2 / MCP1) using Luminex xMAP technology.

[0298] As shown in Figure 16, administration of BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR to cynomolgus monkeys resulted in only small changes in plasma cytokine levels that were considered unrelated to the test compound, whereas administration of bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR resulted in a dose-dependent increase in IL-6 and MCP-1 levels.

[0299] The lower cytokine levels observed after treatment with the bispecific BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR compared to the BsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR antibody may be beneficial in the clinical setting.

[0300] toxicokinetics Plasma concentrations of the CD3xB7H4 bispecifics were determined using a standard IgG PK ECLIA method. Toxicokinetic parameters were estimated using Certara Phoenix WinNonlin pharmacokinetic software version 8.1 using a noncompartmental approach consistent with the route of administration, i.v. infusion injection. Figure 17 shows that the toxicokinetic profiles of both CD3xB7H4 bispecific antibodies were remarkably similar up to 7 days post-dose, and both demonstrated dose-related plasma exposure.

[0301] Pharmacokinetic modeling exercises were performed to assess whether the predicted clinical dose range required by the BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR variant with low CD3 affinity was unsustainably high. A PK model based on observations in cynomolgus monkeys was used. A clinical dose range predicted to elicit a weekly mean plasma exposure equivalent to the EC50-EC90 for T cell-mediated cell killing observed in vitro was derived. The resulting dose range was considered feasible, and this nature did not a priori support one type of bispecific antibody over the other (BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR vs. BsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR).

[0302] Example 15 B7H4 Expression in Various Human Cancer Indications B7H4 mRNA levels were extracted from the TCGA database of Omicsoft and visualized using Oncoland software (Qiagen, USA).

[0303] Figure 18 shows B7H4 mRNA expression levels in a range of primary solid tumors ranked by median expression. mRNA expression was observed across a wide range of cancer indications, with variability within each indication, with the highest median expression observed in uterine carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), invasive breast cancer (BRCA), uterine endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), and cholangiocarcinoma (CHOL).

[0304] B7H4 protein expression was analyzed by immunohistochemistry (IHC) in tissue microarrays (TMA; all purchased from BioMax) for colon, lung (small cell lung cancer [SCLC] and non-small cell lung cancer [NSCLC]), gastric, pancreatic, bladder, cervical, head and neck, breast (including triple-negative breast cancer [TNBC]), ovarian, esophageal, renal, prostate, uterine, and bile duct cancers. Prior to staining, freshly cut TMA sections (5 μm) were deparaffinized and incubated with target retrieval solution pH 9 (DAKO, S2367; 97°C for 30 minutes, cooled for 60 minutes). B7H4 IHC was performed on a LabVision automated stainer platform using a commercially available rabbit anti-human B7-H4 monoclonal antibody (clone D1M8I, #14572, Cell Signaling Technologies) at an optimal dilution (1:25; final concentration 2.6 μg / mL) for 30 minutes at room temperature. Sections were then incubated with an anti-rabbit IgG polymer (Envision™ FLEX+ Rabbit (DAKO, S2022)), washed, and incubated with DAKO Liquid DAB+ Substrate-Chromogen System (DAKO, K3468). Hematoxylin (DAKO, S3301) was used to detect nucleated cells. Cytokeratin IHC (to identify tumor regions of interest (ROIs)) was performed using a mouse anti-cytokeratin antibody mix (clone AE1 / AE3) on a Ventana Benchmark with OptiView detection. Cytokeratin was visualized with DAB, and nuclei were counterstained with hematoxylin using the default Ventana reagent. Stained TMA sections were digitized on an AxioScan (Zeiss) at 20x magnification. Initially, manual scoring was performed to determine the mean B7H4 staining intensity (negative-low-moderate-high) and the percentage of tumor centers with >10% B7H4-positive tumor cells.

[0305] Subsequently, automated scoring was performed. Tumor ROIs were defined using a cytokeratin mask on the surface of TMA sections adjacent to the TMA sections stained for B7H4. B7H4 staining intensity in the tumor ROI was quantified (negative, weak (1), moderate (2), or strong (3)), and the percentage of B7H4-positive tumor cells (range 0-100%) was determined using HALO image analysis software. For each indication, the percentage of tumor center with more than 10% B7H4-positive tumor cells was determined.

[0306] Table 14 shows B7H4 protein expression as determined by IHC analysis of the BioMax TMA. Absent to very low B7H4 expression was observed in colon, prostate, renal, and small cell lung cancer samples. B7H4 expression in samples from other indications varied, with graded increases in B7H4 expression found in gastric, pancreatic, bile duct, esophageal, bladder, non-small cell lung (particularly squamous NSCLC), cervical, head and neck, breast (triple-negative breast cancer [TNBC] and non-TNBC), ovarian, and uterine cancers.

[0307] Table 14. B7H4 protein expression determined by IHC analysis of BioMax TMA. ND = not determined. TIFF0007749575000024.tif240153

Claims

1. An antibody comprising an antigen-binding region capable of binding to human B7H4 and further comprising an antigen-binding region capable of binding to human CD3, wherein the antigen-binding region capable of binding to human B7H4 is CDR1 sequence shown in SEQ ID NO: 26, the CDR2 sequence shown in SEQ ID NO: 30, and CDR3 sequence shown in SEQ ID NO: 28 a heavy chain variable region (VH) comprising: CDR1 sequence shown in SEQ ID NO: 34, a CDR2 sequence having the sequence GAS, and CDR3 sequence shown in SEQ ID NO: 35 An antibody comprising a light chain variable region (VL) comprising:

2. The antibody of claim 1, wherein the antigen-binding region capable of binding to human B7H4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 29 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

33.

3. 3. The antibody of claim 1 or 2, which is a bispecific antibody.

4. The antibody of any one of claims 1 to 3, which is capable of binding to cancer cells and T cells.

5. The antibody described in claim 4, wherein the cancer cells express human B7H4 and / or are solid tumor cells.

6. The antibody according to any one of claims 1 to 5, wherein the antigen-binding region capable of binding to CD3 is capable of binding to human CD3ε (epsilon).

7. An antibody described in any one of claims 1 to 5, wherein the antigen-binding region capable of binding to CD3 is capable of binding to human CD3ε (epsilon) shown in SEQ ID NO:

13.

8. the antigen-binding region that binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL); (a) the VH comprises the CDR1, CDR2, and CDR3 regions of the VH sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, and the VL comprises the CDR1, CDR2, and CDR3 regions of the VL sequence shown in SEQ ID NO: 22; or (b) the VH comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, or the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21, respectively, and the VL comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 23, GTN, and SEQ ID NO: 24, respectively; An antibody described in any one of claims 1 to 7.

9. The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 16 or 17, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 16 or 17; and A light chain variable region (VL) comprising 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 to the sequence of SEQ ID NO:

22. , with the proviso that it does not contain mutations in CDR1, CDR2, and CDR3 of VH and CDR1, CDR2, and CDR3 of VL; An antibody described in any one of claims 1 to 8.

10. the antigen-binding region capable of binding to human B7H4 is capable of binding to the IgC-like constant region of human B7H4 and / or is capable of binding to B7H3-IgV / B7H4-IgC having the sequence of SEQ ID NO: 11 and / or is incapable of binding to B7H4-IgV / B7H3-IgC having the sequence of SEQ ID NO: 10; An antibody described in any one of claims 1 to 9.

11. The antibody of any one of claims 1 to 10, which is a full-length antibody.

12. 12. The antibody of any one of claims 1 to 11, which is of the IgG1 isotype.

13. 13. The antibody of any one of claims 1 to 12, comprising a first heavy chain and a second heavy chain, wherein (a) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in the first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain; or (b) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain; Antibodies, where amino acid positions are numbered according to EU numbering.

14. 14. The antibody of any one of claims 1 to 13, wherein the antibody comprises a first heavy chain and a second heavy chain, and in both the first heavy chain and the second heavy chain, the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively.

15. 15. The antibody of claim 14, wherein the antibody comprises a first heavy chain and a second heavy chain, and in both the first heavy chain and the second heavy chain, the amino acid residue at a position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is A.

16. an antigen-binding region capable of binding to human B7H4 is contained in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a kappa light chain constant region; and an antigen-binding region capable of binding to human CD3 is contained in the heavy chain and the light chain, the heavy chain comprising a VH region and an IgG1 heavy chain constant region, and the light chain comprising a VL region and a lambda light chain constant region; An antibody described in any one of claims 1 to 15.

17. An IgG1 heavy chain constant region comprising: one IgG1 heavy chain constant region as defined in SEQ ID NO: 60; and the other IgG1 heavy chain constant region as defined in SEQ ID NO: 61; and the kappa light chain constant region is as defined in SEQ ID NO: 63, and the lambda light chain constant region is as defined in SEQ ID NO: 64; The antibody of claim 16.

18. 18. The antibody of claim 17, wherein the terminal lysine is deleted in the IgG1 heavy chain constant region defined in SEQ ID NO: 60 and SEQ ID NO:

61.

19. The antigen-binding region that can bind to human B7H4 a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 25, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; b) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; c) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; d) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and a variable light chain region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively; e) the variable heavy chain (VH) region of SEQ ID NO: 25 and the variable light chain region of SEQ ID NO: 33; or f) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33 An antibody comprising an antigen-binding region capable of binding to human B7H4, comprising:

20. A composition comprising an antibody according to any one of claims 1 to 19.

21. 21. The composition of claim 20, which is a pharmaceutical composition comprising the antibody of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

22. A medicament for treating a disease in a subject, comprising the antibody of any one of claims 1 to 19.

23. 23. The pharmaceutical composition of claim 22, wherein the disease is cancer.

24. The pharmaceutical agent of claim 23, wherein the cancer is characterized by expression of B7H4 in cancer cells.

25. The pharmaceutical agent according to claim 23 or 24, wherein the cancer is a solid tumor.

26. The pharmaceutical agent according to any one of claims 23 to 25, wherein the cancer is selected from the group consisting of lung cancer, NSCLC (ADC or SQCC), gastric cancer, pancreatic cancer, bile duct cancer, bladder cancer, cervical cancer, head and neck cancer, breast cancer, ovarian cancer, and uterine cancer.

27. a) a nucleic acid sequence encoding a heavy chain variable region sequence of an antigen binding region capable of binding to B7H4 of any one of claims 1, 2, or 19; and b) a nucleic acid sequence encoding the corresponding light chain variable region sequence of said antigen-binding region capable of binding to B7H4 of any one of claims 1, 2, or 19. A nucleic acid comprising:

28. a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to B7H4 according to any one of claims 1 or 2; b) a nucleic acid sequence encoding the corresponding light chain sequence of an antibody comprising an antigen-binding region capable of binding to B7H4 according to any one of claims 1 or 2; c) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 8 to 10; and d) A nucleic acid sequence encoding the corresponding light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 8 to 10. One or more nucleic acids comprising:

29. 29. The nucleic acid or nucleic acids according to claim 27 or 28, which is RNA or DNA.

30. 30. A cell comprising the nucleic acid or one or more nucleic acids of any one of claims 27 to 29.

31. The following stages: a) providing an antibody capable of binding to B7H4, wherein the antibody comprises an antigen-binding region capable of binding to B7H4 according to any one of claims 1 to 19; b) providing an antibody capable of binding to CD3, said antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 1 to 19; c) incubating the antibody capable of binding to B7H4 with the antibody capable of binding to CD3 under reducing conditions sufficient to cause disulfide bond isomerization at cysteines in the hinge region; and d) obtaining antibodies capable of binding to B7H4 and CD3; 20. A method for producing an antibody capable of binding to both B7H4 and CD3 according to any one of claims 1 to 19, comprising:

32. A kit for use as a companion diagnostic, comprising an antibody according to any one of claims 1 to 19 and instructions for using the kit.

33. A kit for identifying patients within a patient population who have a property that will respond to treatment using an antibody described in any one of claims 1 to 19, the kit comprising an antibody described in any one of claims 1 to 19 and instructions for using the kit.

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