Pharmaceutical compositions comprising bispecific antibodies that bind to B7H4 and CD3

Abstract

The present invention relates to pharmaceutical compositions and unit dosage forms comprising antibodies that bind to B7H4 and CD3. The present invention further provides the use of said pharmaceutical compositions and unit dosage forms for therapeutic and diagnostic procedures, particularly in cancer therapy.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to pharmaceutical compositions and unit dosage forms comprising a bispecific antibody that binds B7H4 and CD3, and to uses of such pharmaceutical compositions or unit dosage forms. [Background technology]

[0002] Introduction B7H4 (B7-H4, T-cell activation inhibitor containing V-set domain 1, or VTCN1) is a member of the B7 family of proteins, which includes 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 that contains a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with IgV-like and IgC-like domains in which there are four conserved cysteine ​​residues and seven sites for N-linked glycosylation. (Sica et al., 2003, Immunity 18: 849-861). To date, a receptor for B7H4 has not been identified.

[0003] While B7H4 expression is very limited in normal mature tissues, it has been found 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] Thus, targeting B7H4 for the treatment of cancer has been proposed (Podojil and Miller, Immunological Reviews, 2017: 276; 40-51). Currently, antibodies that bind to B7H4 are in development for cancer therapy. For example, FPA150 is an afucosylated human antibody that attenuates B7H4-mediated inhibition of T cell activation and exhibits antibody-dependent cellular cytotoxicity (ADCC) activity (Wainberg et al., 2019, Annals of Oncology 30, Suppl. 5, v489 (1198P)). Currently, FPA150 is 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 mouse anti-human B7H4 and mouse anti-human CD3 antibodies (Iizuka et al., 2019, Clin Cancer Res 25: 2925-2934 (Non-Patent Document 6)). Smith et al. described engineered T cells with 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 lymphocytic 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 remains a need to develop antibody-based cancer therapies that target B7H4 that are effective and / or safe for use in humans. There is also a need for pharma- ceutically acceptable formulations of antibodies for use in such therapies. [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]

[0008] It is an object of the present invention to provide novel pharmaceutical compositions comprising 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). A further object is to provide pharmaceutical compositions comprising antibodies, the formulations of which are stable over a wide range of antibody concentrations and / or temperatures. A further object is to provide pharmaceutical compositions comprising antibodies, the formulations of which are stable over a period of at least 3 months, or even longer, such as at least 6 months or at least 12 months. A further object of the present invention is to provide pharmaceutical formulations of antibodies that are well tolerated when infused IV. The antigen-binding regions of such antibodies comprise at least human framework regions, such as, for example, FR1, FR2, FR3, and FR4. Most preferably, all framework regions are of human origin. Such antigen-binding regions are humanized and / or human antigen-binding regions. These pharmaceutical compositions are useful in the treatment of conditions in which it is desirable to specifically target and T cell-mediated kill B7H4-expressing cells, such as, for example, in conditions such as cancer. Preferably, the pharmaceutical composition is suitable for use in humans, for example in medical treatment. The cancer that may be suitable for treatment is a solid tumor. For example, the B7H4 expression and T cell-mediated killing in cancer cells according to the present invention can range from relatively low B7H4 expression, such as in MCF-7 cells, to relatively high B7H4 expression, such as 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 when present. In a preferred embodiment, the pharmaceutical composition of the present invention is suitable for both IV administration.

[0009] In a first aspect, the present invention provides a pharmaceutical composition comprising: a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin; and b) a buffering agent, the composition having a pH of 4.0-8.0, preferably 4.5-6.5, most preferably 5.0-6.0. The buffering agent is preferably selected from the group consisting of histidine, glutamate, and mixtures thereof, and the pharmaceutical composition preferably further comprises a non-ionic excipient. Such pharmaceutical compositions have been found to provide surprisingly high stability, e.g., thermal and storage stability, of the antibody, as well as a high degree of solubility.

[0010] In one embodiment, a pharmaceutical composition of the invention comprises a bispecific antibody comprising 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 wherein 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. Preferably, the CDR regions are determined by the IMGT method described below.

[0011] In a further aspect, the pharmaceutical composition according to the invention is for use in medical therapy. [Brief description 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 the B7H4 antibody was determined using a panel of cells transfected to express human B7H4 (I), 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. [Diagram 2] Binding of B7H4 antibodies to B7H4, B7H3, or B7H4-B7H3 chimeric molecules. 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 change in binding smaller than the mean fold change-1.5×SD were considered as "reduced binding variants". Residues with positive fold change in binding are residues with reduced binding relative to the reference antibody. Numbers below the x-axis refer to amino acid position. (A) Results for C1-N52S with C2 as reference antibody. (B) Results for C2 with C1-N52S as reference antibody. (C) Results for C3 with C2 as reference antibody. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] Binding of B7H4 and CD3xB7H4 bispecific antibodies to human and cynomolgus 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 or cynomolgus B7H4 was determined by flow cytometry. Non-transfected HEK-293F cells (C) were used as a negative control; for these, binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR is shown. [Diagram 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. Non-transfected HEK-293F cells (C) were used as negative controls; 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 untransfected 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 formats 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 (C bsAb), bsIgG1-huCD3-H101G-FEAL×B7H4-C4-FEAR (D bsAb), and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR (E bsAb) was determined by flow cytometry. bsIgG1-huCD3-H101G-FEAL×b12-FEAR (F bsAb) or IgG1-b12-K409R (F homodimer) were used as non-binding control antibodies. [Figure 11] In vitro induction of T cell-mediated cytotoxicity against SK-BR3 cells by CD3xB7H4 bispecific antibody using purified T cells as effector cells at various effector to target ratios (E:T). bsIgG1-huCD3-FEALxb12-FEAR was used as a non-binding control antibody. A=bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR; B=bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR; C=bsIgG1-huCD3-FEALxb12-FEAR. [Figure 12]In vitro induction of T cell-mediated cytotoxicity in various tumor cell lines in the presence of CD3xB7H4 bispecific antibodies with different CD3 arms. bsIgG1-huCD3-FEALxb12-FEAR was used as a non-binding control antibody. A=bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR; B=bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR; C=bsIgG1-huCD3-FEALxb12-FEAR, D=bsIgG1-huCD3-H101G-FEALxb12-FEAR. [Figure 13] B7H4 expression levels and IC50 of 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 of T cell-mediated tumor cell killing against various tumor cell lines in the presence of bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR (I) or bsIgG1-huCD3-H101G-FEAL x 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 value. Cell lines are ranked based on B7H4 expression levels. [Figure 14A] T cell activation by B7H4 bispecific antibodies in co-cultures of T cells and tumor cells. (A) T cell activation 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 (percentage of CD69 on CD8+ cells) as determined by flow cytometry. (B) EC50 of T cell activation using T cells from 3-5 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 supernatants of T cell and tumor cell co-cultures 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 3-4 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 injection 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 TCGA database from Omicsoft 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 carcinoma, HNSC=head and neck squamous cell carcinoma, PRAD=prostate adenocarcinoma, LUAD=lung adenocarcinoma, ESCA=esophageal carcinoma, CESC=cervical squamous cell carcinoma and endocervical 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 carcinoma, UCEC=uterine endometrial carcinoma, OV=ovarian serous cystadenocarcinoma, and CHOL=cholangiocarcinoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Table 1: Amino acid and nucleic acid sequences TIFF2024519212000001.tif194143TIFF2024519212000002.tif221143TIFF2024519212000003.tif230143TIFF2024519212 000004.tif227143TIFF2024519212000005.tif231143TIFF2024519212000006.tif229143TIFF2024519212000007.tif87143

[0014] The CDR regions in the above table (CDR1, CDR2, and CDR3, as well as the underlined sequences in the VH and VL sequences) have been 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 table 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 any derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions and / or tumor-specific conditions, with a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days, or any other suitable period of time that is functionally defined (e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the antibody binding to the antigen and / or a time sufficient for the antibody to be internalized). An antibody includes a binding region (or a binding domain, which may be used herein, both of which have the same meaning) that can interact with an antigen, or a binding region that includes both the heavy and light chain variable regions of an immunoglobulin molecule, and the like. The antibody can include an antibody (Ab) constant region that can mediate 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, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant DNA techniques. The term "antibody" includes bispecific antibodies and / or antibodies with further modifications, such as antibody-drug conjugates thereof.

[0017] The 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 and described as a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90), also known as dAb fragments (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 the Fv fragment, VL and VH, are encoded by separate genes, the VL and VH regions pair together 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 the antibody to be produced as a single protein chain forming a single chain (see, e.g., J. Am. Soc. 423-426 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. In general, such fragments are included within the meaning of antibody, but these fragments, collectively and each independently, are unique features of the invention and exhibit different biological properties and utilities. These and other antibody fragments useful 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 that 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 systems and then mixing the antibodies, or by producing several types of antibodies in the same production system.

[0020] The term "immunoglobulin heavy chain" or "heavy chain of immunoglobulin" as used herein is intended to refer to one of the heavy chains of immunoglobulins. A heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the immunoglobulin isotype. The heavy chain constant region is typically composed of three domains, namely CH1, CH2, and CH3. The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four chains optionally interconnected by disulfide bonds. The structural characteristics of immunoglobulins are well characterized (see, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989). In the structure of an immunoglobulin, the two heavy chains are linked together by disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain is typically composed of several regions, namely the light chain variable region (abbreviated herein as VL) and the light chain constant region. The light chain constant region is typically composed of one domain, namely 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), which are interrupted by regions of high degree of conservation called framework regions (FRs). Each of the VH and VL is typically composed 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 made by recombining half molecules derived from each of the first and second antibodies by any known method to result in a 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 making 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] The term "antigen-binding region" or "binding region" as used herein means a 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 term "blocking binding" or "blocking antibody binding" or "cross-blocking binding" or "cross-blocking binding" refers to the situation where 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 these 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, for example, as described 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, in sandwich epitope binning assay, an antibody in solution is examined for binding to its specific antigen that has been captured via immobilized antibody.In the present invention, if an antibody can bind to an antigen in the presence of a second antibody, the antibody does not interfere with the binding of the second antibody, and vice versa.The terms "interfere with binding" and "interfere with antibody binding" and "cross-interfere with binding" and "cross-interfere with binding" may 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 may also be described as competing with the other antibody to bind to the target.

[0024] "K D " (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 k off Also called the off-value or off-rate.

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

[0027] The term "binding" as used herein refers to the binding of an antibody to a given 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 non-specific 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 corresponding to

[0028] The term "B7H4" as used herein refers to the protein called B7H4, which is also called B7-H4, T-cell activation inhibitor 1 with V-set domain, 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 that contains a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with IgV-like and IgC-like domains with four conserved cysteine ​​residues and seven sites for N-linked glycosylation. (Sica et al., 2003, Immunity 18: 849-861). B7H4 proteins are known from a variety of species, including human (Homo sapiens) B7H4 (Uniprot Accession No. Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7H4 transcript 1 (Uniprot Accession No. A0A2K5U6P5), dog (Canis familiaris) B7H4 (Uniprot Accession No. F1P8R9), rabbit (Lepus argentii) B7H4 (Uniprot Accession No. G1TQE8), rat (Rattus norvegicus) B7H4 (Uniprot Accession No. Q501W4), mouse (Mus musculus) B7H4 (Uniprot Accession No. Q7TSP5), and pig (Sus scrofa) B7H4 (Uniprot Accession No. F1SAY4). Natural 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 thus the term "CD3" need 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 No. P09693, or cynomolgus CD3γ UniProtKB / Swiss-Prot No. Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No. P04234, or cynomolgus CD3δ UniProtKB / Swiss-Prot No. Q95LI8), and two CD3ε (epsilon) chains (human CD3ε: UniProtKB / Swiss-Prot No. P07766, the sequence of which is incorporated herein as SEQ ID NO: 13, in which amino acid residues 1 to 22 correspond to the signal peptide and amino acid residues 23 to 207 correspond to the mature CD3ε polypeptide; cynomolgus CD3ε or rhesus monkey CD3ε UniProtKB / Swiss-Prot No. G7NCB9), and the CD3 ζ chain (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No. P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No. Q09TK0). These chains bind to a molecule known as the T cell receptor (TCR) and generate an activation signal in T lymphocytes. Together, the TCR and CD3 molecules constitute 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 assay (using an antigen construct in which a region of the antigen is exchanged with that of another species and determining 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. An epitope usually consists of a surface collection of molecules such as amino acids, sugar side chains, or a combination thereof, and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational 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 that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are substantially blocked or covered by an antibody when the antibody binds to an antigen (in other words, these amino acid residues are included in or closely adjacent to the binding area of ​​a specific antibody).

[0032] As used herein, terms such as "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 can be 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 that contain B cells fused to immortalized cells, and the B cells are 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 allotypes 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 lambda (λ) light chain.

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

[0035] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody that comprises 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, into 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., the non-human antibody) (backmutation). Structural homology modeling may 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 that are primarily of human origin, optionally including one or more amino acid backmutations to non-human amino acid sequences, and constant regions that are entirely of human origin. 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, e.g., to avoid deamidation, to provide an "inactive 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). A "human antibody" can incorporate VH and VL sequences made 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 to be, for example, human VH and VL sequences fused to a constant domain derived from a human immunoglobulin constant domain.

[0037] Thus, a "human antibody" may be an engineered antibody. A "human antibody" may be further engineered, including, for example, modifications to avoid deamidation, to provide an "inactive Fc region", to allow the generation of bispecific antibodies, and / or to improve manufacturability. A human antibody may also be produced in a non-human cell, such as a CHO cell. 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 that includes at least the hinge, CH2, and CH3 regions of the two heavy chains of an antibody from the N-terminus to the C-terminus. 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 216-230 according to the EU numbering as set forth 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] The term "CH1 region" or "CH1 domain" as used herein means 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 set forth in Kabat, op. cit. However, the CH1 region may also be of any of the other subtypes described herein.

[0041] The term "CH2 region" or "CH2 domain" as used herein means 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, supra. However, the CH2 region may also be of any of the other subtypes described herein.

[0042] The term "CH3 region" or "CH3 domain" as used herein means 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 set out in Kabat, op. cit. 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 resulting 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-gamma 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] The term "inactive", "inactive" or "non-activated" as used herein refers to an Fc region that at least cannot bind to any FcγR, cannot induce Fc-mediated FcγR cross-linking, or cannot induce FcγR-mediated cross-linking of target antigens mediated by two Fc regions of individual antibodies, or cannot bind to C1q. An example of this is the FEA substitution within the constant domain described herein. The inactivity of the Fc region of an antibody can be tested using the 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 contains all of the domains corresponding to a particular isotype as normally found in nature, 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" in the present invention 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 crosslink 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 two identical epitopes on two identical antigens 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 that contain 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 characterization of the R group TIFF2024519212000008.tif49128

[0052] Table 3. Alternative physical and functional classification of amino acid residues. TIFF2024519212000009.tif83128

[0053] The substitution of one amino acid with another amino acid can be classified as conservative 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 above two tables: 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, substitutions in antibodies are indicated as the original amino acid-position-substitute amino acid. In terms of the well-recognized nomenclature for amino acids, three-letter or one-letter symbols, including the code "Xaa" or "X", are used to indicate any amino acid residue. Thus, Xaa or X typically represents any of the 20 natural amino acids. The term "natural" as used herein 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 designation "K409R" or "Lys409Arg" means 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 the case of modifications in which the original and / or replacement amino acids may include multiple, but not all, amino acids, the multiple amino acids may be separated by "," or " / ". For example, 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, the 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 represented 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 is specifically included herein.

[0057] Antibodies according to the invention may also include deletions of amino acid residues. Such deletions may be designated as "del", including, for example, writing 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 is introduced. It should be understood that such terms are not intended to mean only a particular subject cell, but may also include the progeny of such a cell. Since some modifications may occur in subsequent generations, either due to 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 lymphocytic cells, as well as prokaryotic cells such as E. coli, and other eukaryotic hosts such as plant cells and fungi.

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

[0060] For the purposes of the present invention, sequence identity between two amino acid sequences is determined over the entire length of the referenced sequence using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably as implemented in the Needle program of 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, named "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, as 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 higher (e.g., about 99%).

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

[0063] Pharmaceutical compositions and unit dosage forms comprising bispecific antibodies targeting CD3xB7H4 In a first aspect, a pharmaceutical composition or unit dosage form is provided, comprising 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 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, there is provided a pharmaceutical composition or unit dosage form comprising 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 comprise human framework regions.An antibody according to the invention described herein comprising 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, for example, as a CD3xB7H4 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 are cancer cells that originate 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 the following, as shown in the examples: that is, the antibody binds to its target in a binding assay, as shown, for example, by a typical binding curve as shown in Figures 3 and 4 herein, or by determining the binding affinity using, for example, biolayer interferometry as shown in Examples 3 and 4. An antigen-binding region that cannot bind to a specified target, for example, has an undetectable binding affinity to the target, for example, a response below 0.05 nm at the highest concentration used in a typical biolayer interferometry assay as shown in Example 3. In any case, those skilled in the art are familiar with how to determine whether an antigen-binding region can bind to its target.

[0066] In a first aspect, the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin; and b) Buffer A pharmaceutical composition comprising: pH is 4.0 to 8.0; The present invention relates to a pharmaceutical composition.

[0067] The pH is preferably 4.5 to 6.5. In a preferred embodiment, the pH of the composition is 5.0 to 6.5. In a particularly preferred embodiment, the pH of the composition is 5.0 to 6.0. These pH ranges have been found to provide particularly high stability, especially thermal stability, of the antibody. In the pH range of the present invention, the pharmaceutical composition also provides a high degree of solubility of the antibody.

[0068] In a preferred embodiment, the buffering agent is selected from the group consisting of histidine, glutamate, and mixtures thereof.

[0069] In a preferred embodiment, the pharmaceutical composition further comprises c) a non-ionic excipient.

[0070] In a preferred embodiment, the non-ionic excipient is a sugar or sugar alcohol.

[0071] In certain embodiments, the non-ionic excipient is selected from sorbitol, sucrose, or a mixture thereof. In a preferred embodiment, the non-ionic excipient is sorbitol.

[0072] In a preferred embodiment, the non-ionic excipient is present at a concentration of 100-300 mM, for example 125-250 mM, preferably 250 mM. In a preferred embodiment, the non-ionic excipient is sorbitol, present at a concentration of 125-250 mM, preferably 250 mM.

[0073] In a preferred embodiment, the pharmaceutical composition further comprises d) a surfactant.

[0074] In some embodiments, the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbintan fatty acid esters, polyoxyethylene sterarates, polyoxyl hydroxystearates, polyoxyl glycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan ester sucrose palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof.

[0075] In certain embodiments, the surfactant is a polysorbate, preferably polysorbate 20 or polysorbate 80. In a preferred embodiment, the surfactant is polysorbate 80.

[0076] In a preferred embodiment, the surfactant is present in a concentration of about 0.005% to 0.4% w / v, such as about 0.01 to 0.1% w / v, such as about 0.01 to 0.09% w / v, such as about 0.01 to 0.06% w / v, such as about 0.01 to 0.05% w / v, such as 0.02% w / v or 0.03% w / v or 0.04% w / v or 0.05% w / v, preferably 0.02% w / v. In a preferred embodiment, the surfactant is polysorbate 80 and is present in the pharmaceutical formulation at 0.02% w / v to 0.04% w / v. Preferably, the concentration is 0.02% w / v.

[0077] In a preferred embodiment, the concentration of the antibody is 0.5 to 100 mg / ml, for example, 1.0 to 50 mg / ml, or for example, 5 to 30 mg / ml, for example, 5 mg / ml, or 6 mg / ml, or 7 mg / ml, or 8 mg / ml, or 9 mg / ml, or 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or 25 mg / ml, or 26 mg / ml, or 28 mg / ml, or 29 mg / ml, or 30 mg / ml, or 31 mg / ml, or 32 mg / ml, or 33 mg / ml, or 34 mg / ml, or 35 mg / ml, or 36 mg / ml, or 37 mg / ml, or 38 mg / ml, or 39 mg / ml, or 40 mg / ml, or 41 mg / ml, or 42 mg / ml, or 43 mg / ml, or 44 mg / ml, or 45 mg / ml, or 46 mg / ml, or 47 mg / ml, or 48 mg / ml, or 49 mg / ml, or 50 mg / ml, or 51 mg / ml, or 52 mg / ml, or 53 mg / ml, or 54 mg / ml, or 55 mg / ml, or 56 mg / ml, or 57 mg / ml, or 58 mg / ml, or 59 mg / ml, or 60 mg / ml, or 61 mg / ml, or 62 mg / ml, or 63 mg g / ml, or 27 mg / ml, or 28 mg / ml, or 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, or for example 60 mg / ml. The most preferred concentration is 10 to 20 mg / ml, for example 20 mg / ml. However, the antibody may be present at 10 mg / ml. In another embodiment, the antibody is present at 15 mg / ml. In another embodiment, the antibody is present at 25 mg / ml. In another embodiment, the antibody is present at 30 mg / ml. In another embodiment, the antibody is present at 35 mg / ml. In another embodiment, the antibody is present at 40 mg / ml. In another embodiment, the antibody is present at 45 mg / ml. In another embodiment, the antibody is present at 50 mg / ml. In another embodiment, the antibody is present at 55 mg / ml. In another embodiment, the antibody is present at 60 mg / ml.

[0078] In a preferred embodiment, the buffer is present at a concentration of 5-40 mM, such as 10-30 mM, preferably 20 mM. In a preferred embodiment, the buffer is 20 mM glutamate at pH 5.1-5.3, such as pH 5.2. In yet another preferred embodiment, the buffer is 20 mM histidine at pH 5.7-5.9, such as pH 5.8.

[0079] In a preferred embodiment, the pharmaceutical composition is a liquid composition. In a preferred embodiment, the pharmaceutical composition is an aqueous composition.

[0080] In a preferred embodiment, the pharmaceutical composition comprises: a) 5 to 50 mg / ml of antibody, preferably 5 to 25 mg / ml of antibody, and most preferably 10 to 20 mg / ml of antibody; b) 10 to 20 mM glutamate, preferably 15 to 20 mM glutamate, most preferably 20 mM glutamate; c) 150 to 350 mM sorbitol, preferably 200 to 300 mM sorbitol, most preferably 250 mM sorbitol; d) Polysorbate, preferably Polysorbate 80, most preferably 0.02% w / v Polysorbate 80 Including, Here, the pH of the composition is 5.0 to 6.0, preferably 5.1 to 5.3.

[0081] In a preferred embodiment, the pharmaceutical composition comprises: a) 10-20 mg / ml antibody, b) 20 mM glutamate, c) 250 mM sorbitol, d) 0.02% w / v polysorbate 80, and the pH of the composition is 5.1-5.3.

[0082] In another preferred embodiment, the pharmaceutical composition comprises a) 10 to 20 mg / ml of antibody, b) 20 mM histidine, c) 250 mM sorbitol, d) 0.02% w / v polysorbate 80, and the pH of the composition is 5.7 to 5.9.

[0083] In another embodiment, the composition is an intravenous composition and / or the composition is for use in intravenous administration.

[0084] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 comprises a heavy chain variable region (VH), wherein CDR1 is as set forth in SEQ ID NO: 18, CDR2 is as set forth in SEQ ID NO: 19, and CDR3 is as set forth in SEQ ID NO: 21.

[0085] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 comprises a light chain variable region (VL), CDR1 is as set forth in SEQ ID NO:23, CDR2 is GTN, and CDR3 is as set forth in SEQ ID NO:24.

[0086] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 capable of binding to B7H4 comprises a variable heavy (VH) region, wherein CDR1 is as set forth in SEQ ID NO:26, CDR2 is as set forth in SEQ ID NO:30, and CDR3 is as set forth in SEQ ID NO:28.

[0087] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 capable of binding to B7H4 comprises a light chain variable region (VL), CDR1 is as set forth in SEQ ID NO:34, CDR2 is GAS, and CDR3 is as set forth in SEQ ID NO:35.

[0088] In a preferred embodiment, the antibody of the pharmaceutical formulation 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) in which CDR1 is as set forth in SEQ ID NO: 18, CDR2 is as set forth in SEQ ID NO: 19, and CDR3 is as set forth in SEQ ID NO: 21, and A light chain variable region (VL) in which CDR1 is as set forth in SEQ ID NO: 23, CDR2 is GTN, and CDR3 is as set forth in SEQ ID NO: 24. Including, The antigen-binding region capable of binding to B7H4 is a variable heavy chain (VH) region, wherein CDR1 is as set forth in SEQ ID NO: 26, CDR2 is as set forth in SEQ ID NO: 30, and CDR3 is as set forth in SEQ ID NO: 28; and A light chain variable region (VL) in which CDR1 is as set forth in SEQ ID NO: 34, CDR2 is GAS, and CDR3 is as set forth in SEQ ID NO: 35. Includes.

[0089] In a preferred embodiment, the antibody of the pharmaceutical formulation 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: 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 variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 29, and a variable light chain (VL) region comprising the CDR1, CDR2, and CDR3 regions, respectively, of SEQ ID NO: 33; wherein the CDR regions may be numbered according to IMGT.

[0090] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22. and The antigen-binding region capable of binding to B7H4 is A variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 29 and a variable light chain (VL) region comprising the amino acid sequence of SEQ ID NO: 33. Includes.

[0091] In a preferred embodiment, the antibody of the pharmaceutical formulation 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 amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22, and further comprising a constant heavy chain region (CH) comprising the amino acid sequence of SEQ ID NO: 60 and a constant light chain region (CL) comprising the amino acid sequence of SEQ ID NO: 64; The antigen-binding region capable of binding to B7H4 is It comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 29 and a variable light chain (VL) region comprising the amino acid sequence of SEQ ID NO: 33, and further comprises a constant heavy chain region (CH) comprising the amino acid sequence of SEQ ID NO: 61 and a constant light chain region (CL) comprising the amino acid sequence of SEQ ID NO: 63.

[0092] In a particularly preferred embodiment, the antibody of the pharmaceutical formulation is bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR or a biosimilar thereof.

[0093] The present invention also relates to the pharmaceutical composition defined herein for use as a medicament, for example for use in a method for treating a disease.In a preferred embodiment, the disease is cancer.In a preferred embodiment, the cancer is characterized by the expression of B7H4 in cancer cells.Preferably, the expression of B7H4 is determined in cancer cells obtained from a patient.

[0094] In a preferred embodiment, the cancer is a solid tumor.Preferably, 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.

[0095] In another aspect, the present invention relates to a method for treating disease, comprising administering to a subject in need thereof the pharmaceutical composition defined herein.In a preferred embodiment, the method is for treating cancer.In some embodiments, 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).

[0096] In another aspect, the present invention relates to a method of treating cancer in a subject, comprising administering to a subject in need thereof a pharmaceutical composition of the present invention for a period of time sufficient to treat the cancer. In some embodiments, the composition is administered intravenously.

[0097] In another aspect, the present invention relates to the use of the pharmaceutical composition of the present invention for intravenous administration. In a preferred embodiment, the use is for treating cancer.

[0098] In another aspect, the present invention provides a method for producing a composition comprising: a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin, in an amount of 5 pg to 120 mg; and b) a buffering agent, preferably selected from the group consisting of histidine, glutamate, and mixtures thereof; A unit dosage form comprising: The pH is 4.0 to 8.0, preferably 4.5 to 6.5, and more preferably 5.0 to 6.0. The unit dosage form.

[0099] Preferably, the amount of the antibody is 40 pg to 8 g. In some embodiments, the amount of the antibody is 40 pg to 60 mg, for example, 40 pg, 50 pg, 100 pg, 150, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg. , 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27m g, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45 mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 65mg, 70mg, 7 5mg, 80mg, 90mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500 mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1500mg, 2000mg, 3000mg, 4000mg, 5000mg, 6000mg, or for example 7000mg.

[0100] In another preferred embodiment, the total volume of the unit dosage form is between 20 ml and 200 ml, and the dosage form is for intravenous administration.

[0101] In a preferred embodiment, the unit dosage form comprises a non-ionic excipient, preferably a sugar or sugar alcohol. Preferably, the non-ionic excipient is selected from sorbitol, sucrose, or a mixture thereof. In an advantageous embodiment, the non-ionic excipient is present in a concentration of 100 to 300 mM, for example 125 to 250 mM, preferably 250 mM.

[0102] It is preferred that the unit dosage form further comprises d) surfactant.In some embodiments, the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, citrimid, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, polyoxyl hydroxystearic acid, polyoxyl glycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan ester sucrose palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof.In a preferred embodiment, the surfactant is polysorbate, preferably polysorbate 20 or polysorbate 80, most preferably polysorbate 80. In a preferred embodiment the surfactant is present at a concentration of about 0.005% to 0.4% w / v, such as about 0.01 to 0.1% w / v, such as about 0.01 to 0.09% w / v, such as about 0.01 to 0.06% w / v, such as about 0.01 to 0.05% w / v, such as 0.02% w / v or 0.03% w / v or 0.04% w / v or 0.05% w / v, preferably 0.02% w / v.

[0103] In a preferred embodiment, the concentration of the antibody in the unit dosage form is 0.5 to 100 mg / ml, for example 1.0 to 50 mg / ml or for example 5 to 30 mg / ml, for example 5 mg / ml, or 6 mg / ml, or 7 mg / ml, or 8 mg / ml, or 9 mg / ml, or 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or 25 mg / ml, or is 26mg / ml, or 27mg / ml, or 28mg / ml, or 29mg / ml, 30mg / ml, 31mg / ml, 32mg / ml, 33mg / ml, 34mg / ml, 35mg / ml, 36mg / ml, 37mg / ml, 38mg / ml, 39mg / ml, 40mg / ml, 41mg / ml, 42mg / ml, 43mg / ml, 44mg / ml, 45mg / ml, 46mg / ml, 47mg / ml, 48mg / ml, 49mg / ml, 50mg / ml, 51mg / ml, 52mg / ml, 53mg / ml, 54mg / ml, 55mg / ml, 56mg / ml, 57mg / ml, 58mg / ml, 59mg / ml, or for example 60mg / ml.

[0104] In a preferred embodiment, the buffer is present in the unit dosage form at a concentration of 5 to 40 mM, for example 10 to 30 mM, preferably 20 mM.

[0105] In a preferred embodiment, the unit dosage form is a liquid unit dosage form.

[0106] In another aspect, the present invention relates to a method of treating cancer in a subject, comprising administering to a subject in need thereof a unit dosage form of the present invention for a period of time sufficient to treat the cancer. In another aspect, the present invention relates to a unit dosage form of the present invention for use in the treatment of cancer.

[0107] In yet another aspect, the present invention relates to a container comprising a unit dosage form of the present invention or a pharmaceutical composition of the present invention. The container may be made of glass or one or more polymeric materials.

[0108] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a pharmaceutical composition of the invention or any unit dosage form of the invention, b) a container for said pharmaceutical composition or said unit dosage form, and c) instructions for dilution and / or use. This relates to a kit of parts.

[0109] In another aspect, the present invention provides a method for producing a composition comprising: a) a pharmaceutical composition of the invention or any unit dosage form of the invention, b) a diluent, c) a container for said unit dosage form, and d) instructions for dilution and / or use. This relates to a kit of parts.

[0110] In another aspect, the present invention relates to a kit of parts, e.g., for use as a companion diagnostic / for identifying patients within a patient population who have a propensity to respond to treatment with a pharmaceutical composition defined herein, comprising a pharmaceutical composition defined herein and instructions for using the kit.

[0111] In another aspect, the present invention relates to a method for preparing a pharmaceutical composition as defined herein, comprising the steps of: a) mixing 0.5-120 mg / ml of an antibody and b) a buffer in water for injection, and adjusting the pH to 4.0-8.0, preferably 5.0-6.0.

[0112] In another aspect, the present invention provides a method for preparing a unit dosage form as defined herein, comprising the steps of: a) preparing a pharmaceutical composition by the steps of the method for preparing a pharmaceutical composition described above or providing a pharmaceutical composition as defined herein; b) providing a diluent; and c) mixing the pharmaceutical composition with the diluent to obtain a desired antibody concentration. The present invention relates to a method comprising the steps of:

[0113] In another aspect, the invention relates to a pharmaceutical composition or unit dosage form obtainable by any of the aforementioned methods.

[0114] Bispecific morphology The present invention provides pharmaceutical compositions or unit dosage forms comprising bispecific CD3xB7H4 antibodies that efficiently promote T-cell-mediated killing of B7H4-expressing tumor cells. A particular antigen-binding region can be selected from the set 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.

[0115] Examples of bispecific antibody molecules that can be used in the present invention include (i) a single antibody with two arms that contain 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; (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(iv) chemically linked bispecific (Fab')2 fragments; (v) Tandabs, a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) Flexibodies, a combination of scFv and diabody resulting in a multivalent molecule; (vii) so-called "dock and lock" molecules based on the "dimerization and docking domain" in protein kinase A, which when added to a Fab can result in a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (ix) diabodies.

[0116] 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)), also known as a DuoBody®.

[0117] 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 a stabilized diabody is fused to the constant domain of a heavy chain, Fc region, or a portion thereof; (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; (vi) recombinant IgG-like dual targeting molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of an Fab fragment; (vii) Fc fusion molecules, in which a single-chain Fv molecule or a stabilized diabody is fused to the constant domain of a heavy chain, Fc region, or a portion thereof; and (vi) ScFv- 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.

[0118] 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).

[0119] 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 Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al 2007. Bioorg. Med. Chem. Lett. 17, 501-506.)

[0120] Examples of IgG fusion molecules include dual variable domain (DVD)-Ig molecules (Abbott, US7,612,181), 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, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246), and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0121] 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 for Antibody Drugs, China).

[0122] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86.), dual acting or bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), dock-and-lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0123] 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 Dual targeting heavy chain-only domain antibodies include, but are not limited to, dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010), dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010), and dual targeting heavy chain-only domain antibodies.

[0124] The bispecific antibody used in the composition 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 antibody can be selected to be of human IgG1 isotype as shown in the examples. Any of the human light chain constant regions can be used, namely kappa or lambda. In one embodiment, both heavy chains of the antibody of the present invention are of IgG1 isotype. In one embodiment, the two heavy chains of the bispecific antibody are of IgG1 and IgG4 isotypes, respectively. Preferably, the bispecific antibody can be selected to be of human IgG1 isotype as shown in the examples. Optionally and preferably, the heavy chain of the selected isotype and its Fc sequence can be modified in the hinge region and / or CH3 region as described herein to allow the creation of bispecific antibodies and introduce inactivation.

[0125] 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. More details regarding these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0126] 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 antibody containing only a small number of fairly conservative (asymmetric) mutations in the CH3 region. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain one or more amino acid substitutions at positions that are not identical.

[0127] Antigen-binding region capable of binding to CD3 As mentioned above, the present invention provides a pharmaceutical composition or unit dosage form of an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3. Additionally, the present invention provides a pharmaceutical composition of 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 capable of binding to CD3 is capable of binding to human CD3ε(epsilon), e.g., human CD3ε(epsilon) designated in SEQ ID NO: 13. Such an antigen-binding region is capable of binding to human CD3ε(epsilon) displayed on T cells, such as primary human T cells.

[0128] 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:

[0129] CDR1, CDR2 and CDR3 regions can be identified from the variable heavy and light chain regions using methods known in the art. The CDR regions derived from the variable heavy and light chain regions 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)). Thus, 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 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:

[0130] 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 a 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 a sequence of SEQ ID NO: 16; and optionally A light chain variable region (VL) comprising a 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 a sequence of SEQ ID NO: 22. Also disclosed is the antibody, comprising:

[0131] Such antigen-binding regions capable of binding to human CD3 are generally described in WO2015001085 and WO2017009442. Further antigen-binding regions capable of binding to human CD3 are disclosed and described in WO2015001085 and WO2017009442, which are incorporated herein by reference, and may further be contemplated and serve as building blocks for generating antibodies according to the present invention.

[0132] 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 within the range of 1 to 1000 nM. D And it can be combined.

[0133] 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 100 nM, for example, in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, in the range of 10 to 80 nM, in the range of 10 to 60 nM, in the range of 10 to 40 nM, or in the range of 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.

[0134] In another aspect of the invention, the antibody has a lower binding affinity to 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, such as at least 50 fold lower.

[0135] 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, which is in the range of 200 to 1000 nM, for example, in the range of 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, in the range of 600 to 700 nM. D Exemplary 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.

[0136] The binding affinity can be determined by biolayer interferometry, as optionally described herein in Example 4. Thus, an antibody according to the invention, having a binding affinity for human CD3 as defined herein, may have its binding affinity determined using biolayer 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).

[0137] Furthermore, binding affinity may be determined using an antibody, such as a monospecific, bivalent antibody, for example an antibody that is a full-length IgG1.

[0138] 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, the heavy chain variable (VH) region having 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 shown in SEQ ID NO: 16, the positions being 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.

[0139] 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, Y114V.

[0140] Furthermore, the antibody according to the 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.

[0141] 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 said amino acid substitutions preferably include the amino acid substitutions defined above.

[0142] Further disclosed is a pharmaceutical formulation comprising an antibody, wherein the antibody 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 comprises: a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 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: 17; and optionally A light chain variable region (VL) comprising a 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.

[0143] Antigen-binding region capable of binding to B7H4 Specifically, the present invention provides a pharmaceutical composition or unit dosage form comprising 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 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.

[0144] Specifically, the antibody used in the composition of the present invention is an antibody whose 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 a cell, more preferably a human cell.

[0145] In a further embodiment, the antibody used in the composition of the 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 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, which 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 invention is an antibody in which the antigen-binding region capable of binding to human B7H4 is unable to bind 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.

[0146] Suitable antigen-binding regions capable of binding to human B7H4 that are 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.

[0147] In a preferred embodiment, the antibody of the pharmaceutical formulation of the invention comprises an antigen-binding region capable of binding to human B7H4, the antigen-binding region capable of binding to human B7H4 comprising 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.

[0148] CDR1, CDR2 and CDR3 regions can be identified from the variable heavy and light chain regions using methods known in the art. The CDR regions derived from the variable heavy and light chain regions 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)). Thus, 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: 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.

[0149] In a preferred embodiment, the antibody of the pharmaceutical formulation of the invention comprises an antigen-binding region capable of binding to human B7H4, the antigen-binding region capable of binding to human B7H4 comprising 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.

[0150] Further suitable antigen-binding regions capable of binding to human B7H4 and contemplated according to the present invention as described herein include: 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.

[0151] In a preferred embodiment, the antibody of the pharmaceutical formulation of the invention comprises an antigen-binding region capable of binding to human B7H4, wherein the antigen-binding region capable of binding to human B7H4 comprises the variable heavy chain (VH) region of SEQ ID NO:29 and the variable light chain region of SEQ ID NO:33.

[0152] Optionally, the antigen binding region that binds to B7H4 comprises a heavy chain variable region and a light chain variable region (VH) having 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.

[0153] In preferred embodiments, the antigen-binding region that binds to B7H4 comprises a heavy chain variable region and a light chain variable region (VH) having 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:29 and the variable light chain region of SEQ ID NO:33.

[0154] The antibody according to the invention has a K of 5E-7M or less, for example 1E-7M or less. D and an antigen-binding region capable of binding to human B7H4 having a binding affinity corresponding to a K value for human B7H4, e.g., the binding affinity is in the range of 5E-7 to 2E-10 M, e.g., in the range of 2E-7 to 1E-10 M or 1E-7 to 5E-9 M. D Equivalent to the value.

[0155] The binding affinity may be determined by biolayer interferometry, as optionally described herein in Example 3. Thus, an antibody according to the invention, having a binding affinity for human B7H4 as defined herein, may have its binding affinity determined using biolayer 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 300 seconds and the dissociation over a period of 1000 seconds of human recombinant His-tagged B7H4 protein (Sino Biological Catalogue No. 10738-H08H; a protein with a polyhistidine tag at the C-terminus expressed from a construct of a DNA sequence encoding human VTCN1 (Uniprot Accession No. 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).

[0156] Furthermore, binding affinity may be determined using an antibody, such as a monospecific, bivalent antibody, for example an antibody that is a full-length IgG1.

[0157] 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 a variable heavy chain (VH) region of SEQ ID NO: 29 and a 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 a variable heavy chain (VH) region of SEQ ID NO: 50 and a variable light chain region of SEQ ID NO: 54; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 65 and a variable light chain region of SEQ ID NO: 69. cannot be cross-blocked.

[0158] 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 a variable heavy chain (VH) region of SEQ ID NO: 50 and a variable light chain region of SEQ ID NO: 54; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 65 and a variable light chain region of SEQ ID NO: 69. can be cross-hindered, and The antigen-binding region comprises: An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 29 and a 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. The antibody is unable to cross-block antibodies containing

[0159] 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 from the set of samples in an amount of 20 μg / mL 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 no. 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 no. Q7Z7D3) (Phe29 to Ala258)) to the ARG2 biosensor with immobilized antibody; iv) determining binding for a second antibody in an amount of 10 μg / mL from said set of samples for 300 seconds.

[0160] 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 the techniques suitable for determining the ability of an antibody to cross-block the binding of another antibody to a target, and the present application discloses the procedures suitable for determining the blocking and displacement of binding.In a further embodiment, the cross-blocking described herein is determined as described in Example 5.

[0161] 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, said antigen-binding region capable of binding to human B7H4 being capable of binding to B7H3-IgV / B7H4-IgC (SEQ ID NO:11) and, optionally, not capable of binding to B7H4-IgV / B7H3-IgC (SEQ ID NO:10).

[0162] Combination of CD3 and B7H4 antigen-binding regions The disclosure further provides a pharmaceutical composition or unit dosage form comprising 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: 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.

[0163] Also, CDR1, CDR2 and CDR3 regions can be identified from variable heavy and variable light chain regions by using methods known in the art.The CDR regions derived from variable heavy and variable light chain regions 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)).In a preferred embodiment, the CDR regions derived from variable heavy and variable light chain regions of the present invention are annotated according to IMGT.

[0164] The disclosure further provides pharmaceutical compositions and unit dosage forms comprising 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 capable of binding 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.

[0165] In a preferred embodiment, the present invention provides pharmaceutical compositions and unit dosage forms comprising 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 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; Includes.

[0166] The disclosure also provides pharmaceutical compositions and unit dosage forms comprising 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 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.

[0167] The disclosure further provides pharmaceutical compositions and unit dosage forms comprising 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 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.

[0168] In a preferred embodiment, the present invention provides pharmaceutical compositions and unit dosage forms comprising 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 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 They include variable heavy chain (VH) regions comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 28, respectively, and variable light chain regions comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 34, GAS, and SEQ ID NO: 35, respectively.

[0169] Further disclosed are pharmaceutical compositions and unit dosage forms comprising 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 The antigen-binding region capable of binding to B7H4 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, 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:

[0170] Also disclosed are pharmaceutical compositions and unit dosage forms comprising 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:

[0171] In a preferred embodiment, the present invention provides pharmaceutical compositions and unit dosage forms comprising 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 It contains a variable heavy chain (VH) region of SEQ ID NO:29 and a variable light chain region of SEQ ID NO:33.

[0172] 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 in 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 in 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 CD3xB7H4 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, optionally, the terminal lysine of the IgG1 heavy chain constant region defined in SEQ ID NO:60 and SEQ ID NO:61 can be deleted.

[0173] Thus, in a preferred embodiment of the present invention, the CD3×B7H4 bispecific antibody in a pharmaceutical formulation in unit dosage form comprises: A CD3 binding domain, said CD3 binding region comprising 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 further comprising an IgG1 heavy chain constant region as defined in SEQ ID NO: 60 and a lambda light chain constant region as defined in SEQ ID NO: 64; A B7H4 binding domain, said B7H4 binding region comprising 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, and further comprising an IgG1 heavy chain constant region defined in SEQ ID NO: 61 and a kappa light chain constant region defined in SEQ ID NO: 63; Including, wherein, optionally, the IgG1 heavy chain constant region defined in SEQ ID NO:60 and SEQ ID NO:61 may lack a terminal lysine.

[0174] 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 antibody according to the invention. Furthermore, the antibody according to the 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.

[0175] In certain embodiments, the antibody used in the pharmaceutical composition or unit dosage form of the present invention comprises a first heavy chain and a second heavy chain, for example, the first heavy chain and the 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 that contain only a small number of 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 invention, the antibody comprises a first heavy chain in which at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, and a second heavy chain in which at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, where the substitutions for 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. For example, constant domains with 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.

[0176] The term "amino acid corresponding to a position" as used herein 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 of constant region sequences are numbered herein according to the EU index numbering (as described in Kabat, EA et al., 1991, Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689). Thus, an amino acid or segment in one sequence that "corresponds 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 similar, 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 corresponding positions in the sequence for amino acid positions according to the present invention are deemed to be well known in the art.

[0177] In certain embodiments, the invention provides pharmaceutical compositions or unit dosage forms of antibodies, wherein the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in a first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in a second heavy chain, or vice versa.

[0178] In some embodiments, the antibody according to the present invention also comprises an Fc region having the Fc sequences of two heavy chains in addition to the antigen-binding region. The first Fc sequence and the second Fc sequence can each be of any isotype, including any human isotype, for example, IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotype, or mixed isotype. Preferably, the Fc region is of human IgG1, IgG2, IgG3, IgG4 isotype, or 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 IgG1 type.

[0179] The antibody according to the present invention may comprise modifications in the Fc region that make the antibody inactive or inactivated.Thus, in the antibody 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 that comprises unmodified first and second heavy chains.Fc-mediated effector function can be assessed by determining Fc-mediated CD69 expression (i.e., CD69 expression as a result of Fcγ receptor-dependent CD3 cross-linking via CD3 antibody) on T cells by binding to Fcγ receptor, by binding to C1q, or by inducing Fc-mediated FcγR cross-linking. Specifically, the heavy chain constant sequence can be modified so that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to wild-type (unmodified) antibody, and the Fc-mediated CD69 expression is determined in a functional assay using PBMC, for example, as described in Example 3 of WO2015001085. The modification of the heavy chain constant sequence and the light chain constant sequence can also result in a reduction in the binding of C1q 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 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 an unmodified antibody, where T cell proliferation is determined in a functional assay using PBMCs.

[0180] 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 abolish 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).

[0181] Examples of amino acid positions that may be modified, for example 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 in both the first and second heavy chains are F and E, respectively. It will be appreciated that in addition to modifying amino acid positions L234 and L235, further positions may be modified.

[0182] 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 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 in both the first heavy chain and the second heavy chain is A. A further aspect of the invention provides an antibody in which the amino acids at the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain in at least one, e.g. both, of the first heavy chain and the second heavy chain are F, E, and A, respectively. In the present application, antibodies having a 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 suffixes "FEAR" or "FEAL", respectively.

[0183] 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 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 have the amino acid sequence set forth in SEQ ID NO: 62.

[0184] The amino acid sequence of the IgG1 heavy chain constant region having the L234F, L235E, and D265A substitutions is identified herein as SEQ ID NO: 59. The amino acid sequence of the IgG1 heavy chain constant region having the L234F, L235E, D265A, and F405L substitutions is identified herein as SEQ ID NO: 60. The amino acid sequence of the IgG1 heavy chain constant region having the L234F, L235E, D265A, and K409R substitutions is identified herein as SEQ ID NO: 61.

[0185] The constant region sequences listed in SEQ ID NOs: 57-62 describe a terminal lysine (K), and such sequences were used in the Examples section herein. The origin of this lysine is the natural sequence found in humans from which these Fc regions are derived. During recombinant antibody production in cell culture, this terminal lysine can be cleaved by proteolysis 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 so that an antibody without a lysine is produced. For example, with antibodies produced in CHO-based production systems, there is typically a high degree of processing of the terminal lysine, so that antibodies produced from nucleic acid sequences that either do or do not encode the terminal lysine are substantially identical in 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 and do not have a terminal lysine as recited herein. Thus, for manufacturing purposes, antibodies can be made that do not have a terminal lysine.

[0186] The present invention further provides a pharmaceutical composition or unit dosage form of an antibody, the antibody comprising: 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.

[0187] The present invention further provides a pharmaceutical composition or unit dosage form of an antibody, the antibody comprising: 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 has been humanized.

[0188] 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.

[0189] In a further embodiment of the invention, the antibody comprises a lambda (λ) light chain. In a particular embodiment of the invention relating to a bispecific antibody, the lambda light chain comprises the CDR1, CDR2 and CDR3 sequences of the CD3 antibody light chain disclosed above, in particular the CDR1, CDR2 and CDR3 sequences of the 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.

[0190] 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.

[0191] Thus, in a further embodiment, in the bispecific antibody defined herein, the antigen-binding region capable of binding to human B7H4 is comprised in the heavy chain and in 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 region capable of binding to human CD3 is comprised in the heavy chain and in 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 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, 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. It is understood that the IgG1 heavy chain constant regions defined in SEQ ID NO: 60 and SEQ ID NO: 61 may lack a terminal lysine.

[0192] Binding, cytotoxicity, and T cell activation The 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 broad range of cancers with varying levels of B7H4 expression.

[0193] As mentioned above, preferably the antibody according to the invention lacks or has reduced Fc-mediated effector function, and furthermore the antibody a) capable of binding to B7H4-expressing human tumor cells, as described in Examples 9 and 10 herein; b) are capable of mediating 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, for example when purified PBMCs or T cells are used as effector cells; d) T cells can be activated in vitro in the presence of B7H4-expressing human tumor cells, for example as analyzed 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, e.g., when analyzed as described in Examples 11 and 12 herein; and / or g) For example, when analyzed as described in Examples 11 and 12 herein, it is 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.

[0194] Furthermore, the antibodies according to the invention may lack or have reduced Fc-mediated effector function and may further induce T cell-mediated cytotoxicity, where cytotoxicity is assessed in an in vitro IC50 assay comprising the following steps: i) providing peripheral blood mononuclear cells (PBMCs) isolated from the buffy coat of a healthy human donor or purified T cells; 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 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 the IC50.

[0195] Instead of isolated peripheral blood mononuclear cells (PBMCs), purified T cells may be provided in step i).

[0196] 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 the PBMCs with a plurality of samples of B7H4-expressing tumor cells, wherein the ratio of numbers of T cells derived from the PBMCs to 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 the IC50.

[0197] 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) isolated from the buffy coat of a healthy human donor or purified T cells; 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 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 the IC50.

[0198] 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 as described in Example 12.

[0199] 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) providing a dilution series of the 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.

[0200] 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.

[0201] B7H4 antibody

[0202] In another aspect, the invention provides a pharmaceutical composition or unit dosage form comprising an antibody comprising an antigen-binding region capable of binding to human B7H4, wherein the antigen-binding region capable of binding to human 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: 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. Including, the pharmaceutical composition or unit dosage form further comprises a buffer; and The pH of the composition is 4.0 to 8.0. The present invention relates to a pharmaceutical composition or unit dosage form.

[0203] Such antibodies do not necessarily contain the 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 treating cancer.Thus, such antibodies may be monospecific antibodies that bind to B7H4.Such antibodies may be bivalent antibodies.

[0204] The pH of the pharmaceutical composition or unit dosage form is preferably 4.5 to 6.5. In a preferred embodiment, the pH of the pharmaceutical composition or unit dosage form is 5.0 to 6.5. In a particularly preferred embodiment, the pH of the pharmaceutical composition or unit dosage form is 5.0 to 6.0. In a preferred embodiment, the buffer is selected from the group consisting of histidine, glutamate, and mixtures thereof. In a preferred embodiment, the pharmaceutical composition further comprises c) a non-ionic excipient. In a preferred embodiment, the non-ionic excipient is a sugar or a sugar alcohol. In a preferred embodiment, the non-ionic excipient is selected from sorbitol, sucrose, or a mixture thereof. In a preferred embodiment, the non-ionic excipient is present in a concentration of 100 to 300 mM, for example 125 to 250 mM, preferably 250 mM. In a preferred embodiment, the pharmaceutical composition further comprises d) a surfactant. In a preferred embodiment, the surfactant is a polysorbate, preferably polysorbate 20 or polysorbate 80, most preferably polysorbate 80. In a preferred embodiment the surfactant is present at a concentration of about 0.005% to 0.4% w / v, such as about 0.01 to 0.1% w / v, such as about 0.01 to 0.09% w / v, such as about 0.01 to 0.06% w / v, such as about 0.01 to 0.05% w / v, such as 0.02% w / v or 0.03% w / v or 0.04% w / v or 0.05% w / v, preferably 0.02% w / v. Any of the features or embodiments described above for the first aspect of the invention may apply to this aspect of the invention as well.

[0205] Preferably, such an antibody is an antibody that comprises a heavy chain constant region that is a human IgG1 constant region, such as those listed in SEQ ID NOs: 57 to 62. A preferred light chain constant region is a kappa light chain, such as those listed in SEQ ID NO: 63.

[0206] In one embodiment, the antibody of the pharmaceutical composition or unit dosage form 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 antibody 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.

[0207] In another embodiment, the antibodies provided herein can bind to an epitope or antibody binding region on human B7H4 that includes 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 amino acid residues V157, D158, Y159, E164, L166; the numbering of each amino acid residue refers to its position in SEQ ID NO: 1.

[0208] 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, for example, through non-covalent interactions with amino acid residues within the CDR sequences of the antibody.

[0209] 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 the 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 be specifically performed as described or essentially as described in Example 7 herein.

[0210] Furthermore, alanine scanning can be carried out by a procedure comprising the steps of: 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 a corresponding wild-type B7H4 polypeptide, in human embryonic kidney cells, e.g., HEK293 cells, such that a sample comprising 40-60,000 cells, e.g., 50,000 cells, for each of the mutant B7H4 or wild-type B7H4 is provided; ii) incubating the cells in each sample with 20 μl of an antibody, which consists of one heavy chain and one light chain and is labeled with a suitable label for flow cytometry analysis, such as, for example, mNeogreen label, and incubating at room temperature for 1 hour; followed by washing with FACS buffer (e.g., phosphate-buffered saline [PBS; Lonza, Cat. No. BE17-517] + 0.1% [w / v] BSA [Roche, Cat. No. 10735086001] + 0.02% [w / v] sodium azide [NaN3; EMELCA Bioscience, Cat. No. 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 of 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: In the formula, "aa position" refers to the position mutated to alanine, To represent the decrease or increase in antibody binding, the fold change or Z score is calculated based on the following formula: TIFF2024519212000011.tif9128Here, 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", increased binding resulted in ">0", decreased binding resulted in "<0", and only B7H4 amino acid residues with a fold change in binding smaller 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 as "decreased binding mutants", and data were excluded from analysis if the gMFI of the reference antibody against a particular B7H4 mutant was smaller than the mean gMFI-2.5 x (SD of the mean gMFI of the control Ab).

[0211] Furthermore, such antibodies may also be bispecific antibodies, comprising, in addition to the antigen-binding region capable of binding to B7H4, 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 an antigen-binding region capable of binding to CD3 as described and disclosed herein.

[0212] In a further embodiment, the pharmaceutical composition or unit dosage form of the invention comprises a bispecific antibody, wherein the antigen-binding region capable of binding to human B7H4 is 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 region capable of binding to human CD3 is 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 is understood that, optionally, the terminal lysine of the IgG1 heavy chain constant region defined in SEQ ID NO:60 and SEQ ID NO:61 can be deleted.

[0213] A highly preferred bispecific antibody in the pharmaceutical composition or unit dosage form of the invention is as described and used in the Examples section and is called BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR.

[0214] Thus, in a preferred embodiment, there is provided a pharmaceutical composition or unit dosage form comprising a bispecific antibody capable of binding to human CD3 and human B7H4, 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 as 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.

[0215] 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).

[0216] In a further preferred embodiment, there is provided a pharmaceutical composition or unit dosage form comprising a bispecific antibody capable of binding to human CD3 and human B7H4, 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.

[0217] Similarly, it is understood that the human IgG1 heavy chain constant region may lack the terminal lysine (K).

[0218] In yet another further preferred embodiment, there is provided a pharmaceutical composition or unit dosage form comprising a bispecific antibody capable of binding to human CD3 and human B7H4, 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.

[0219] In another further preferred embodiment, there is provided a pharmaceutical composition or unit dosage form comprising a bispecific antibody capable of binding to human CD3 and human B7H4, 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, which lacks a terminal lysine (K), 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 constant region is deleted of 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.

[0220] Methods for preparing bispecific antibodies Conventional methods such as hybrid hybridoma and chemical ligation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used in preparing the bispecific antibodies of the invention. Co-expression of two antibodies consisting of different heavy and light chains in a host cell results in 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.

[0221] Strategies that promote the formation of functional bispecific products when co-expressing various antibody constructs can also be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusing rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to preferential heavy / light chain pairing that is species-constrained. Another strategy that promotes the formation of heterodimers over homodimers is the "knob-into-hole" strategy, in which a protuberance is introduced on the surface of a first heavy chain polypeptide and a corresponding recess is introduced on a second heavy chain polypeptide, so that at the interface of these two heavy chains, the protuberance fits into the recess, 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. A compensatory "dimple" of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (US Pat. No. 5,731,168). EP1870459 (Chugai) and WO 2009089004 (Amgen) describe other strategies to promote heterodimer formation when different antibody domains are co-expressed in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced with charged amino acids, so that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describes yet another strategy that exploits the differences in the CH3 domains of IgA and IgG to promote heterodimerization.

[0222] Another in vitro method for making bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (switching of light chains bound to heavy chains) between two monospecific IgG4 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.

[0223] Preferred methods for preparing the bispecific CD3xB7H4 antibodies of the invention include those described in WO2011131746 and WO13060867 (Genmab), comprising the following steps: a) providing a first antibody comprising an Fc region that comprises 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 a 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.

[0224] 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, where 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.

[0225] Without being limited by theory, in step c) the 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, such as a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercapto-ethanol, preferably 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 be non-reducing or less reducing, for example by removing the reducing agent by desalting, etc.

[0226] For this method, any of the CD3 and B7H4 antibodies described herein can be used. In a particular embodiment, the CD3 and B7H4 antibodies can each be selected to obtain a bispecific CD3xB7H4 antibody described herein.

[0227] In one embodiment of this method, the first and / or second antibody is a full-length antibody.

[0228] 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 functions.

[0229] In a further embodiment, one of the antibody starting proteins is engineered not to bind to Protein A, thus allowing separation of the heterodimeric protein from the homodimeric starting protein by passing the product over a Protein A column.

[0230] As mentioned above, the sequences of the first CH3 region and the second CH3 region of the homodimeric starting antibody 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.

[0231] Specifically, stable bispecific CD3xB7H4 antibodies can be obtained in high yields using the above-described method of the invention based on two homodimeric starting antibodies that bind to CD3 and B7H4, respectively, and that contain only a small number of fairly conservative asymmetric mutations in the CH3 region, meaning that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at positions that are not identical.

[0232] In an embodiment, a method for producing an antibody according to the invention capable of binding to both B7H4 and CD3 is provided, the method comprising the steps of: a) providing an antibody capable of binding to B7H4, the antibody comprising an antigen-binding region capable of binding to B7H4 as defined herein; b) providing an antibody capable of binding to CD3, said antibody comprising an antigen-binding region capable of binding to CD3 as defined herein; c) incubating said antibody capable of binding to B7H4 with said antibody capable of binding to CD3 under reducing conditions sufficient to cause disulfide bond isomerization at cysteines in the hinge region; and d) obtaining an antibody capable of binding to B7H4 and CD3.

[0233] In such methods, the step of providing an antibody capable of binding to B7H4 and / or CD3 may comprise the steps of: - providing a cell comprising an expression vector for producing said antibody or antibodies; and - causing said cells to produce said antibody or antibodies; and then - Obtaining said antibody or antibodies, thereby providing said antibody or antibodies.

[0234] Preferably, the antibody is included in the pharma- ceutically acceptable carrier in the pharmaceutical composition or unit dosage form of the present invention. The pharmaceutical composition of the present invention may include a bispecific antibody of the present invention that targets both B7H4 and CD3. The pharmaceutical composition may also include an antibody that targets B7H4. The pharmaceutical composition may also include a combination of antibodies, including an antibody that targets B7H4 and / or a bispecific antibody according to the present invention.

[0235] The pharmaceutical composition according to the present invention is preferably for use as a medicament. The pharmaceutical composition according to the present invention is preferably for use in the treatment of 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 can be of solid tumor type.

[0236] 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 via T cell mediation.

[0237] In one embodiment, the present invention provides a method for treating cancer in a subject comprising administering a therapeutically effective amount of a bispecific B7H4xCD3 antibody of the pharmaceutical composition of the present invention. In a further embodiment, the present 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 present invention.

[0238] 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.

[0239] As mentioned above, the suitable disease that can be considered in the method and use according to the present invention is cancer.Most preferably, the cancer is characterized by the expression of B7H4.The expression of B7H4 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 transcript and / or B7H4 protein.The antibody described herein that can bind to human B7H4 can be used, for example, in immunostaining and / or FACS analysis.

[0240] 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), squamous cell carcinoma of the head and neck, bladder cancer, esophageal cancer, bile duct cancer, pancreatic cancer, gastric cancer, renal cancer, and prostate cancer.

[0241] Cancers that may express B7H4 include cancers such as 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, and uterine cancer. Preferred possible cancer types are selected from 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).

[0242] In further embodiments, patients diagnosed with cancer may be subjected to evaluation for B7H4 expression in cancer cells, and if B7H4 is detected, it may range from low to high, and such patients may be selected for treatment with the 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 further embodiments, patients diagnosed with uterine carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), invasive breast cancer (BRCA), endometrial endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), or cholangiocarcinoma (CHOL) may be subjected to such testing. However, including such evaluation in selecting patients for treatment may not necessarily be a requirement.

[0243] kit The present invention further provides a kit-of-parts comprising a pharmaceutical composition or unit dosage form comprising the antibody disclosed above, e.g. 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 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 the antibody as disclosed above and instructions for using the kit.

[0244] A kit of parts, for example 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.

[0245] 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.

[0246] In another aspect, the present invention relates to a kit for detecting cross-linking of CD3-expressing cells and B7H4-expressing cells in a sample derived from a patient, the kit comprising: i) a bispecific antibody according to any one of the embodiments disclosed herein; and ii) Instructions for using the kit.

[0247] 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 with CD3-expressing cells. The reagents may include, for example, fluorescent, enzymatic, or other detectable tags. The reagents may also include secondary or tertiary antibodies, or reagents for enzymatic reactions that result in a product that can be visualized.

[0248] 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 a 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 was formed.

[0249] Specific Aspects of the Invention 1. a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin; and b) Buffer Including, A pharmaceutical composition having a pH of 4.0 to 8.0.

[0250] 2. The pharmaceutical composition described in embodiment 1, wherein the pH of the composition is 4.5 to 6.5.

[0251] 3. The pharmaceutical composition described in embodiment 1, wherein the pH of the composition is 5.0 to 6.0.

[0252] 4. The pharmaceutical composition of any one of the preceding aspects, wherein the buffering agent is selected from the group consisting of histidine, glutamate, and mixtures thereof.

[0253] 5. The pharmaceutical composition of any one of the preceding aspects, further comprising a non-ionic excipient.

[0254] 6. The pharmaceutical composition of embodiment 5, wherein the non-ionic excipient is a sugar or sugar alcohol.

[0255] 7. The pharmaceutical composition of embodiment 5 or 6, wherein the non-ionic excipient is selected from sorbitol, sucrose, or a mixture thereof.

[0256] 8. A pharmaceutical composition according to any one of aspects 5 to 7, wherein the non-ionic excipient is present in a concentration of 100 to 300 mM, such as 125 to 250 mM, preferably 250 mM.

[0257] 9. The pharmaceutical composition of any one of the preceding aspects, further comprising d) a surfactant.

[0258] 10. The pharmaceutical composition of embodiment 9, wherein the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, docusate sodium, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, cytrimide, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, polyoxyl hydroxystearates, polyoxylglycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan ester sucrose palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof.

[0259] 11. The pharmaceutical composition of embodiment 9 or 10, wherein the surfactant is a polysorbate.

[0260] 12. The pharmaceutical composition of embodiment 11, wherein the polysorbate is polysorbate 20 or polysorbate 80, preferably polysorbate 80.

[0261] 13. A pharmaceutical composition according to any one of aspects 9 to 12, wherein the surfactant is present in a concentration of about 0.005% to 0.4% w / v, such as about 0.01 to 0.1% w / v, such as about 0.01 to 0.09% w / v, for example about 0.01 to 0.06% w / v, such as about 0.01 to 0.05% w / v, for example 0.02% w / v or 0.03% w / v or 0.04% w / v or 0.05% w / v, preferably 0.02% w / v.

[0262] 14. The concentration of the antibody is 0.5 to 100 mg / ml, for example 1.0 to 50 mg / ml, or for example 5 to 30 mg / ml, for example 5 mg / ml, or 6 mg / ml, or 7 mg / ml, or 8 mg / ml, or 9 mg / ml, or 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or 25 mg / ml, or 26 mg / ml, or 27 mg / ml, or 28mg / ml, or 29mg / ml, 30mg / ml, 31mg / ml, 32mg / ml, 33mg / ml, 34mg / ml, 35mg / ml, 36mg / ml, 37mg / ml, 38mg / ml, 39mg / ml, 40mg / ml, 41mg / ml, 42mg / ml, 43mg / ml, 44mg / ml, 45mg / ml, 46mg / ml, 47mg / ml, 48mg / ml, 49mg / ml, 50mg / ml, 51mg / ml, 52mg / ml, 53mg / ml, 54mg / ml, 55mg / ml, 56mg / ml, 57mg / ml, 58mg / ml, 59mg / ml, or for example 60mg / ml.

[0263] 15. The pharmaceutical composition according to any one of the previous aspects, wherein the buffering agent is present at a concentration of 5 to 40 mM, such as 10 to 30 mM, preferably 20 mM.

[0264] 16. The pharmaceutical composition of any one of the preceding aspects, which is an aqueous composition.

[0265] 17. a) 5 to 50 mg / ml of antibody; b) 10-20 mM glutamate or histidine; c) 150-350 mM sorbitol or sucrose; d) Polysorbate Including, 4. The pharmaceutical composition according to any one of the preceding aspects, wherein the pH is 5.0 to 6.0.

[0266] 18. The pharmaceutical composition of any one of the preceding embodiments, selected from the group consisting of: a) an antibody at 10-20 mg / ml, b) 20 mM glutamate, c) 250 mM sorbitol, d) 0.02% w / v polysorbate 80, wherein the pH of the composition is 5.1-5.3, and 1. A pharmaceutical composition comprising: a) 10-20 mg / ml of an antibody; b) 20 mM histidine; c) 250 mM sucrose; d) 0.02% w / v polysorbate 80; wherein the pH of the composition is 5.4-5.6.

[0267] 19. The pharmaceutical composition of any one of the preceding aspects, which is a liquid composition.

[0268] 20. The pharmaceutical composition of any one of aspects 1-19, which is an intravenous composition and / or is for use in intravenous administration.

[0269] 21. The pharmaceutical composition of any one of the preceding aspects, for use in the treatment of cancer.

[0270] 22. A pharmaceutical composition described in any one of the preceding aspects, in a unit dosage form.

[0271] 23. A pharmaceutical composition according to any one of the preceding aspects, which is stable for pharmaceutical use at a storage temperature of 2 to 8°C, such as 5°C, for at least 6 months, such as at least 9 months or at least 12 months.

[0272] 24. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is a bispecific antibody.

[0273] 25. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is capable of binding to cancer cells and T cells.

[0274] 26. The pharmaceutical composition of embodiment 25, wherein the cancer cells express human B7H4.

[0275] 27. The pharmaceutical composition of embodiment 25 or 26, wherein the cancer cells are from a solid tumor.

[0276] 28. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is capable of inducing T cell-mediated cell killing.

[0277] 29. The pharmaceutical composition according to any one of the preceding aspects, wherein the antigen-binding region capable of binding to CD3 is capable of binding to human CD3ε (epsilon), such as human CD3ε (epsilon) designated in SEQ ID NO: 13.

[0278] 30. The pharmaceutical composition of any one of the preceding embodiments, wherein the antigen-binding region that binds to CD3 comprises: 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.

[0279] 31. The pharmaceutical composition according to any one of aspects 1 to 29, wherein the antigen-binding region that binds 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: 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.

[0280] 32. The pharmaceutical composition according to any one of aspects 1 to 29, wherein the antigen-binding region that binds to CD3 comprises: a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 17, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to a sequence of SEQ ID NO: 17; and A light chain variable region (VL) comprising a 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.

[0281] 33. Dissociation equilibrium constant K between the antigen-binding region that binds to CD3 and CD3 D is in the range of 1 to 100 nM, for example in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, in the range of 10 to 80 nM, in the range of 10 to 60 nM, in the range of 10 to 40 nM, or for example in the range of 10 to 20 nM.

[0282] 34. The pharmaceutical composition of any one of the previous embodiments, wherein 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, such as at least 10-fold, such as at least 20-fold, at least 30-fold, at least 40-fold, at least 45-fold, or such as at least 50-fold lower.

[0283] 35. The antigen-binding region that binds to CD3 has an equilibrium dissociation constant K in the range of 200-1000 nM, e.g., in the range of 300-1000 nM, in the range of 400-1000 nM, in the range of 500-1000 nM, in the range of 300-900 nM, in the range of 400-900 nM, in the range of 400-700 nM, in the range of 500-900 nM, in the range of 500-800 nM, in the range of 500-700 nM, in the range of 600-1000 nM, in the range of 600-900 nM, in the range of 600-800 nM, or in the range of 600-700 nM, for example. D 2. The pharmaceutical composition of any one of the preceding aspects, having the following structure:

[0284] 36. 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 an amino acid substitution at a position 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; The pharmaceutical composition according to any one of the preceding aspects.

[0285] 37. The pharmaceutical composition of embodiment 36, 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, Y114V.

[0286] 38. A pharmaceutical composition according to any one of aspects 30 to 32, wherein the CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 comprise in total at most 1, 2, 3, 4 or 5 amino acid substitutions compared to the CDR1, CDR2 and CDR3 of SEQ ID NO: 16, which amino acid substitutions preferably comprise the amino acid substitutions defined in aspect 36 or aspect 37.

[0287] 39. The pharmaceutical composition of any one of the preceding aspects, wherein the human B7H4 is human B7H4 of SEQ ID NO: 1.

[0288] 40. The pharmaceutical composition of any one of the preceding aspects, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to the extracellular domain of human B7H4.

[0289] 41. The pharmaceutical composition of any one of the preceding aspects, wherein the antigen-binding region capable of binding to human B7H4 is capable of binding to the IgC-like constant region of human B7H4.

[0290] 42. The pharmaceutical composition of any one of the preceding aspects, 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.

[0291] 43. The pharmaceutical composition of embodiment 42, 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.

[0292] 44. The pharmaceutical composition of any one of the preceding embodiments, 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: 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.

[0293] 45. The pharmaceutical composition of any one of the preceding embodiments, 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: 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.

[0294] 46. ​​The pharmaceutical composition of any one of the preceding embodiments, wherein the antigen-binding region capable of binding to human B7H4 comprises: 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.

[0295] 47. 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 has a binding affinity corresponding to a K value, for example, in the range of 5E-7 to 2E-10 M, for example, in the range of 2E-7 to 1E-10 M or 1E-7 to 5E-9 M D 23. The pharmaceutical composition of any one of the preceding embodiments, having a binding affinity corresponding to a value.

[0296] 48. The pharmaceutical composition of embodiment 47, wherein the binding affinity is determined by biolayer interferometry, optionally as set forth in Example 3 herein.

[0297] 49. The binding affinity is in the following stages: I) immobilizing an amount of 1 μg / mL of antibody on an anti-human IgG Fc capture biosensor for 600 seconds; II) determining the association over a period of 300 seconds and the dissociation over a period of 1000 seconds of human recombinant His-tagged B7H4 protein (Sino Biological Catalogue No. 10738-H08H; a protein with a polyhistidine tag at the C-terminus expressed from a construct of a DNA sequence encoding human VTCN1 (Uniprot Accession No. Q7Z7D3) (Phe29-Ala258)) using a two-fold dilution series ranging from 1.56 nM to 100 nM; III) Relating data to the buffer control (0 nM) Determined using biolayer interferometry, including The pharmaceutical composition according to embodiment 47 or embodiment 48.

[0298] 50. A pharmaceutical composition according to any one of aspects 47 to 49, wherein the binding affinity is determined using an antibody defined in any one of the preceding aspects that is a monospecific, bivalent antibody, e.g., an antibody that is a full-length IgG1.

[0299] 51. The antibody 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: 29 and a 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 a variable heavy chain (VH) region of SEQ ID NO: 50 and a variable light chain region of SEQ ID NO: 54; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 65 and a variable light chain region of SEQ ID NO: 69. Can not be crossed and interfered with, The pharmaceutical composition according to any one of the preceding aspects.

[0300] 52. The antibody 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 a variable heavy chain (VH) region of SEQ ID NO: 50 and a variable light chain region of SEQ ID NO: 54; and An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 65 and a variable light chain region of SEQ ID NO: 69. can be cross-hindered, and The antigen-binding region is An antibody comprising a variable heavy chain (VH) region of SEQ ID NO: 29 and a 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. 5, which is unable to cross-block antibodies containing The pharmaceutical composition according to any one of the preceding aspects.

[0301] 53. The pharmaceutical composition of any one of the preceding aspects, 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.

[0302] 54. The pharmaceutical composition according to any one of the preceding embodiments, 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.

[0303] 55. The pharmaceutical composition of any one of the preceding aspects, wherein the antigen-binding region capable of binding to B7H4 is of human origin, and the antigen-binding region capable of binding to CD3 is humanized.

[0304] 56. The pharmaceutical composition of any one of the preceding aspects, wherein 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.

[0305] 57. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0306] 58. The pharmaceutical composition of embodiment 57, wherein the two heavy chain constant domains and the two light chain constant regions are derived from a human.

[0307] 59. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is a full-length antibody.

[0308] 60. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is of the IgG1 isotype.

[0309] 61. The pharmaceutical composition of any one of the preceding aspects, 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 in the first heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, and in the second heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, wherein the substitutions for the first heavy chain and the second heavy chain are not at the same position, and wherein these amino acid positions are numbered according to the EU numbering system.

[0310] 62. The pharmaceutical composition of embodiment 61, 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.

[0311] 63. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody comprises a first heavy chain and optionally a second heavy chain, and 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 relative to the identical unmodified antibody.

[0312] 64. The pharmaceutical composition of embodiment 63, 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.

[0313] 65. The pharmaceutical composition of embodiment 63 or embodiment 64, 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 the position corresponding to position D265 in a human IgG1 heavy chain based on EU numbering is A.

[0314] 66. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody comprises a kappa (κ) light chain.

[0315] 67. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody comprises a lambda (λ) light chain.

[0316] 68. The pharmaceutical composition of any one of the preceding aspects, wherein 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 comprises a binding region capable of binding to CD3, and a heavy chain and a kappa light chain that comprises a binding region capable of binding to B7H4.

[0317] 69. The pharmaceutical composition of any one of the preceding embodiments, wherein an antigen-binding region capable of binding to human B7H4 is 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 an antigen-binding region capable of binding to human CD3 is 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.

[0318] 70. The pharmaceutical composition of embodiment 69, 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.

[0319] 71. The pharmaceutical composition of embodiment 70, wherein the IgG1 heavy chain constant region as defined in SEQ ID NO: 60 and SEQ ID NO: 61 lacks a terminal lysine.

[0320] 72. The antibody lacks Fc-mediated effector function or has 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) is capable of 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 as analyzed 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, e.g., 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 analyzed, for example, as described in Examples 11 and 12 herein; The pharmaceutical composition according to any one of the preceding aspects.

[0321] 73. The antibody has an IC50 in the range of 0.001 to 5 μg / ml, wherein the IC50 is determined to be in the range of: i) providing peripheral blood mononuclear cells (PBMCs) isolated from the buffy coat of a healthy human donor or purified T cells; 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 or purified T cells derived from the PBMCs to the selected tumor cells is 8:1; iv) providing the antibody in a dilution series, for example ranging from 0.0128 ng / mL to 10,000 ng / mL, to the sample; and v) incubating the sample, for example for 72 hours at 37° C.; followed by vi) assessing the viability of the B7H4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample; and viii) determining IC50 In an in vitro cytotoxicity assay comprising: The pharmaceutical composition according to any one of the preceding aspects.

[0322] 74. The pharmaceutical composition of embodiment 73, having an IC50 in the range of 0.001 to 0.03 μg / ml.

[0323] 75. The pharmaceutical composition of embodiment 73, having an IC50 in the range of 0.05 to 5 μg / ml.

[0324] 76. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody comprises an antigen-binding region capable of binding to human B7H4, the antigen-binding region capable of binding to human B7H4 comprising: 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.

[0325] 77. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is a bivalent antibody.

[0326] 78. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody 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 comprises a heavy chain variable region (VH), wherein CDR1 is set forth in SEQ ID NO: 18, CDR2 is set forth in SEQ ID NO: 19, and CDR3 is set forth in SEQ ID NO: 21.

[0327] 79. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody 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 comprises a light chain variable region (VL), CDR1 is as set forth in SEQ ID NO: 23, CDR2 is GTN, and CDR3 is as set forth in SEQ ID NO: 24.

[0328] 80. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody 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 capable of binding to B7H4 comprises a variable heavy (VH) region, wherein CDR1 is set forth in SEQ ID NO: 26, CDR2 is set forth in SEQ ID NO: 30, and CDR3 is set forth in SEQ ID NO: 28.

[0329] 81. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody 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 capable of binding to B7H4 comprises a light chain variable region (VL), CDR1 is as set forth in SEQ ID NO: 34, CDR2 is GAS, and CDR3 is as set forth in SEQ ID NO: 35.

[0330] 82. 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) in which CDR1 is as set forth in SEQ ID NO: 18, CDR2 is as set forth in SEQ ID NO: 19, and CDR3 is as set forth in SEQ ID NO: 21, and a light chain variable region (VL) in which CDR1 is as set forth in SEQ ID NO: 23, CDR2 is GTN, and CDR3 is as set forth in SEQ ID NO: 24; and The antigen-binding region capable of binding to B7H4 is a variable heavy chain (VH) region, wherein CDR1 is as set forth in SEQ ID NO: 26, CDR2 is as set forth in SEQ ID NO: 30, and CDR3 is as set forth in SEQ ID NO: 28; and A light chain variable region (VL) in which CDR1 is as set forth in SEQ ID NO: 34, CDR2 is GAS, and CDR3 is as set forth in SEQ ID NO: 35. Including, The pharmaceutical composition according to any one of the preceding aspects.

[0331] 83. 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 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; 20. The pharmaceutical composition of any one of the preceding aspects, comprising:

[0332] 84. The pharmaceutical composition of any one of the preceding aspects, wherein the antibody is bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR or a biosimilar thereof.

[0333] 85. A pharmaceutical composition according to any one of aspects 1 to 84, for use as a medicament.

[0334] 86. A pharmaceutical composition for use as a medicament according to embodiment 85, for use in a method for the treatment of a disease.

[0335] 87. The pharmaceutical composition for use according to embodiment 86, wherein the disease is cancer.

[0336] 88. The pharmaceutical composition for use according to embodiment 87, wherein the cancer is characterized by expression of B7H4 in cancer cells.

[0337] 89. The pharmaceutical composition for use according to embodiment 88, wherein expression of B7H4 is determined in cancer cells obtained from the patient.

[0338] 90. A pharmaceutical composition for use according to any one of embodiments 87 to 89, wherein the cancer is a solid tumor.

[0339] 91. The pharmaceutical composition for use according to any one of aspects 87 to 90, 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.

[0340] 92. A method for treating a disease, comprising administering to a subject in need thereof a pharmaceutical composition described in any one of aspects 1 to 84.

[0341] 93. The method of embodiment 92, for treating cancer.

[0342] 94. The method of embodiment 93, 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).

[0343] 95. A method of treating cancer in a subject, comprising administering to a subject in need thereof a pharmaceutical composition described in any one of aspects 1-84 for a period of time sufficient to treat the cancer.

[0344] 96. The method of embodiment 95, wherein the composition is administered intravenously.

[0345] 97. Use of a pharmaceutical composition according to any one of aspects 1 to 84 for the treatment of cancer.

[0346] 98. Use of a pharmaceutical composition according to any one of aspects 1 to 84 or 97, which is for intravenous administration.

[0347] 99. a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin, in an amount of 5 pg to 1200 mg; and b) a buffering agent, preferably selected from the group consisting of histidine, glutamate, and mixtures thereof; Including, A unit dosage form having a pH of 4.0 to 8.0, preferably 4.5 to 6.5, and more preferably 5.0 to 6.0.

[0348] 100. The unit dosage form according to embodiment 99, wherein the antibody is defined in any one of embodiments 24 to 84.

[0349] 101. The unit dosage form of aspect 99 or 100, wherein the amount of the antibody is between 20 mg and 1000 mg.

[0350] 102. The unit dosage form according to any one of aspects 99 to 101, wherein the amount of the antibody is between 40 mg and 1000 mg, e.g., 40 mg, 50 mg, 100 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or, for example, 1 g.

[0351] 103. The unit dosage form of any one of aspects 99 to 102, wherein the total volume is 20 ml to 200 ml and the dosage form is for intravenous administration.

[0352] 104. A method of treating cancer in a subject, comprising administering to a subject in need thereof a unit dosage form described in any one of aspects 99-103 for a period of time sufficient to treat the cancer.

[0353] 105. A unit dosage form according to any one of aspects 99 to 103 for use in treating cancer.

[0354] 106. A container comprising a unit dosage form according to any one of aspects 99 to 103 or a pharmaceutical composition according to any one of aspects 1 to 84.

[0355] 107. a) a pharmaceutical composition according to any one of embodiments 1 to 84 or a unit dosage form according to any one of embodiments 99 to 103, b) a container for said pharmaceutical composition or said unit dosage form; c) Instructions for dilution and / or use Kit of parts including:

[0356] 108. a) a pharmaceutical composition according to any one of embodiments 1 to 84 or a unit dosage form according to any one of embodiments 99 to 103, b) a diluent; c) a container for said unit dosage form; and d) Instructions for dilution and / or use Includes kit of parts.

[0357] 109. A kit for use as a companion diagnostic / for identifying patients in a patient population who have a propensity to respond to treatment with a pharmaceutical composition according to any one of aspects 1 to 84, comprising a pharmaceutical composition according to any one of aspects 1 to 84 and instructions for using the kit. A kit of parts, such as:

[0358] 110. The following: a) 0.5 to 120 mg / ml of an antibody, and b) Buffer in water for injection; and Adjusting the pH to 4.0 to 8.0, preferably 5.0 to 6.0 A method for preparing a pharmaceutical composition according to any one of aspects 1 to 84, comprising:

[0359] 111. The following: a) preparing a pharmaceutical composition by the steps of the method according to embodiment 110 or providing a pharmaceutical composition according to any one of embodiments 1 to 84; b) providing a diluent; c) mixing said pharmaceutical composition with said diluent to obtain a desired antibody concentration. A method for preparing a unit dosage form according to any one of aspects 99 to 103, comprising:

[0360] 112. A pharmaceutical composition or unit dosage form obtainable by any of the methods according to embodiment 110 or 111. EXAMPLES

[0361] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0362] Example 1: Generation and Screening of B7H4 Antibody Materials Expression of B7H4 constructs Constructs were generated encoding various full-length B7H4 variants: 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 argenteus) 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).

[0363] Additionally, a construct was made for the extracellular domain (ECD of human B7H4 (amino acids 25-259 of Uniprot Accession No. 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, and thus the mature B7H4ECD-FcHisC protein corresponds to amino acid residues 25-259 of SEQ ID NO: 1.

[0364] 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 ECD construct of B7H4 were cloned into pSB, a mammalian expression vector containing Sleeping Beauty inverted terminal sequences flanking an expression cassette consisting of the CMV promoter and HSV-TK polyA signal.

[0365] 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.

[0366] Purification of His-tagged B7H4 B7H4ECD-FcHisC was expressed using the Expi293F expression platform (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. A14527) essentially as described by the manufacturer.

[0367] The His tag allows 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 weakly compared to the His-tagged proteins and are eluted in the flow-through fraction. To remove weakly bound proteins, the column was washed. 2+ Tightly bound His-tagged proteins were eluted with an imidazole-containing buffer, which competes with His binding to the eluent. The eluent was removed by buffer exchange using a desalting column.

[0368] 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 a week 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 number S6322) or CFA, complete Freund's adjuvant (first injection) and IFA, incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA, catalog number F5881 / F5506) (subsequent injections), followed by a final subcutaneous injection of antigen in PBS without adjuvant.

[0369] Antibody production Three days after the final boost, 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 with 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 binding to antigen. After performing antigen-specific screening assays, 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 the amino acid position numbers are according to Eu numbering (corresponding to SEQ ID NO: 60), and into expression vectors containing a human kappa light chain or a human lambda light chain. For some of the antibodies, variants were generated with point mutations in the variable domains 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).

[0370] 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 in a homogeneous binding assay. Samples were analyzed for antibody binding to HEK-293F cells expressing human B7H4, cynomolgus monkey B7H4, or mouse B7H4, or HEK-293F wild-type cells (negative control) that were transiently transfected with constructs made to express full-length B7H4 variants. Samples were added to the cells to allow the antibody to bind 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 backbone, 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). Plates were incubated at room temperature for 9 h, 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 x fluorescence intensity was at least 3-fold greater than the negative control.

[0371] 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 human B7H4-transfected HEK-293F cells as described above. Thirty-five antibodies were selected: 26 with the original sequence and 9 variants with point mutations in the variable domain. The antibodies were generated as monovalent binding antibodies (as CD3 bispecifics) and bivalent binding antibodies (as IgG1 molecules) and tested for binding to tumor cells 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.

[0372] Further B7H4 Antibodies In the examples, further 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 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 relevant sequences of these variable domains are set out in Table 1 herein, including SEQ ID NO: 36 and SEQ ID NO: 40; those described in WO2009073533 (referenced therein as SEQ ID NO: 2 and SEQ ID NO: 7), which corresponds to B7H4-C4 herein and the relevant sequences of these variable domains are set forth in Table 1 herein, including SEQ ID NO: 50 and SEQ ID NO: 54; and those described in US20190085080A1, which corresponds to B7H4-C5 herein and the relevant sequences of these variable domains 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 domain and human kappa constant domain or human lambda constant domain or variants thereof to generate monospecific and bispecific antibodies. When referring to the antibody IgG1-B7H4-CX-FEAL, this refers to an antibody having the 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.

[0373] IgG1-b12 antibody Antibody b12, an HIV-1 gp120 specific antibody (Barbas, CF. 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 with codon-optimized human IgG1m(f) constant domain and human kappa constant domain or variants thereof. The sequences of the variable heavy (VH) and variable light (VL) regions are included herein as SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0374] Example 2 Humanized CD3 antibody for generating CD3×B7H4 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 have no effect on target binding of the antibody into which they are introduced (see, e.g., US 2015 / 0337049 and Engelberts et al., 2020, EBioMedicine 52: 102625).

[0375] The production of humanized antibody IgG1-huCD3-H1L1-H101G (whose variable heavy and light region sequences are set forth herein in SEQ ID NO: 17 and SEQ ID NO: 22) is described in Example 2 of WO2017 / 009442. IgG1-huCD3-H1L1-H101G is referred to as "IgG1-huCD3-H101G". This variant contains the substitution H101G in the variable heavy 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 the amino acid substitutions L234F, L235E, D265A, and F405L.

[0376] 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 affinity of IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C4-FEAR to human B7H4 and mouse B7H4 was determined using BLI. Anti-human IgG Fc capture (AHC) biosensors (ForteBio, Cat. No. 18-5060) were pre-primed by exposure to 10 mM glycine (Sigma-Aldrich, Cat. No. 15527) buffer, pH 1.7 for 5 seconds, followed by neutralization in sample diluent (ForteBio, Cat. No. 18-1048) for 5 seconds. Both steps were repeated twice. Antibodies (1 μg / mL in sample diluent) were then added to the AHC sensor for 600 s. After a baseline measurement (100 s) in sample diluent, the binding (300 s) and dissociation (1,000 s) of human B7H4 (Sino Biological, Cat. No. 10738-H08H-100) or mouse B7H4 (R&D Systems, Cat. No. 2154-B7-050) were determined using human B7H4 and mouse B7H4 in the concentration ranges of 1.56–100 nM (0.04–2.68 μg / mL) and 5.9–375 nM (0.16–10 μg / mL), respectively, by two-fold dilution steps in sample diluent. The theoretical molecular weights of human B7H4 and mouse B7H4 (as ECD-His tagged molecules) based on amino acid sequence (26.8 kDa and 26.6 kDa, respectively) were used for calculation. 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, and both steps were repeated twice. The antibody was then added back to the sensor for the next cycle of kinetic measurements.

[0377] Data were acquired using Data Acquisition Software v9.0.0.49d (ForteBio) and analyzed by Data Analysis Software v9.0.0.12 (ForteBio). Data traces were corrected for each antibody by subtracting the reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and step-to-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 by a 1:1 Global Full fit model with the time windows of interest for association and dissociation times set at 300 and 200 seconds, respectively.

[0378] In the 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. The experiment was performed as previously described with some minor exceptions. The preconditioning steps were repeated five times. Binding (200 s) and dissociation (1,000 s) of human B7H4 or mouse B7H4 were determined using a concentration range of 0.78–800 nM by 2-fold dilution steps in diluent. Data were acquired using Data Acquisition Software v12.0.1.8 (ForteBio) and analyzed by 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 s for association times and 200 s for dissociation times, except for IgG1-B7H4-C2-FEAR, for which a dissociation time of 1,000 s was used. Dissociation times were calculated using the R 2 The data were selected based on the values, visual inspection of the curves, and a signal decay of at least 5% during the dissociation step. Data traces generated with antigen concentrations above 100 nM were excluded from the analysis for antibodies with affinities below 50 nM.

[0379] In addition, the affinity to cynomolgus 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 to cynomolgus 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. Recombinant hIgG1 Fc-tagged cynomolgus B7H4 (Creative BioMart, Catalog No. VTCN1-1517R) at 10 μg / mL in 10 mM sodium acetate, pH 4.0 (ForteBio, Catalog No. 18-1068) was added to the activated sensor for 600 seconds and the reaction was stopped with 1 M ethanolamine, pH 8.5 (ForteBio, Catalog No. 18-1071) for 300 seconds. After baseline measurements in sample diluent (300 seconds; ForteBio, Catalog No. 18-1048), association (100 seconds) and dissociation (1,000 seconds) 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) by 2-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.

[0380] Data were acquired using Data Acquisition Software v9.0.0.49d (ForteBio) and analyzed by Data Analysis Software v9.0.0.12 (ForteBio). Data traces were corrected for each antibody by subtracting the reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and step-to-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 by a 1:1 Global Full fit model with the time windows of interest for association and dissociation times set at 100 and 200 seconds, respectively.

[0381] In a second experiment to determine the affinity of the B7H4 antibody to cynomolgus B7H4, the affinity of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR was determined. This experiment was performed as described above with some minor exceptions. After a baseline determination of 600 seconds in sample diluent, the association (200 seconds) and dissociation (1,000 seconds) for functionally monovalent B7H4 binding by the CD3×B7H4 bispecific antibody (shown in Table 9) were determined using a concentration range of approximately 0.1-116 μg / mL (0.78-800 nM) by two-fold dilution steps in sample diluent. The intrinsic molecular weight of each antibody (approximately 145 kDa) was used for the calculations. Data were acquired using Data Acquisition Software v12 (ForteBio) and analyzed by Data Analysis Software v12 (ForteBio). Data traces with responses below 0.03 nm were excluded from the analysis. Data were fitted by a 1:1 Global Full fit model using a time window of interest of 200 s for association and dissociation times. Dissociation times were calculated using R 2The selection was based on the R value, visual inspection of the curves, and at least 5% signal decay 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.

[0382] "K D " (M) refers to the equilibrium dissociation constant of the antibody-antigen interaction, 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. k d is k off It is also called the k value or off rate. a " " -1 ×sec -1 ) refers to the binding rate constant of the antibody-antigen interaction. a is k on It is also sometimes called the value or on-rate.

[0383] Tables 4 and 5 show the results of the first and second experiments, which show the binding rate constants k of 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.

[0384] Table 4: Binding affinity of antibodies to human B7H4 extracellular domain as determined by label-free biolayer interferometry. ND = not determined. TIFF2024519212000012.tif61128

[0385] Table 5. Binding affinity of antibodies to human B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF2024519212000013.tif61128 1 The results shown are the average of three experiments.

[0386] Tables 6 and 7 show the results of two experiments in which the k of the antibodies shown against mouse B7H4 was a (1 / Ms), k d (1 / s), and K D (M) was determined by biolayer interferometry.

[0387] Table 6: Binding affinity of antibodies to mouse B7H4 extracellular domain as determined by label-free biolayer interferometry. ND = not determined. - = no binding (response < 0.05 nM at highest concentration used). TIFF2024519212000014.tif61128

[0388] Table 7: Binding affinity of antibodies to mouse B7H4 extracellular domain as determined by label-free biolayer interferometry. - = no binding (response below 0.05 nM at highest concentration used). TIFF2024519212000015.tif61128

[0389] Tables 8 and 9 show the results of two experiments in which the k values ​​of the antibodies shown in the tables against cynomolgus B7H4 were a (1 / Ms), k d (1 / s), and K D (M) was determined by biolayer interferometry.

[0390] Table 8. Binding affinity of functionally monovalent antibodies to the cynomolgus monkey B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF2024519212000016.tif65144

[0391] Table 9. Binding affinity of functionally monovalent antibodies to the cynomolgus B7H4 extracellular domain as determined by label-free biolayer interferometry. TIFF2024519212000017.tif35154 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.

[0392] Example 4 Determination of CD3 binding affinity using biolayer interferometry The binding affinity of IgG1-huCD3-FEAL and IgG1-huCD3-H101G-FEAL was determined as described in Example 7 of WO2017 / 009442.

[0393] Briefly, the binding affinity of selected CD3 antibodies in the IgG1-huCD3-FEAL form to recombinant soluble CD3ε (CD3E27-GSKa) (mature protein of SEQ ID NO: 13) was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio). hIgG (1 μg / mL) was added to an anti-human Fc capture biosensor (ForteBio, Cat. No. 18-5060) for 600 s. After a baseline measurement (200 s), the binding (1000 s) and dissociation (2000 s) 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) by 3-fold dilution steps (sample diluent, ForteBio, Cat. No. 18-5028). The theoretical molecular weight of CD3E27-GSKa based on the amino acid sequence, i.e. 27.11 kDa, was used for the calculation. The experiments were performed at 30°C with shaking at 1000 rpm. Each antibody was tested in at least two independent experiments. Data were analyzed by 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 with the antibody loaded on the biosensor and using only sample diluent). 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.

[0394] Table 10 shows the binding rate constants 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 IgG1-huCD3-FEAL, IgG1-huCD3-H101G-FEAL (K D : 683 nM) is relatively high compared to D : 15 nM), showing binding affinity to recombinant CD3ε.

[0395] Table 10. Binding affinity of monospecific bivalent CD3 antibodies to recombinant CD3ε determined by label-free biolayer interferometry. TIFF2024519212000018.tif35128

[0396] 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 with 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.

[0397] Amine-reactive second generation (AR2G) biosensors (ForteBio, Cat. No. 18-5092) were activated with a solution of 20 mM EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (Sigma-Aldrich, Cat. No. 03449) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (Sigma-Aldrich, Cat. No. 56485) for 300 seconds. 20 μg / mL of the first antibody in 10 mM sodium acetate, pH 6.0 (ForteBio, Cat. No. 18-1070) was added to the activated AR2G sensor for 600 seconds and the reaction was stopped with 1 M ethanolamine, pH 8.5 (ForteBio, Cat. No. 18-1071) for 300 seconds. After a baseline measurement in sample diluent (50 s; ForteBio, Cat. No. 18-1048), human B7H4 (100 nM or 2.68 μg / mL diluted in sample diluent; Sino Biological, Cat. No. 10738-H08H) was added for 300 s to the AR2G biosensor containing immobilized antibody. The theoretical molecular weight of human B7H4 (26.8 kDa) based on the amino acid sequence was used for calculations. Binding (300 s) of the second antibody (10 μg / mL in sample diluent) was determined. The sensor was regenerated by exposure to 10 mM glycine (Riedel-de Haen, Cat. No. 15527) buffer, pH 2.5, for 5 s, followed by neutralization in sample diluent for 5 s. Both steps were repeated twice. The sensor containing immobilized first antibody was then used again, starting from the baseline step.

[0398] Data were acquired using Data Acquisition Software v9.0.0.49d (ForteBio) and analyzed by Data Analysis HT Software v10.0.17 (ForteBio). Data traces were corrected by subtracting a reference curve (sample diluent instead of the second antibody) to correct for dissociation of B7H4 from the immobilized first 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 second antibodies were presented in a matrix format. Typically, responses above 0.05 nM were considered to be non-cross-interfering antibodies, whereas responses below 0.05 nM were considered to be interfering antibody pairs.

[0399] The cross-blocking experiment was repeated to include IgG1-B7H4-C5-FEAR and performed as described above with minor modifications. The experiment was performed at 22°C with shaking at 1,000 RPM. Data was acquired using Data Acquisition Software v12.0.1.8 (ForteBio) and analyzed by Data Analysis HT Software v12.0.1.55 (ForteBio). Responses above 0.1 nm were generally considered to be non-cross-blocking antibodies, whereas responses below 0.1 nm were considered to be blocking antibody pairs.

[0400] A first set of cross-blocking experiments was performed with 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 grey, non-blocking antibody combinations are not marked (clear background). It is shown that IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C5-FEAR cross-block each other and do not cross-block with IgG1-B7H4-C4-FEAR and IgG1-B7H4-C2-FEAR, and vice versa.

[0401] 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 grey; non-interfering antibody combinations are not marked (clear background). TIFF2024519212000019.tif73128

[0402] 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 grey; non-interfering antibody combinations are not marked (clear background). TIFF2024519212000020.tif80139

[0403] 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: F405L mutation in one parent IgG1 antibody (i.e., CD3 antibody in this application) and K409R mutation in the other parent IgG1 antibody (i.e., B7H4 antibody or 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).

[0404] To generate bispecific antibodies, equal masses of the 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 500 mM NaCl (pH 7.4) at 37 °C for 2 h. 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 h. 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 molecular weight cut-off of 10 kDa (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0405] The following antibodies were used in the examples: B7H4 antibody IgG1-B7H4-C1-FEAR (having VH and VL sequences as set forth in SEQ ID NO: 25 and SEQ ID NO: 33). IgG1-B7H4-C1-N52S-FEAR (having VH and VL sequences as set forth in SEQ ID NO: 29 and SEQ ID NO: 33). IgG1-B7H4-C2-FEAR (having VH and VL sequences as set forth in SEQ ID NO: 43 and SEQ ID NO: 47). IgG1-B7H4-C3-FEAR (having VH and VL sequences set forth in SEQ ID NO: 36 and SEQ ID NO: 40). IgG1-B7H4-C4-FEAR (having VH and VL sequences as set forth in SEQ ID NO: 50 and SEQ ID NO: 54). IgG1-B7H4-C5-FEAR (having 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 IgG1 isotype was made, 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).

[0406] 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 made, 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).

[0407] Control antibodies 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 IgG1 isotype was made, 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).

[0408] bispecific antibody The aforementioned CD3 and B7H4 antibodies were combined to generate 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

[0409] 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 differences in the extracellular domain), or two different human B7H4-B7H3 chimeric molecules. Expression constructs were prepared encoding human B7H4, human B7H3 (Uniprot accession number Q5ZPR3-1; SEQ ID NO: 9), or 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). HEK cells were transiently transfected to express these constructs.

[0410] Cells (3 × 10 ) were cultured in polystyrene 96-well round-bottom plates (Greiner bio-one, Cat. No. 650101) containing serial dilutions of antibodies (ranging from 0.0046 to 10 μg / mL in 3-fold dilution steps) in 50 μL of PBS / 0.1% BSA / 0.02% azide (FACS buffer). 4The cells (1:100 cells / well) were incubated at 4°C for 30 min. After washing twice in FACS buffer, the cells were incubated with secondary antibody at 4°C for 30 min. R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (1:500 in staining buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, Cat. No. 109-116-098) was used as the secondary antibody. The cells were then washed twice in FACS buffer, resuspended in 20 μL of FACS buffer, and analyzed by 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 for binding of the following agents at 10 μg / mL: 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.

[0411] FIG. 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 FIG. 1 indicate that the IgC domain is also involved in binding (WO2014159835 and Leong et al 2015, Mol. Pharmaceutics 12, 1717-1729).

[0412] 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 analysis of a full dose-response curve. In these experiments, the domain specificity of bsIgG1-huCD3-H101G-FEAL×B7H4-C5-FEAR was also determined. bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, bsIgG1-huCD3-H101G-FEAL×B7H4-C2 ... Binding of serial dilutions (0.014 to 30 μg / mL in 3-fold dilution steps) of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, 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 involved in the binding of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, in agreement 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 appear to be involved in the binding of bsIgG1-huCD3-H101GFEAL×B7H4-C3-FEAR.

[0413] Determining the Contribution of B7H4 Amino Acid Residues to Binding of B7H4 Antibodies Using a B7H4 Alanine Scanning Library Library Design A human B7H4 (Uniprot Q7Z7D3-1) single residue alanine library was synthesized (GeneArt) in which all amino acid residues in the extracellular domain of human B7H4 were individually mutated 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 a pMAC expression vector containing a CMV / TK-polyA expression cassette, an Amp resistance gene, and a pBR322 origin of replication.

[0414] Library construction and screening Antibodies C1-N52S, C2, and C3 were produced as recombinant monovalent antibodies as described in WO2007059782 using mNeonGreen tags. Wild-type B7H4 and alanine mutants were expressed separately 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. Cells were incubated for 1 hour at room temperature. Subsequently, 150 μL of FACS buffer was added and cells were washed twice with FACS buffer. Cells were resuspended in 30 μL of fresh FACS buffer and analyzed by flow cytometry using an iQue screener (Intellicyt Corporation, USA).

[0415] The entire experiment was performed twice in duplicate.

[0416] 4. Data Analysis For each sample, the average antibody binding per cell was determined as the geometric mean of the fluorescence intensity (gMFI) of the ungated cell population. The gMFI is affected by the affinity of the antibody to the B7H4 variant and the expression level of the B7H4 variant per cell. Because individual alanine mutations may affect the surface expression level of mutant B7H4, to correct for differences in expression across each B7H4 variant, the data were normalized to the binding intensity of a non-cross-blocking B7H4-specific reference antibody using the formula: TIFF2024519212000021.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.

[0417] To express the decrease or increase in antibody binding on a linear scale of fold change, the following calculation was used: TIFF2024519212000022.tif9128

[0418] The increased binding is in most cases due to decreased binding of the reference antibody to the individual ala mutants.

[0419] In these calculations, amino acid positions that result in neither a decrease nor an increase in binding with a particular antibody when the amino acid is substituted with alanine are given 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 as "reduced binding mutants".

[0420] The gMFI of the reference antibody for a particular B7H4 variant is the mean gMFI-2.5×(mean gMFI 対照Ab If the mean expression level was smaller than 1 SD, data were excluded from the analysis (assuming that expression levels were not sufficient for these B7H4 variants).

[0421] Figure 3 shows the fold change in binding of the B7H4 antibody to B7H4 variants with ala mutations in the ECD, with amino acid residues for which the fold change in binding was less than the mean fold change - 1.5 x SD annotated. Fold changes are shown as Z scores in Figure 3. These results demonstrate the following: the binding of the antibody C1-N52S is dependent at least on the amino acids S151, V157, D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242, and I245, which are present in the IgC domain of human B7H4; the binding of antibody C2 is dependent at least on the amino acids R98, G99, R116, K118, N119, and D124, which are present in the IgV of human B7H4; and 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.

[0422] Example 8 Binding of B7H4 Monospecific and CD3xB7H4 Bispecific Antibodies to B7H4 from Various Species First, the binding of bispecific CD3xB7H4 and monospecific B7H4 antibodies to HEK-293F cells transiently transfected with human B7H4 or cynomolgus monkey (Macaca fascicularis) B7H4 was analyzed by flow cytometry. Untransfected HEK-293F cells were used as a negative control; these cells were confirmed not to express CD3.

[0423] Cells (3 × 10 ) were cultured in polystyrene 96-well round-bottom plates (Greiner bio-one, Cat. No. 650180) containing serial dilutions of antibodies (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 / well) were incubated for 30 min at 4°C. Experiments were performed in technical duplicates. After washing twice in staining buffer, cells were incubated in 50 μL of secondary antibody for 30 min at 4°C. R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (1:500 in FACS buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, Cat. No. 109-116-098) was used as secondary antibody. Cells were washed twice in staining buffer, resuspended in 30 μL of FACS buffer containing Topro-3 (1:10,000 dilution), and analyzed by 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).

[0424] FIG. 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.

[0425] Next, binding to HEK-293F cells transiently transfected with B7H4 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 from dog, rabbit, rat, and mouse to various degrees; for each, the apparent affinity (EC50) of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR was less 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.

[0426] 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.

[0427] 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 between the antibodies tested. Similar results were obtained with cells expressing rabbit B7H4 and dog 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. Also, the binding of gG1-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, and binding of IgG1-B7H4-C3-FEAR to porcine B7H4 was undetectable.

[0428] Example 9 Binding of B7H4 Monospecific 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 ... 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). We determined 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). Furthermore, 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 were administered to MDA-MB-468 and HCC1954 cells. 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. Typically, solid tumor cell lines 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.

[0429] FIG. 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.

[0430] 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 B7H4 non-expressing cell line HeLa.

[0431] 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 had similar maximum binding levels.

[0432] 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 between the C1-N52S, C2, C4, and C5-based antibodies, but was at a low level for the C3-based antibody.

[0433] 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 4 Cells were plated / well, centrifuged, and incubated for 30 minutes at 4° C. with 50 μl of fixable viability stain FVS-BV510 (BD Biosciences, Cat. No. 564406) diluted 1:1000 in PBS. After washing in staining buffer, cells were incubated with FITC-labeled IgG1-B7H4-C1-N52S-FEAR and a panel of CD3 (EF450 labeled; eBioscience, Cat. No. 48-0037-42), CD45 (BV786 labeled; Biolegend, Cat. No. 304048), CD14 (PE-Cy7 labeled; BD Biosciences, Cat. No. 557742), CD86 (PerCP-Cy5.5 labeled; Biolegend, Cat. No. 305420), CD163 (APC-Cy7 labeled; Biolegend, Cat. No. 333622), and EpCAM (AF700 labeled; R&D Systems, Cat. No. FAB9601N) specific antibodies for 30 min 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.

[0434] 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.

[0435] Example 11 In vitro induction of T cell-mediated cytotoxicity by CD3xB7H4 bispecific antibodies 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 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.

[0436] 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, Cat. 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, Cat. No. 655180) and left 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 plates were incubated for 72 hours at 37°C. Plates were washed three times with PBS and cells were incubated with 150 μl / well of 10% alamarBlue® solution (Invitrogen, Cat. 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, Cat. No. P3075; dissolved in dimethylsulfoxide [DMSO; Sigma-Aldrich, Cat. No. D2438]). AlamarBlue fluorescence, an indicator of the metabolic activity of tumor cell cultures and therefore of 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 target cells treated with PAO] / [absorbance of untreated target cells - absorbance of target cells treated with PAO]) x 100

[0437] 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.

[0438] FIG. 11 shows that T cell-mediated cytotoxicity was observed at all E:T ratios, with maximal tumor cell killing (<10% viable tumor cells) observed at an E:T ratio of 8:1.

[0439] 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 as described above, using an E:T ratio of 8:1. 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 supernatant containing T cells was transferred to U-bottom 96-well culture plates (CellStar, catalog no. 650180) prior to washing and alamarBlue incubation (to determine T cell activation and cytokine release, as described below).

[0440] 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.

[0441] Figure 12 shows that both bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL x B7H4-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% viable tumor cells) was achieved for both bsAb variants, but this occurred at lower concentrations for bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR compared to bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (Table 13).

[0442] No significant relationship was observed between tumor cell lysis and B7H4 expression levels (Figure 13A) for either bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (Figure 13B). Figure 13B shows the IC50 of T cell-mediated killing for each cell line in the presence of bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR using T cells from 4-6 donors. Cell lines are ordered from lowest to highest B7H4 expression levels. This means that T cell-mediated killing can occur over a wide range of B7H4 expression levels.

[0443] Table 13 summarizes the results from a panel of five cell lines and four donors.

[0444] Table 13. In vitro induction of cytotoxicity in various tumor cell lines by CD3xB7H4 bispecific antibodies. TIFF2024519212000023.tif55128bsIgG1-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.

[0445] Example 13 In Vitro Induction of T Cell Activation and Cytokine Production by CD3xB7H4 Bispecific Antibody 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 min at 4° C., after which 75 μL of supernatant was transferred to a new plate for cytokine production determination and T cells were stored for assessment of T cell activation (described below). Cytokine production was analyzed by a multiplex U-plex assay (MeSo Scale Discovery, USA, Cat. No. K15049K) according to the manufacturer's instructions.

[0446] 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 with Ultracomp beads (5 μL; Invitrogen, Cat. No. 01-2222-42) was included and used for flow cytometer compensation adjustment. After 30 min incubation at 4° C., plates were washed three times with PBS / 0.1% BSA / 0.02% azide (staining buffer). Cells were resuspended in 120 μL staining buffer and analyzed using a FACS Fortessa (BD Biosciences). Data was processed using FlowJo (BD Biosciences).

[0447] 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).

[0448] Figure 14A shows T cell activation against B7H4 positive tumor cell lines in the presence of bsIgG1-huCD3-FEAL x B7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEAL x 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.

[0449] 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 usually 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).

[0450] Cytokine production in the supernatants of tumor-T cell cultures was assessed by Mesoscale Discovery Uplex multiplex ELISA. Among the 10 cytokines analyzed across the cell line panel with T cells from four donors, significant increases in cytokine levels were observed primarily 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 usually below 50 pg / ml. We present data on IFN-γ because changes in this cytokine were robustly and consistently detected and because it is one of the central cytokines increased in the serum of patients with cytokine release syndrome.

[0451] Figure 15 shows IFN-γ levels in the supernatants of T cell-tumor cell co-cultures 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 x B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (EC50, EC90, EC99, respectively). Cytokine production levels varied depending on the donor and the target tumor cell line. Nevertheless, at antibody concentrations that induced the same percentage of tumor cell killing, the levels of cytokine production were usually lower after exposure of T cell-tumor cell co-cultures to bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR compared to the levels after exposure of the co-cultures to bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR. Thus, at the same level 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.

[0452] Example 14. Non-clinical safety study of CD3×B7H4 bispecific antibodies in cynomolgus monkeys The non-clinical safety profile of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR in non-human primates (cynomolgus monkeys, Macaca fascicularis, Mauritius) was evaluated at Citoxlab (France). Based on the species specificity of the CD3 arm of bsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR and bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR, and furthermore due to the similar binding of the B7H4 arm to human B7H4 and cynomolgus B7H4, as well as additional pharmacological findings, cynomolgus monkeys were considered the only appropriate species for non-clinical 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).

[0453] The goal of these studies was to clarify the potential toxicity and toxicokinetics of the CD3xB7H4 bispecific antibody. Herein, only the results of the toxicokinetics and determination of plasma cytokine levels are described.

[0454] In two separate studies, animals were treated with a single dose 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) by intravenous (IV) infusion. The day of infusion was designated as day 1 of the study. Blood samples were taken twice pre-dose and at 0.5, 2, 4, 12, 24, and 48 hours post-dose to evaluate toxicokinetic profiles and plasma cytokine levels, and additionally at 168, 336, and 504 hours post-dose for toxicokinetics.

[0455] 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.

[0456] As shown in FIG. 16, administration of BsIgG1-huCD3-H101G-FEAL×B7H4-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-FEAL×B7H4-C1-N52S-FEAR resulted in a dose-dependent increase in IL-6 and MCP-1 levels.

[0457] The lower cytokine levels following 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.

[0458] toxicokinetics Plasma concentrations of 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 non-compartmental approach consistent with the route of administration, intravenous 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 showed dose-related plasma exposure.

[0459] A pharmacokinetic modeling exercise was performed to evaluate whether the predicted clinical dose range required by the BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR variant with lower CD3 affinity was unsustainably high. A PK model based on information from observations in cynomolgus monkeys was used. A clinical dose range expected to cause a weekly mean plasma exposure equal 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 provide a basis for preferring one type of bispecific antibody over the other (BsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR vs. BsIgG1-huCD3-FEAL×B7H4-C1-N52S-FEAR).

[0460] 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).

[0461] Figure 18 shows B7H4 mRNA expression levels in a panel of primary solid tumors ranked based on median expression. mRNA expression was observed across a broad 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).

[0462] B7H4 protein expression in 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 was analyzed by immunohistochemistry (IHC) on tissue microarrays (TMA; all purchased from BioMax). 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 min, cooled for 60 min). B7H4 IHC was performed 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 min (room temperature) on a LabVision autostainer platform. Sections were then incubated with 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 with a mouse anti-cytokeratin antibody mix (clone AE1 / AE3) on a Ventana Benchmark using OptiView detection. Cytokeratin was visualized with DAB and nuclei were counterstained with hematoxylin using default Ventana reagent. Stained TMA sections were digitized in 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 core with >10% B7H4 positive tumor cells.

[0463] Subsequently, automated scoring was performed. Tumor ROIs were defined using a cytokeratin mask of the surface of the TMA section adjacent to the TMA section stained for B7H4. B7H4 staining intensity in the tumor ROI was quantified (negative, weak (1), moderate (2), or string (3)) and the percentage of B7H4 percentage-positive tumor cells (range 0-100%) was determined using HALO image analysis software. For each indication, the percentage of the tumor center with >10% B7H4-positive tumor cells was determined.

[0464] Table 14 shows B7H4 protein expression as revealed by IHC analysis of 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 (especially squamous NSCLC), cervical, head and neck, breast (triple negative breast cancer [TNBC] and non-TNBC), ovarian, and uterine cancers.

[0465] Table 14. B7H4 protein expression determined by IHC analysis of BioMax TMA. ND = not determined. TIFF2024519212000024.tif226143

[0466] Example 16 Thermal and colloidal stability at various pH values Various formulations of bsIgG1-huCD3-H101G-FEAL×B7H4-C1-N52S-FEAR (20 mg / mL) were prepared at various pH values ​​using a series of buffers (acetate, glutamate, succinate, citrate, histidine, phosphate, and Tris) spanning the pH range of 4.0 to 8.0, as shown in Table 15.

[0467] Table 15: Test formulations with various pH values TIFF2024519212000025.tif93134

[0468] Differential scanning fluorimetry (DSF) was performed to determine the effect of each combination of pH and buffer on the thermal stability of the antibody. DSF analysis utilizes a heat ramp from 20 to 95 °C to induce protein unfolding and evaluates thermal state transitions by measuring the fluorescence profile of the sample during the process of increasing thermal stress. A conformational state transition can be revealed if a clear change in the internal fluorescence profile is observed. This clear change is primarily due to changes in the local chemical environment around hydrophobic aromatic residues (e.g., tryptophan) due to conformational modification. Data are reported by comparing the centroid mean (BCM), which is the average wavelength of fluorescence emission, at each temperature. As aromatic residues become exposed due to unfolding, the energy of the fluorescence emission tends to decrease. This leads to the lowest unfolding temperature (T ) observed at temperatures where the BCM increases and exceeds noise fluctuations. 開始 ), and the melting temperature value (T m ) can be measured.

[0469] Static light scattering (SLS) measurements are determined at 266 nm and 473 nm to assess protein colloid stability. The intensity of static light scattering from the laser light used to irradiate the sample is proportional to the presence of particles that are of the same order of magnitude as the incident wavelength. Therefore, this analysis is highly sensitive to protein aggregation during the temperature increase. Static light scattering is measured at 266 nm to detect small aggregate particle sizes and at 473 nm to detect large aggregate species. The aggregation onset temperature (T 凝集) is determined from these data. These data are best analyzed based on large changes in count intensity; more counts indicate more light scattered by protein aggregates clumping together. Changes in SLS counts that occur during the stepwise temperature increase are typically attributed to significant protein aggregation, minimal changes in SLS counts are attributed to partial aggregation, and no change in SLS counts during the temperature increase indicates negligible protein aggregation. SLS analysis was performed using Notebook Method NB9828p9, differential scanning fluorimetry, static light scattering, and dynamic light scattering using a capillary.

[0470] As shown in Table 16, T correlates with pH. 開始 and T m Both trends of thermal stability and thermal stability can be seen, with the higher the pH, the greater the tendency for thermal stability to increase. At pH 5.0 and above, most formulations showed T 開始 The temperature of the base formulation was ≒50°C or higher than 50°C, indicating that the base formulation was relatively stable. 開始 By comparing the values, it was found that the higher the pH, the more heat energy is required to induce protein unfolding, and this tendency was confirmed by the Tm and T 開始 It is clear that this persists up to about pH 6.5, where both values ​​plateau. Low pH can alter the charges of surface, solvent-accessible amino acid side chains, namely Asp (pK = 3.9), Glu (pK = 4.2), and His (pK = 6.0), resulting in the induction and disruption of electrostatic interactions that destabilize the native folding of proteins. SLS T 凝集 The values ​​were recorded at two incident wavelengths, 266 nm and 473 nm, respectively, and were measured simultaneously with the DSF during the temperature increase from 20° C. to 95° C. This allowed characterization of the protein aggregation and colloidal stability of the antibody in each formulation. At both wavelengths, differences were observed, with higher T values ​​at higher pH. 凝集 see Table 16.

[0471] Table 16. DSF and SLS results TIFF2024519212000026.tif107156

[0472] Overall, the DSF data show that pH values ​​below 5.0 result in a T below 50°C (above which the advantage starts to disappear). 開始 The T obtained from SLS is 凝集 The values ​​indicate that formulations containing acetate, glutamate, succinate, and histidine (which also correspond to a pH range below 6.5) had a lower tendency to aggregate and aggregation began at higher temperatures compared to other buffer systems. Also, higher pH values ​​tend to correlate with deamidation, suggesting that a pH range of 5.0 to 6.5 is particularly preferred.

[0473] Example 17 Thermal and colloidal stability with additional excipients Various formulations of bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (20 mg / mL) were prepared with different pH (glutamate at pH=5.0, succinate at pH=5.5, histidine at pH=5.5, and histidine at pH=6.0) and different sets of excipients (NaCl, arginine, sorbitol, and sucrose). See Table 17.

[0474] Table 17. Test formulations with various buffers, pH values, and excipients TIFF2024519212000027.tif93128

[0475] Differential scanning fluorimetry (DSF) and static light scattering (SLS) measurements were performed as described above in Example 16. As summarized in Table 18 below, formulations containing charged excipients (arginine and NaCl; Formulations A, B, E, F, I, J, M, N) showed significantly higher T than their respective counterparts containing non-ionic excipients (sorbitol, sucrose). 開始 and T m Both showed low values.

[0476] Table 18. DSF and SLS results TIFF2024519212000028.tif115151

[0477] Similarly, non-ionic excipients showed a lower degree of aggregation at 266 nm and no aggregation was detected at 473 nm, as revealed by SLS (with the exception of succinate-based formulations).Overall, formulations containing non-ionic excipients showed superior thermal stability, with the buffers ranked in the order of histidine, glutamate, and succinate among the formulations tested.

[0478] Example 18 Solubility, thermal stability, and colloidal stability of various formulations Various formulations of bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (20 mg / mL) were prepared according to Table 19 below.

[0479] Table 19. Test formulations with various buffers, pH values, and excipients TIFF2024519212000029.tif80153

[0480] The formulations in Table 19 were evaluated by dynamic light scattering (DLS) to measure size and size uniformity.

[0481] These data are summarized in Table 20. The mean hydrodynamic diameters ranged from 6.4 nm (20 mM glutamate, 250 mM sorbitol, pH 5.0) to 19.6 nm (20 mM histidine, 250 mM sorbitol, pH 6.0). The monomer hydrodynamic diameters ranged from 5.3 nm (20 mM glutamate, 250 mM sorbitol, pH 5.0) to 13.1 (20 mM succinate, 150 mM NaCl, pH 5.5). The mean polydispersity index (PDI) values ​​ranged from 0.06 (20 mM histidine, 125 mM sorbitol, 75 mM NaCl, pH 5.5) to 2.79 (20 mM histidine, 250 mM sorbitol, pH 6.0). The %PD of the monomers ranged from 19.6% to 31.4%, indicating relatively low polydispersity.

[0482] Table 20: DLS results TIFF2024519212000030.tif93147

[0483] Four of the top five average PDI values ​​were also observed in the four formulations with the lowest and highest pH (pH 5.0 and pH 6.0), respectively, indicating that pH value plays an important role in antibody solubility.

[0484] Differential scanning fluorimetry (DSF) and static light scattering (SLS) measurements were performed as described above in Example 16. The results are summarized in Table 21.

[0485] Table 21. DSF and SLS results TIFF2024519212000031.tif89142

[0486] T 開始 Value and T mComparing the values, the uncharged excipients sucrose and sorbitol tend to improve stability. For SLS, it is clear that all formulations containing uncharged excipients and either glutamate or histidine show no aggregation at 473 nm and a very small relative magnitude of aggregation at 266 nm compared to the succinate-based formulations containing charged excipients.

[0487] These data, in addition to the results of the DSF measurements, further support the role of sucrose and sorbitol as thermally stabilizing excipients. Formulations containing non-ionic excipients and either glutamate or histidine showed an overall better characterized profile throughout the solubility study. Furthermore, formulations buffered with histidine and glutamate containing sorbitol or sucrose as excipients showed the fastest exchange and concentration rates during the sample preparation stage, suggesting that non-ionic excipients play a role in antibody solubilization.

[0488] Example 19 Storage stability of various pharmaceutical compositions A total of 36 formulations of bsIgG1-huCD3-H101G-FEAL x B7H4-C1-N52S-FEAR (20 mg / mL) were prepared and subjected to two storage conditions, namely 5 ± 3 °C and 40 ± 2 °C / 75 ± 5% RH, respectivel...

Claims

1. a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3; b) Buffer Including, A pharmaceutical composition having a pH of 4.0 to 8.

0.

2. 2. The pharmaceutical composition of claim 1, wherein the antigen-binding region capable of binding to human B7H4 and the antigen-binding region capable of binding to human CD3 comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are humanized and / or of human origin, and optionally the pharmaceutical composition has a pH of 4.5 to 6.5, or 5.0 to 6.0, or 5.2 to 5.

5.

3. 2. The pharmaceutical composition of claim 1, wherein the buffering agent is selected from the group consisting of histidine, glutamate, and mixtures thereof.

4. 2. The pharmaceutical composition of claim 1, further comprising: c) a non-ionic excipient which is a sugar or a sugar alcohol, optionally selected from sorbitol, sucrose, or a mixture thereof, and optionally present at a concentration of 100 to 300 mM, such as 125 to 250 mM, preferably 250 mM.

5. 5. The pharmaceutical composition of claim 4, further comprising: d) a surfactant, the surfactant is selected from the group consisting of glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin, benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids, alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbintan fatty acid esters, polyoxyethylene sterarates, polyoxyl hydroxystearates, polyoxyl glycerides, polysorbates, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan ester sucrose palmitate, sucrose stearate, tricaprylin and TPGS, and mixtures thereof; or A pharmaceutical composition wherein the surfactant is a polysorbate, optionally wherein the polysorbate is polysorbate 20 or polysorbate 80, preferably polysorbate 80.

6. 6. The pharmaceutical composition of claim 5, wherein the surfactant is present in a concentration of about 0.005% to 0.4% w / v, such as about 0.01 to 0.1% w / v, such as about 0.01 to 0.09% w / v, such as about 0.01 to 0.06% w / v, such as about 0.01 to 0.05% w / v, such as 0.02% w / v or 0.03% w / v or 0.04% w / v or 0.05% w / v, preferably 0.02% w / v.

7. The antibody concentration may be from 0.5 to 100 mg / ml, for example from 1.0 to 60 mg / ml, or for example from 5 to 60 mg / ml, for example from 5 mg / ml, or 6 mg / ml, or 7 mg / ml, or 8 mg / ml, or 9 mg / ml, or 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or 25 mg / ml, or 26 mg / ml, or 27 mg 2. The pharmaceutical composition of claim 1, wherein the concentration of said compound is 28mg / ml, or 29mg / ml, 30mg / ml, 31mg / ml, 32mg / ml, 33mg / ml, 34mg / ml, 35mg / ml, 36mg / ml, 37mg / ml, 38mg / ml, 39mg / ml, 40mg / ml, 41mg / ml, 42mg / ml, 43mg / ml, 44mg / ml, 45mg / ml, 46mg / ml, 47mg / ml, 48mg / ml, 49mg / ml, 50mg / ml, 51mg / ml, 52mg / ml, 53mg / ml, 54mg / ml, 55mg / ml, 56mg / ml, 57mg / ml, 58mg / ml, 59mg / ml, or for example 60mg / ml.

8. 2. The pharmaceutical composition of claim 1, wherein the buffering agent is present in a concentration of 5 to 40 mM, such as 10 to 30 mM, preferably 20 mM.

9. a) 5 to 60 mg / ml of an antibody; b) 10-20 mM glutamate or histidine; c) 150-350 mM sorbitol or sucrose; d) Polysorbate Including, pH is between 5.0 and 6.0, or a) 20-60 mg / ml of an antibody, b) 20 mM glutamate, c) 250 mM sorbitol, d) 0.02% w / v polysorbate 80, the pharmaceutical composition having a pH of 5.1-5.3, and A pharmaceutical composition comprising: a) 20-60 mg / ml antibody; b) 20 mM histidine; c) 250 mM sucrose; d) 0.02% w / v polysorbate 80; and a pH of 5.4-5.

6. Selected from:

2. The pharmaceutical composition of claim 1.

10. 2. The pharmaceutical composition of claim 1, which is a liquid or aqueous composition, optionally an intravenous composition and / or for use in intravenous administration.

11. 2. The pharmaceutical composition according to claim 1, which is stable for pharmaceutical use at a storage temperature of 2-8°C, such as 5°C, for at least 6 months, such as at least 9 months or at least 12 months.

12. The pharmaceutical composition of claim 1, wherein the antibody is a bispecific or bivalent antibody.

13. The antigen-binding region that binds to CD3 is as follows: A heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 16 or 17; and A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22; or 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 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; 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; or a heavy chain variable region (VH) comprising a 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 a 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 a sequence of SEQ ID NO:

22.

2. The pharmaceutical composition of claim 1, comprising:

14. An antigen-binding region capable of binding to human B7H4 comprising: a) 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; b) 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; 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; 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; or 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; 2. The pharmaceutical composition of claim 1, comprising:

15. An antigen-binding region capable of binding to human B7H4, 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; 2. The pharmaceutical composition of claim 1, comprising:

16. An antigen-binding region capable of binding to human B7H4 comprising: a) 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; b) 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; 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; 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; or 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; 2. The pharmaceutical composition of claim 1, 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: 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.

17. An antigen-binding region capable of binding to human B7H4 comprising: a) the variable heavy chain (VH) region of SEQ ID NO: 29 and the variable light chain region of SEQ ID NO: 33; b) the variable heavy chain (VH) region of SEQ ID NO: 25 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; f) the variable heavy chain (VH) region of SEQ ID NO: 31 and the variable light chain region of SEQ ID NO: 33; or g) the variable heavy chain (VH) region of SEQ ID NO: 65 and the variable light chain region of SEQ ID NO: 69 2. The pharmaceutical composition of claim 1, comprising:

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

33.

19. the antibody 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), wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 18, CDR2 comprises the amino acid sequence of SEQ ID NO: 19, and CDR3 comprises the amino acid sequence of SEQ ID NO: 20 or 21; and A light chain variable region (VL), wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 23, CDR2 comprises the amino acid sequence of GTN, and CDR3 comprises the amino acid sequence of SEQ ID NO:

24. and The antigen-binding region capable of binding to B7H4 is a variable heavy chain (VH) region, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 26, CDR2 comprises the amino acid sequence of SEQ ID NO: 30, and CDR3 comprises the amino acid sequence of SEQ ID NO: 28; and A light chain variable region (VL), wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 34, CDR2 comprises the amino acid sequence of GAS, and CDR3 comprises the amino acid sequence of SEQ ID NO:

35. Contains, or 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 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 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; Including, 2. The pharmaceutical composition of claim 1.

20. the antibody 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 comprises a variable heavy chain (VH) region of SEQ ID NO: 16 or 17 and a variable light chain region of SEQ ID NO: 22; and the antigen-binding region capable of binding to human B7H4 comprises a variable heavy chain (VH) region of SEQ ID NO: 29 and a variable light chain region of SEQ ID NO: 33; 2. The pharmaceutical composition of claim 1.

21. 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.

2. The pharmaceutical composition of claim 1.

22. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL), preferably wherein the two heavy chain constant regions and the two light chain constant regions are derived from a human.

23. 2. The pharmaceutical composition of claim 1, wherein the antibody is a full-length antibody, optionally wherein the antibody is of the IgG1 isotype.

24. 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 in the first heavy chain, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted; and in said second heavy chain, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted; wherein the substitutions for said first heavy chain and said second heavy chain are not at the same position, and these amino acid positions are numbered according to EU numbering; optionally 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; 2. The pharmaceutical composition of claim 1.

25. an antibody comprising a first heavy chain, and optionally a second heavy chain, wherein the first heavy chain, and, if present, the second heavy chain, are modified such that the antibody induces Fc-mediated effector functions to a lesser extent than the same unmodified antibody; or 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; or 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; or 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 that can bind to CD3, and a heavy chain and a kappa light chain that includes a binding region that can bind to B7H4, or and wherein the heavy and light chains comprise antigen binding regions capable of binding to human B7H4, 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 heavy and light chains comprise antigen binding regions capable of binding to human CD3, 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, preferably 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, optionally in the IgG1 heavy chain constant regions defined in SEQ ID NO: 60 and SEQ ID NO: 61, the terminal lysines are deleted.

2. The pharmaceutical composition of claim 1.

26. 2. The pharmaceutical composition of claim 1, wherein the antibody is bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR or bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR or a biosimilar thereof.

27. A pharmaceutical composition according to any one of claims 1 to 26 for use as a medicament.

28. 27. Use of the pharmaceutical composition of any one of claims 1 to 26 in the manufacture of a medicament for treating cancer, optionally wherein the cancer is characterized by expression of B7H4 in cancer cells, and optionally wherein the cancer is a solid tumor.

29. 29. The use of claim 28, 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.

30. a) an antibody comprising an antigen-binding region capable of binding to human B7H4 and an antigen-binding region capable of binding to human CD3, the antigen-binding region comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being humanized and / or of human origin, in an amount of 5 pg to 1200 mg; and b) a buffering agent, preferably selected from the group consisting of histidine, glutamate, and mixtures thereof; Including, The unit dosage form has a pH of 4.0 to 8.0, or 4.5 to 6.5, or 5.0 to 6.

0.

31. 31. The unit dosage form according to claim 30, wherein the antibody is as defined in any one of claims 20 to 26, optionally wherein the amount of antibody is between 20 mg and 1000 mg, such as between 200 mg and 600 mg, such as between 300 mg and 400 mg.

32. a) a pharmaceutical composition according to any one of claims 1 to 26, or a unit dosage form according to claim 30, b) a container for said pharmaceutical composition or said unit dosage form, and c) Instructions for dilution and / or use Includes kit of parts.

33. In water for injection: a) 0.5-120 mg / ml of an antibody, and b) Buffer mixing the Adjusting the pH to 4.0-8.0, preferably 5.0-6.

0. A method for preparing the pharmaceutical composition of any one of claims 1 to 26, comprising:

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