Antibody

Abstract

The present invention relates to antibodies that bind to 5T4, including bispecific antibodies that bind to 5T4 and CD3. The present invention further provides pharmaceutical compositions comprising the antibodies, and the use of the antibodies in therapeutic and diagnostic procedures, particularly in cancer therapy.

Description

Field of the Invention

[0001] The present invention relates to antibodies that bind 5T4, including bispecific antibodies that bind 5T4 and CD3. The present invention further provides pharmaceutical compositions comprising the antibodies, and the use of the antibodies in therapeutic and diagnostic procedures, particularly in cancer therapy. Background of the Invention

[0003] 5T4 (also known as trophoblast glycoprotein [TPBG] or Wnt-activated inhibitor factor 1 [WAIF1]) is a 72 kDa single-pass transmembrane protein that contains eight leucine-rich repeats (LRRs) and seven potential N-glycosylation sites (Zhao et al., 2014 Structure 22, 612-620).

[0004] Except for the placenta, 5T4 expression is restricted in normal adult tissues (Southall et al., 1990 Br J Cancer 61, 89-95). 5T4 is expressed in many human cancers, including renal cancer, cervical cancer, ovarian cancer, lung cancer, prostate cancer, and colon cancer (Stern and Harrop, 2017 Cancer Immunol Immunother 66, 415-426; Southall et al., 1990 Br J Cancer 61, 89-95). Expression of 5T4 in tumor cells drives tumor development by 1) promoting epithelial-to-mesenchymal transition (Damelin et al., 2011 Cancer Res 71, 4236-4246; Carsberg et al., 1996 Int J Cancer 68, 84-92), and 2) inhibiting the canonical Wnt / beta-catenin signaling pathway and activating the non-canonical Wnt pathway (Kagermeier-Schenk et al., 2011 Dev Cell 21, 1129-1143).

[0005] 5T4-targeted antibodies and 5T4-targeted therapies have clinical activity in several cancers known to express 5T4, including colorectal cancer, lung cancer, and renal cancer. For example, naptumomab estafenatox is a recombinant fusion protein consisting of a 5T4-Fab portion genetically fused to an engineered superantigen variant SEA / E-120. Currently, it is in clinical trials as an immunotherapy for non-small cell lung cancer (NSCLC), renal cell (RCC), and pancreatic cancer (see, e.g., Eisen, et al., 2014 Curr Oncol Rep 16, 370). In addition, Modified vaccinia Ankara expressing the 5T4 construct (MVA-5T4), which has shown clinical benefit in colorectal, prostate, and renal cancer (see, e.g., Stern and Harrop, 2017 Cancer Immunol Immunother 66, 415-426; Scurr et al., 2017 JAMA Oncol 12, 10). Other anti-5T4 antibodies are described in WO2007106744, WO03038098, WO2011048369, WO2013041687, WO2017072207.

[0006] Despite significant progress in eradicating cancer, there is still a need for further improvement in antibody-based cancer therapies. Summary of the Invention

[0008] The object of the present invention is to provide an antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), wherein said antibody is capable of blocking the binding of an antibody

[059] comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region to 5T4, said heavy-chain variable (VH) region comprising the sequence shown in SEQ ID NO:5, and said light-chain variable (VL) region comprising the sequence shown in SEQ ID NO:9.

[0009] The antibody can in particular be a bispecific antibody and can further comprise an antigen-binding region that binds to CD3, such as human CD3ε (epsilon), such as the antigen-binding region of an antibody to human CD3ε (epsilon) as defined in SEQ ID NO:4.

[0010] In another aspect, the present invention relates to a nucleic acid construct comprising

[0011] a) a nucleic acid sequence encoding a heavy-chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein, and / or

[0012] b) a nucleic acid sequence encoding a light-chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein.

[0013] In another aspect, the present invention relates to an expression vector comprising

[0014] a) a nucleic acid sequence encoding a heavy-chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein, and / or

[0015] b) a nucleic acid sequence encoding a light-chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein.

[0016] In another aspect, the invention relates to a cell comprising a nucleic acid construct or expression vector as defined herein.

[0017] In another aspect, the invention relates to a composition comprising an antibody according to the invention.

[0018] In another aspect, the invention relates to a pharmaceutical composition comprising an antibody as defined herein and a pharmaceutically acceptable carrier.

[0019] In another aspect, the invention relates to an antibody as defined herein for use as a medicament, such as for treating a disease.

[0020] In another aspect, the invention relates to a method of treating a disease or disorder, the method comprising administering to a subject in need thereof an antibody, composition or pharmaceutical composition as defined herein.

[0021] In another aspect, the invention relates to a method for producing an antibody as defined herein.

[0022] In another aspect, the invention relates to a kit comprising an antibody as defined herein; and instructions for use of the kit.

[0023] In another aspect, the invention relates to an anti-idiotypic antibody that binds to the antigen-binding region of an antibody as defined herein that is capable of binding 5T4. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : IgG1-5T4-059-FEAR and IgG1-5T4-207-FEAR for use in combination with IgG1-5T4-A3-F405L Antibody replacement of IgG1-5T4-226-FEARAntibody displacement was determined by biolayer interferometry on an Octet HTX instrument (ForteBio). IgG1-5T4-A3-F405L was immobilized on the biosensor and loaded with human 5T4ECDHis (the mature protein of SEQ ID NO. 99). Subsequently, the loaded biosensor was exposed to IgG1-5T4-A3-F405L, IgG1-5T4-H8-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, or IgG1-5T4-226-FEAR. The figure shows the association response (500 s) after exposure to the second antibody. A-C. IgG1-5T4-A3-F405L showed no binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex, indicating cross-blocking (self-blocking) with IgG1-5T4-A3-F405L. The IgG1-5T4-H8-FEAR antibody showed an increase in mass (indicating binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex) and thus no cross-blocking with IgG1-5T4-A3-F405L. A. IgG1-5T4-059-FEAR, B. IgG1-5T4-207-FEAR, and C. IgG1-5T4-226-FEAR all showed an initial increase in mass (indicating antibody binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex), followed by a rapid decrease in mass. This behavior of the antibodies indicates antibody displacement (Abdiche YN, et al. (2017) Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another. PLoS ONE 12(1):e0169535. doi:10.1371 / journal.pone.0169535).

[0026] Figure 2 : Simultaneous binding of 5T4 antibody to membrane-bound 5T4 measured by flow cytometry.The 5T4 antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR were conjugated to fluorescein isothiocyanate (FITC) and added to SK-OV-3 cells expressing 5T4 at a concentration of 2 μg / mL in the presence of 10 μg / mL of unconjugated IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12, IgG1-5T4-207-FEAR, or IgG1-5T4-226-FEAR. The percentage of binding of the FITC-labeled antibody was calculated and depicted as mean percentage binding ± standard deviation (SD).

[0027] Figure 3 : Binding of the 5T4 antibody to HEK-293 cells transfected with full-length human and chicken 5T4. HEK-293 cells transiently transfected with full-length human 5T4 (SEQ ID NO:1) (A) or chicken 5T4 (SEQ ID NO:3) (B) were incubated with various concentrations of IgG1-5T4-A3-F405L, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, or IgG1-5T4-226-FEAR antibodies. After incubation with goat anti-human IgG F(ab')2 conjugated to R-phycoerythrin (PE), the mean fluorescence intensity (MFI) was determined by flow cytometry. As a negative control, IgG1-b12-K409R (10 μg / mL) was included.

[0028] Figure 4 : Internalization ability of monovalent 5T4 antibody. Conjugated toxin bispecific antibodies were generated by controlled Fab arm exchange of unconjugated 5T4 antibodies with (HIV-1 gp120-specific) IgG1-b12 antibodies in which each antibody had been conjugated to one Duostatin-3 molecule, which recognize 5T4 with one Fab arm and an irrelevant antigen (HIV-1 gp120, which is not expressed on tumor cells) with a second Fab arm. As indicated, MDA-MB-468 (A) and HCC1954 (B) cells were incubated with increasing concentrations of the antibody. Cell viability was measured after 5 days. Data are represented as the mean percentage of viable cells from three replicate experiments. As a negative control, monospecific bivalent IgG1-b12 conjugated to Duostatin-3 (IgG1-b12-vcDuo3) was included.

[0029] Figure 5(I): Binding of CD3x5T4 bispecific antibody to full-length human and cynomolgus monkey (cynomolgus monkey) 5T4 transfected into HEK-293 cells. Analysis of the binding of monovalent and bivalent 5T4 antibodies was performed using HEK-293 cells transiently transfected with full-length human (left panel) or cynomolgus monkey 5T4 (right panel). Cells were incubated with increasing concentrations of antibody as indicated. After secondary labeling with FITC-conjugated goat anti-human IgG F(ab’)2, binding was analyzed by flow cytometry. As a negative control antibody, IgG1-b12-K409R (3 μg / mL) was included. Data are represented as mean fluorescence intensity (MFI) values ± SD of two technical replicates. A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR.

[0030] B. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR. C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR. D. Binding of bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T4-H8-FEAR.

[0031] Figure 5(II): Binding of bispecific CD3x5T4 antibodies to cynomolgus monkey and human 5T4 transfected into HEK-293 cells. Monovalent and bivalent binding of 5T4 antibodies was analyzed using HEK-293 cells transiently transfected with human 5T4 (left panel) or cynomolgus monkey 5T4 (right panel). Cells were incubated with increasing concentrations of antibody as indicated. Binding was analyzed by flow cytometry after secondary labeling with phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2. A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; B. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; D. Binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; E. Binding of bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR and IgG1-5T4-076-FEAR; F. Binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; G. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR;

[0032] H. Binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; I. Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR.

[0033] Figure 6(I): Binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to 5T4-positive human tumor cells. The monovalent and bivalent binding of 5T4 antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel) was determined by flow cytometry. Cells were incubated with increasing concentrations of the antibody. After secondary labeling with FITC-conjugated goat anti-human IgG F(ab')2, the MFI was determined by flow cytometry. A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). B. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). IgG1-b12-K409R (3 μg / mL) was included as a negative control (open circles). Figure 6 (II): Binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to HeLa cells. The monovalent and bivalent binding of 5T4 antibodies to HeLa cells was determined by flow cytometry. Cells were incubated with increasing concentrations of the antibody. After secondary labeling with phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2, the mean fluorescence intensity (MFI) was determined by flow cytometer.

[0034] A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; B. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR;

[0035] D. Binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; E. Binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; F. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR;

[0036] Binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; H. Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR

[0037] Figure 6(III): Binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to MDA-MB-231 cells. The monovalent and bivalent binding of 5T4 antibodies to MDA-MB-231 cells was determined by flow cytometry. Cells were incubated with increasing concentrations of antibodies. After secondary labeling with PE-conjugated goat anti-human IgG F(ab′)2, the mean fluorescence intensity (MFI) was determined by flow cytometry. A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; B. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; D. Binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR;

[0038] E. Binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; F. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR; G. Binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR;

[0039] H. Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR.

[0040] Figure 7(I): Using purified T cells as effector cells, CD3x5T4 bispecific antibody induces cytotoxicity in MDA-MB-231 cells in vitro. MDA-MB-231 cells were incubated with increasing concentrations of CD3x5T4 bispecific antibody or monospecific bivalent 5T4 antibody and isolated T cells as effector cells at an effector cell:target cell (E:T) ratio of 8:1. Purified T cells obtained from two different donors were used for the experiment, donor A (left panel) and donor B (right panel). Cytotoxicity was determined by measuring the percentage of viable MDA-MB-231 cells after 72 h of incubation (% viable cells = [sample absorbance – absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells – absorbance of staurosporine-treated target cells] x 100).

[0041] A. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR,

[0042] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; B. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR.

[0043] Figure 7(II): Using purified T cells as effector cells, IC50 value of cytotoxicity induced by CD3x5T4 bispecific antibody in MDA-MB-231 cells in vitro. . MDA-MB-231 cells were analyzed for bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,

[0044] bsIgG1-huCD3-FEALx5T4-226-FEAR,

[0045] bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,

[0046] bsIgG1-huCD3-FEALx5T4-059-FEAR or

[0047] IC50 value of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR-induced T cell-mediated cytotoxicity. Data are represented as mean IC50 values ± SD for two different donors.

[0048] Figure 8(I): In vitro, using T cells as effector cells, the CD3x5T4 bispecific antibody induces cytotoxicity in MDA-MB-231 cells. MDA-MB-231 cells were incubated with increasing concentrations of the CD3x5T4 bispecific antibody or 5T4 homodimer and isolated T cells as effector cells at an E:T ratio of 8:1. Three different donors were used for this experiment. Data shown are mean percent survival ± standard error of the mean (SEM) of the three donors tested.

[0049] A. T cell-mediated cytotoxicity (decreased survival) induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR,

[0050] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR;

[0051] B. In the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR,

[0052] T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; D. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; E. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; F. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR.

[0053] Figure 8 (II): IC50 values of cytotoxicity induced by CD3x5T4 bispecific antibody in MDA-MB-231 cells using T cells as effector cells in vitro. The IC50 values of T cell-mediated cytotoxicity induced by CD3x5T4 bispecific antibody in MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are represented as mean IC50 values ± SD from three different donors. A. IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR, and bsIgG1-huCD3-FEALx5T4-A3-FEAR; B. IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.

[0054] Figure 9(I): In vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. MDA-MB-231 cells were incubated with increasing concentrations of CD3x5T4 bispecific antibody and monospecific bivalent 5T4 antibody (as indicated) and isolated T cells as effector cells at an E:T ratio of 8:1. Expression of three T cell activation markers (PD1 [upper panel], CD25 [middle panel] and CD69 [lower panel]) was analyzed by flow cytometry. Two different donors were used for this experiment, namely donor A (solid symbols) and donor B (open symbols). A. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; B. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; C. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR.

[0055] Figure 9(II): EC50 value of in vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. Figure 11 EC50 values of in vitro T cell activation markers (PD1, CD25 and CD69) induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR in the presence of MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are presented as mean ± SD for two different donors.

[0056] Figure 10 (I): In vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. MDA-MB-231 cells were incubated with increasing concentrations of CD3x5T4 bispecific antibody and 5T4 homodimer and isolated T cells as effector cells at an E:T ratio of 8:1. By CD4 + (left panel) and CD8 +(Right panel) T cell activation was measured by the increase in % CD69+ cells within the T cell population. Three different donors were used for this experiment; the data shown are the mean % CD69 upregulation ± SEM of the three donors tested. A. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, and IgG1-5T4-207-FEAR; B. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, and IgG1-5T4-226-FEAR; C. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, and IgG1-5T4-059-FEAR; D. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, and IgG1-5T4-106-FEAR; E. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, and IgG1-5T4-A1-FEAR; F. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR, and IgG1-5T4-A3-FEAR.

[0057] Figure 10 (II): EC50 values of in vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of MDA-MB-231 cells. The EC50 values of T cell activation markers (increase in % CD69 50 and CD8 + T cell populations [A-B], CD25 + in the CD4 + [C-D], and PD1 + [E-F], increase in % CD25 and CD69 cells) induced by CD3x5T4 bispecific antibodies in vitro in the presence of MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. + [E-F], CD25 and CD69 cell % increase) of the EC50 50Values. Data are presented as mean ± SD of three different donors. A. EC50 values for CD69 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR;

[0058] B. EC 50 values for CD69 upregulation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR;

[0059] C. EC 50 values for CD25 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR; D. EC 50 values for CD25 upregulation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR;

[0060] E. EC values of PD1 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR 50 value;

[0061] F. EC values of PD1 upregulation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR 50 value.

[0062] Release. : T cell cytokines induced by a CD3x5T4 bispecific antibody in the presence of 5T4-positive tumor cells Figure 12MDA-MB-231 cells were incubated with 0.2 μg / mL of CD3x5T4 bispecific antibodies (bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR) and 5T4 monospecific antibodies (IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR or IgG1-5T4-059-FEAR) and isolated T cells as effector cells at an E:T ratio of 8:1. Release of cytokines was analyzed by U-PLEX assay. A. Concentrations of IL-10, IL-13 and TNF in the supernatants of T cell (derived from donor A) - tumor cell co-cultures after incubation with CD3x5T4 bispecific antibodies or 5T4 monospecific antibodies for 72 hours. B. Concentrations of IL-10, IL-13 and TNF in the supernatants of T cell (derived from donor B) - tumor cell co-cultures after incubation with CD3x5T4 bispecific antibodies or 5T4 monospecific antibodies for 72 hours.

[0063] Inducing cytotoxicity in vitro in OV-3 cells. : Using PBMC as effector cells at different E:T ratios, by CD3x5T4 bispecific antibody in SK- Figure 13 SK-OV-3 cells were incubated with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right panel) and PBMC as effector cells at E:T ratios of 1:2, 1:1, 2:1, 4:1, 8:1 and 12:1. Cytotoxicity was determined by measuring the percentage of viable SK-OV-3 cells after 72 hours of incubation (% viable cells = [absorbance of sample – absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells – absorbance of staurosporine-treated target cells]] x 100). PBMC from two different donors were used for this experiment: A. Donor C and B. Donor D.

[0064] Inducing cytotoxicity in vitro in OV-3 cells. : Using T cells as effector cells at different E:T ratios by CD3x5T4 bispecific antibody in SK- Figure 14SK-OV-3 cells were incubated with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right panel) and isolated T cells as effector cells at E:T ratios of 1:2, 1:1, 2:1, 4:1, and 8:1. Cytotoxicity was determined by measuring the percentage of viable SK-OV-3 cells after 72 hours of incubation (% viable cells = [sample absorbance – absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells – absorbance of staurosporine-treated target cells]] x 100). PBMCs from two different donors were used for this experiment: A. Donor E and B. Donor F.

[0065] Antitumor activity. . In the MDA-MB-231 xenograft model in NSG-HIS mice, the CD3x5T4 bispecific antibody Figure 15 A. Mean tumor size in the MDA-MB-231 xenograft model in NSG-HIS mice after treatment with PBS (vehicle control), 0.5 mg / kg bsIgG1-huCD3-FEALx5T4-207-FEAR, or 0.5 mg / kg bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR. Tumor size was evaluated by caliper measurement. Error bars indicate SEM. B. Percentage of NSG-HIS mice injected with MDA-MB-231 cells with tumor size < 500 mm 3 after treatment with PBS, bsIgG1-huCD3-FEALx5T4-207-FEAR, or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR.

[0066] Definition : Binding of directly FITC-labeled 5T4-specific antibody to human 5T4 variants having a single alanine mutation at positions 32 to 355 of the human 5T4 ECD, as determined by flow cytometry. Binding is expressed as Z-score (fold change) compared to a non-cross-blocking 5T4-specific control antibody (bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC) used for normalization, as a measure of binding change. The numbers on the x-axis refer to the amino acid positions in human 5T4 (SEQ ID: 1). Residues with a Z-score of binding below -1.5 (indicated by the dashed line) are considered "binding-loss variants". Residues with a positive Z-score in binding are binding-loss residues of the non-cross-blocking 5T4-specific control antibody (bsIgG1-5T4-A1-67F-F405Lxb12-FEAR-FITC). Residues at aa positions 38, 45, 49, 51, 54, 62, 64, 66, 68, 71, 72, 77, 91, 104, 108, 110, 112, 118, 121, 122, 135, 137, 155, 161, 167, 171, 201, 202, 205, 208, 218, 231, 269, 279, 298, 300, 303, 323, 324, 340 and 344 are not evaluated because these positions contain endogenous alanine or cysteine. The data shown are the Z-scores of binding of (A) bsIgG1-b12-FEALx5T4-059-FEAR-FITC, (B) bsIgG1-b12-FEALx5T4-207-FEAR-FITC, (C) bsIgG1-b12-FEALx5T4-226-FEAR-FITC and (D) bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC. Buried residues with a Z-score exactly below -1.5 and predicted to be spatially separated from most surface-exposed binding-loss residues are excluded (for bIgG1-b12-FEALx5T4-207-FEAR-FITC: L281 [Z-score: -1.57] and P326 [Z-score: 1.54]; for bsIgG1-b12-FEALx5T4-226-FEAR-FITC: L273 [Z-score: -1.58], L281 [Z-score: -1.65], N294 [Z-score: -1.57], L309 [Z-score: -1.63] and P326 [Z-score: -1.67]).

[0067] Figure 16(I): In vitro induction of cytotoxicity by the CD3x5T4 bispecific antibody in tumor cells of different indications using T cells as effector cells. Tumor cells were incubated with increasing concentrations of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) and isolated T cells as effector cells at an E:T ratio of 4:1. Cytotoxicity (decrease in survival) was determined by measuring the percentage of viable tumor cells after 72 hours of incubation. The data shown are the mean % survival ± SEM of duplicate wells from one representative donor out of at least three tested donors. A. Cytotoxicity (decrease in survival) induced in pancreatic cancer cell lines; B. Cytotoxicity (decrease in survival) induced in cervical cancer cell lines.

[0068] Figure 16(II): IC50 values of in vitro cytotoxicity induced by the CD3x5T4 bispecific antibody in tumor cell lines of different indications using T cells as effector cells. The IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in tumor cells of the indicated indications were analyzed using GraphPad Prism V7.02 software. The data are presented as the mean IC50 values (see Table 10) ± SD of at least three different donors.

[0069] Figure 17(I): In vitro T cell activation by the CD3x5T4 bispecific antibody in the presence of tumor cells of different indications. Tumor cells were incubated with increasing concentrations of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) and isolated T cells as effector cells at an E:T ratio of 4:1 for 72 hours. By CD4 + (left panel) and CD8 +(Right panel) T cell activation was measured by upregulation of CD69 (percentage of CD69+ cells) within the T cell population. The data shown are the mean % CD69+ cells ± SD of duplicate wells from one representative donor out of at least three tested donors. A. T cell activation induced by the CD3x5T4 bispecific antibody in the presence of the pancreatic cancer cell line BxPc-3; B. T cell activation induced by the CD3x5T4 bispecific antibody in the presence of the pancreatic cancer cell line PANC-1; C. T cell activation induced by the CD3x5T4 bispecific antibody in the presence of the cervical cancer cell line SiHa; D. T cell activation induced by the CD3x5T4 bispecific antibody in the presence of the cervical cancer cell line CaSki.

[0070] Figure 17 (II): EC50 values of in vitro T cell activation by the CD3x5T4 bispecific antibody in the presence of tumor cell lines of different indications. The EC50 values of T cell activation (CD4 + and CD8 + percentage of CD69+ cells within the T cell population) induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR co-cultured with tumor cell lines of different indications were analyzed using GraphPad Prism V7.02 software. The data are presented as the mean EC50 values (see Table 10) ± SD of at least three different donors. A. EC50 values of CD4+ T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of the indicated tumor cell lines; B. EC50 values of CD8+ T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of the indicated tumor cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0072] Category

[0073] As used herein, the term "antibody" (Ab) is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either having the ability to specifically bind an antigen with a half-life of a relatively long period of time under typical physiological conditions and / or tumor-specific conditions, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functional definition of a period of time (e.g., a time sufficient to induce, promote, enhance, and / or regulate a physiological response associated with the binding of the antibody to the antigen and / or a time sufficient for the antibody to be internalized). The binding region that interacts with the antigen (or binding domain, which can be used interchangeably herein) includes the variable regions of the heavy and light chains of the immunoglobulin molecule. The constant region of an antibody (Ab) 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 (e.g., C1q), i.e., the first component in the classical pathway of complement activation.

[0074] In the context of the present invention, the term "antibody" includes monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and "antibody fragments" or "fragments thereof" provided by any known technique such as enzymatic cleavage, peptide synthesis, and recombinant techniques that retain the ability to specifically bind an antigen (antigen-binding fragment) and retain the ability to conjugate with a toxin. Unless the disclosure herein is otherwise limited, the antibodies defined according to the present invention can have any isotype.

[0075] As indicated above, unless otherwise stated or clearly inconsistent with the context, the term antibody as used herein includes antibody fragments that retain the ability to specifically interact with an antigen, such as bind. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of the variable domain of the light chain (VL), the variable domain of the heavy chain (VH), the constant region of the light chain (CL), and the first constant region of the heavy chain (CH1) domain or monovalent antibodies as described in WO 2007 / 059782; (ii) F(ab')2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting essentially of the VH domain, also known as domain antibodies Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90 Ward et al., Nature341 , 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). In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be joined by recombinant methods via a synthetic linker that enables them to be prepared as a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (referred to as a single - chain antibody or single - chain Fv (scFv), see, for example, Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1): 111 - 24 and Bird et al., Science 242 , 423 - 426 (1988). Unless otherwise specified or clearly indicated by the context, such single - chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of an antibody, they are each and collectively unique features of the present invention, exhibiting different biological properties and utilities. In the context of the present invention, these and other useful antibody fragments are further discussed herein.

[0076] Antibodies can be produced and collected from different in vitro or ex vivo expression or production systems, such as from recombinant - modified host cells, hybridomas, or systems using cell extracts that support in vitro transcription and / or translation of the nucleic acid sequence encoding the antibody. It should be understood that a variety of different antibodies (as defined in the context of the present invention) can be provided by separately generating each antibody in the production systems described above and then mixing the antibodies, or by generating several antibodies in the same production system.

[0077] As used herein, the term "immunoglobulin heavy chain" or "heavy chain of an immunoglobulin" refers to one of the heavy chains of an immunoglobulin. A heavy chain typically consists of a variable region of the heavy chain (abbreviated herein as VH) that defines the immunoglobulin isotype and a constant region of the heavy chain (abbreviated herein as CH). The constant region of the heavy chain typically consists of three domains, CH1, CH2, and CH3. As used herein, the term "immunoglobulin" means a class of structurally related glycoproteins composed of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four potentially linked to each other by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989))). Within the structure of an immunoglobulin, the two heavy chains are linked to each other by disulfide bonds in the so-called "hinge region". Similar to the heavy chain, each light chain typically consists of several regions: a variable region of the light chain (abbreviated herein as VL) and a constant region of the light chain. The constant region of the light chain typically consists of one domain, CL. In addition, the VH and VL regions can be further subdivided into hypervariable regions (or regions of high variability, which can be hypervariable in the sequence and / or structure of a structurally defined loop), also known as complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences are defined 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)).

[0078] When used herein, the terms "half-molecule", "Fab arm", and "arm" refer to a heavy chain-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" means that the bispecific antibody is produced by recombining the half-molecules from each of the first antibody and the second antibody into the resulting bispecific antibody by any known method. In this context, "recombinant" is not intended to be limited by any particular recombinant method and thus includes, for example, all methods of producing bispecific antibodies described below, including, for example, recombination by half-molecule exchange and recombination at the nucleic acid level and / or by co-expression of the two half-molecules in the same cell.

[0079] As used herein, the terms "antigen-binding region" or "binding region" refer to the region of an antibody that is capable of binding an antigen. The antigen can be any molecule, such as a polypeptide, for example, present on a cell, bacterium, or virion. Unless the context is contradictory, the terms "antigen" and "target" can be used interchangeably in the context of the present invention. Unless the context is contradictory, the terms "antigen-binding region" and "antigen-binding site" can be used interchangeably in the context of the present invention.

[0080] The term "blocking binding" or "blocking the binding of an antibody" or "cross-blocking binding" refers to a situation in which the binding of one antibody to a specific antigen prevents 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 the antigen, as determined by a significant binding response assay, while in the presence of the other antibody, one of the antibodies lacks a binding response. The ability of one antibody to block the binding of another antibody can be determined by biolayer interferometry in a classical sandwich epitope binning assay format, for example, as described in Example 3 of this application and 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 the sandwich epitope binning assay, the binding of an antibody in solution to its specific antigen is tested, and the specific antigen is first captured by an immobilized antibody. In the context of the present invention, according to the definition of "displacement" below, if an antibody is able to "displace" another antibody, it does not block the binding of the other antibody. Unless the context is contradictory, the terms "blocking binding" and "blocking the binding of an antibody" and "cross-blocking binding" can be used interchangeably in the context of the present invention. Preferably, the ability of one antibody to block the binding of another antibody is determined using full-length antibodies.

[0081] The term "displacement" or "displacement ability" refers to the following situation, wherein two antibodies interfere with each other's binding to the antigen by kinetically changing each other's binding to their specific antigens via the formation of a transient trimolecular complex, which rapidly decomposes by retaining one antibody directed to the antigen and displacing another. Antibody displacement is defined in Abdiche et al., 2017 (Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morishige W, et al. (2017) Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another. PLoS ONE 12 (1): e0169535. doi: 10.1371 / journal. pone.0169535). As described in Abdiche et al. 2017 and Example 4 of the present application, the displacement of antibodies can be determined by using a real-time label-free biosensor in a classical sandwich assay format by biolayer interferometry. Preferably, antibody displacement is determined using the IgG form of the antibody.

[0082] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, typically measured by biolayer interferometry using the antibody as a ligand and the antigen as an analyte at a concentration corresponding to 1E. -6 M or smaller, such as 5E -7 M or smaller, 1E -7 M or smaller, such as 5E -8 M or smaller, such as 1E -8 M or smaller, such as 5E -9 M or smaller, or 1E -9 M or smaller K D The binding affinity of D The affinity of the antigen is determined by the K D At least 10 times lower, such as at least 100 times lower, such as at least 1000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen.

[0083] As used herein, the term "K D ” (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction and is expressed by k d Divide by k a get.

[0084] As used herein, the term "k d " (sec -1 ) refers to the dissociation rate constant of a specific antibody-antigen interaction. The value is also referred to as the k off value or dissociation rate.

[0085] As used herein, the term "k a " (M -1 x sec -1 ) refers to the association rate constant of a specific antibody-antigen interaction. The value is also referred to as the k on value or association rate.

[0086] As used herein, the term "5T4" refers to the protein named 5T4, which is also known as trophoblast glycoprotein, 5T4 carcinoembryonic antigen, 5T4 carcinoembryonic trophoblast glycoprotein, TPBG, WAIF1, and M6P1. It is a 72-80 kDa transmembrane protein with an extensive N-linked glycosylation core. In humans (Homo sapiens), the 5T4 protein has the amino acid sequence shown in SEQ ID NO:1 (human trophoblast glycoprotein: Uniprot accession number Q13641). In the amino acid sequence shown in SEQ ID NO:1, amino acid residues 1-31 are the signal peptide, and amino acid residues 32-420 are the mature polypeptide. In cynomolgus monkeys (Macaca fascicularis), the 5T4 protein has the amino acid sequence shown in SEQ ID NO:2 (Uniprot accession number Q4R8Y9). In the amino acid sequence shown in SEQ ID NO:2, amino acid residues 1-34 are the signal peptide, and amino acid residues 35-420 are the mature polypeptide. In chickens (Gallus gallus), the 5T4 protein has the amino acid sequence shown in SEQ ID NO:3 (Uniprot accession number R4GM46). In the sequence shown in SEQ ID NO:3, amino acid residues 1-27 are the signal peptide, and amino acid residues 28-379 are the mature polypeptide.

[0087] As used herein, the term "CD3" refers to the human cluster of differentiation 3 protein, which is part of the T cell co-receptor protein complex and consists of four distinct chains. CD3 also exists in other species, and thus, unless the context is contradictory, the term "CD3" is not limited to human CD3. In mammals, the complex contains the CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No P09693 or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No Q95LI7), the CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No P04234 or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No Q95LI8), two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No P07766; amino acid residues 1-22 are the signal peptide, and amino acid residues 23-207 are the mature CD3ε polypeptide, which is identified herein as SEQ ID NO:4; cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No Q95LI5; or rhesus monkey CD3ε UniProtKB / Swiss-Prot No G7NCB9) and the CD3ζ (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No Q09TK0). These chains associate with a molecule called the T cell receptor (TCR) and generate activation signals in T lymphocytes. The TCR and CD3 molecules together constitute the TCR complex.

[0088] The term "antibody binding region" refers to the region of an antigen that contains the epitope to which an antibody binds. The antibody binding region can be determined by epitope mapping using biolayer interferometry, by alanine scanning, or by shuffling assays (using antigen constructs that exchange regions of one antigen with regions of another antigen and determining whether the antibody still binds the antigen). The amino acids involved in the interaction with the antibody within the antibody binding region can be determined by hydrogen / deuterium exchange mass spectrometry and by crystallography of the antibody bound to its antigen.

[0089] The term "epitope" refers to the antigenic determinant that is specifically bound by an antibody. Epitopes typically consist of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof, and generally have specific three-dimensional structural features as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that in the presence of a denaturing solvent, binding to the former is lost but not to the latter. An epitope can include amino acid residues that are directly involved in binding, as well as other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the antibody when the antibody binds to the antigen (in other words, the amino acid residues are within or in close proximity to the footprint of a particular antibody).

[0090] As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", etc. refer to preparations of antibody molecules consisting of a single molecule. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomic non-human animals, such as transgenic mice, that are fused with immortalized cells, the B cells having a genome comprising a human heavy chain transgene and a light chain transgene. Monoclonal antibodies can also be produced from recombinant modified host cells or using systems that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0091] As used herein, the term "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f). In addition, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain.

[0092] As used herein, the term "full-length antibody" refers to an antibody (e.g., a parental or variant antibody) that comprises one or two pairs of heavy and light chains, each heavy and light chain containing all of the heavy and light chain constant and variable domains that are normally present in the heavy-chain / light-chain pair of a wild-type antibody of that isotype. In a full-length variant antibody, the heavy and light chain constant and variable domains may particularly contain amino acid substitutions that improve the functional properties of the antibody. Full-length antibodies according to the invention can be produced by a method comprising the steps of: (i) cloning CDR sequences into a suitable vector containing a complete heavy-chain sequence and a complete light-chain sequence, and (ii) expressing the complete heavy-chain and light-chain sequences in a suitable expression system. Producing full-length antibodies starting from CDR sequences or complete variable region sequences is within the knowledge of those skilled in the art. Thus, those skilled in the art will know how to produce full-length antibodies according to the invention.

[0093] As used herein, the term "human antibody" is intended to include antibodies having variable regions and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences from the germline of another non-human species such as a mouse have been grafted onto human framework sequences.

[0094] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that contains human antibody constant domains and non-human variable domains that have been modified to have a high level of sequence homology with human variable domains. This can be achieved by grafting six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site onto a homologous human receptor framework region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parental antibody, it may be necessary to replace framework residues from the parental antibody (i.e., the non-human antibody) with human framework regions (backmutations). Structural homology modeling can assist in identifying amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, predominantly human framework regions, and fully human constant regions, the human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences. Optionally, other amino acid modifications that are not necessarily backmutations may be applied to obtain a humanized antibody having preferred properties such as affinity and biochemical properties.

[0095] As used herein, the term "Fc region" refers to the region that includes at least the hinge region, CH2 region, and CH3 region in the direction from the N-terminal to the C-terminal end of an antibody. The Fc region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0096] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 - 230 according to the EU numbering as set forth in Kabat, Kabat, E.A. 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 can also be any other subtype as described herein.

[0097] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118 - 215 according to the Eu numbering as set forth in Kabat (supra). However, the CH1 region can also be any other subtype as described herein.

[0098] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231 - 340 according to the Eu numbering as set forth in Kabat (supra). However, the CH2 region can also be any other subtype as described herein.

[0099] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341 - 447 according to the Eu numbering as set forth in Kabat (supra). However, the CH3 region can also be any other subtype as described herein.

[0100] As used herein, the term "Fc-mediated effector function" is intended to refer to a function that results from the binding of a polypeptide or antibody to its target or antigen on a cell membrane, where the Fc-mediated effector function can be attributed to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-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 opsonizing antibodies to complement receptors mediated by the antibody, (xi) opsonization, and (xii) a combination of any of (i) to (xi).

[0101] As used herein, the terms "inert", "inactive", or "non-activated" refer to an Fc region that is at least unable to bind any FcγR, induce Fc-mediated FcγR cross-linking, or induce FcγR-mediated target antigen cross-linking via the two Fc regions of an individual antibody, or is unable to bind C1q. Monospecific or bispecific forms of an antibody can be used to test the inertness of the Fc region of the antibody.

[0102] When used in the context of an antibody, the term "full-length" means that the antibody is not a fragment, but contains all of the domains of that particular isotype that are typically found in nature for that isotype, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0103] In the context of the present invention, the term "monovalent antibody" refers to an antibody molecule that can interact with a specific epitope on an antigen by virtue of only one antigen-binding domain (e.g., one Fab arm). In the context of a bispecific antibody, "monovalent antibody binding" refers to the binding of a bispecific antibody to a specific epitope on an antigen by virtue of only one antigen-binding domain (e.g., one Fab arm).

[0104] In the context of the present invention, the term "monospecific antibody" refers to an antibody that has binding specificity for only one epitope. The antibody can be a monospecific monovalent antibody (i.e., carrying only one antigen-binding region) or a monospecific bivalent antibody (i.e., an antibody having two identical antigen-binding regions).

[0105] The term "bispecific antibody" refers to an antibody having two different antigen-binding domains, such as two different Fab arms or two Fab arms having different CDR regions. In the context of the present invention, a bispecific antibody is specific for at least two different epitopes. Such epitopes may be on the same or different antigens or targets. If the epitopes are on different antigens, such antigens may be on the same cell or different cells, cell types or structures, such as on the extracellular matrix or vesicles and soluble proteins. Thus, bispecific antibodies may be able to crosslink multiple antigens, e.g., two different cells.

[0106] The term "bivalent antibody" refers to an antibody having two antigen-binding regions that bind to one or two epitopes on a target or antigen or to one or two epitopes on the same antigen. Thus, a bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0107] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. An amino acid is an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group as well as a side chain (R group) unique to each amino acid. In the context of the present invention, amino acids can be classified based on their structure and chemical properties. Thus, amino acid classes may be reflected in one or both of the following tables:

[0108] Major classification based on the structure and general chemical characterization of the R group

[0109] Amino acid Acidic residue D and E Basic residue K, R and H Hydrophilic uncharged residue S, T, N and Q Aliphatic uncharged residue G, A, V, L and I Non-polar uncharged residue C, M and P Aromatic residue F, Y and W Immunoconjugate

[0110] Alternative physical and functional classification of amino acid residues

[0111]

[0112] Replacing one amino acid with another can be classified as a conservative or non-conservative substitution. In the context of the present invention, a "conservative substitution" is the replacement of one amino acid with another having similar structure and / or chemical characteristics, such as replacing one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above: e.g., leucine can be replaced with isoleucine because they are both aliphatic, branched-chain hydrophobes. Similarly, aspartic acid can be replaced with glutamic acid because they are both small, negatively charged residues.

[0113] In the context of the present invention, substitutions in an antibody are represented as:

[0114] Original amino acid - position - substituted amino acid;

[0115] Referring to the well - recognized amino acid nomenclature, three - letter codes or one - letter codes are used, including the codes "Xaa" or "X" to represent any amino acid residue. Thus, Xaa or X can generally represent any one of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any one of the following amino acid residues; glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the symbol "K409R" or "Lys409Arg" means that the antibody contains a substitution of lysine by arginine at amino acid position 409.

[0116] The substitution of an amino acid at a given position by any other amino acid is called:

[0117] The original amino acid - position; or for example "K409"

[0118] For modifications where one or more of the original amino acids and / or one or more of the substituted amino acids can contain more than one but not all amino acids, more than one amino acid can be separated by "," or " / ". For example, the substitution of lysine at position 409 by arginine, alanine, or phenylalanine is:

[0119] "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409 to R,A or F".

[0120] Such names can be used interchangeably in the context of the present invention, but have the same meaning and purpose.

[0121] In addition, the term "substitution" includes substitution into any one or other 19 natural amino acids, or substitution into other amino acids, such as non - natural amino acids. For example, the substitution of 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. Incidentally, this is equivalent to the name 409X, where X represents any amino acid other than the original amino acid. These substitutions can also be called K409A, K409C, etc. or K409A,C, etc. or K409A / C / etc. Similarly, this applies to each position mentioned herein to specifically include any one of such substitutions in this text.

[0122] Antibodies according to the invention may also comprise deletions of amino acid residues. Such deletions may be denoted as "del", and include, for example, written as K409del. Thus, in such embodiments, the lysine at position 409 has been deleted from the amino acid sequence.

[0123] As used herein, the term "host cell" is intended to refer to a cell that has received an expression vector. It should be understood that such term is not only intended to refer to a particular subject cell, but also to the progeny of such cell. Since certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may actually be different from the parental cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfected cells such as CHO cells, HEK 293 cells, Expi293F cells, PER.C6 cells, NS0 cells and lymphocytes, as well as prokaryotic cells such as Escherichia coli and other eukaryotic hosts such as plant cells and fungi.

[0124] As used herein, the term "transfected cell" includes recombinant eukaryotic host cells that express 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.

[0125] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used, 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), preferably version 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5 and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of Needle marked "longest identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0126] (Number of identical residues x 100) / (Alignment length - Total number of gaps in the alignment).

[0127] Retention of similar residues can also or alternatively be measured by a similarity score, such as determined by using the BLAST program (e.g., BLAST 2.2.8 obtained via NCBI, using the standard settings BLOSUM62, open gap = 11 and extend gap = 1). Suitable variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95% or more (e.g., about 99%) similarity to the parental sequence.

[0128] As used herein, the term "internalization" or "internalizing" refers to the biological process by which a molecule such as an antibody according to the invention is engulfed by the cell membrane and drawn into the interior of the cell. Internalization can also be referred to as "endocytosis".

[0129] antibody

[0130] In a first aspect, the invention provides an antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), wherein the antibody is capable of blocking the binding of an antibody selected from the group consisting of:

[0131] a) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:5 and a VL region containing the sequence shown in SEQ ID NO:9

[059] ,

[0132] b) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:12 and a VL region containing the sequence shown in SEQ ID NO:16

[076] ,

[0133] c) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:19 and a VL region containing the sequence shown in SEQ ID NO:23

[085] ,

[0134] d) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:26 and a VL region containing the sequence shown in SEQ ID NO:30

[106] ,

[0135] e) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:33 and a VL region containing the sequence shown in SEQ ID NO:37

[127] ,

[0136] f) an antibody comprising a VH region containing the sequence shown in SEQ ID NO:40 and a VL region containing the sequence shown in SEQ ID NO:44

[207] ; and

[0137] g) An antibody comprising a VH region having the sequence shown in SEQ ID NO: 47 and a VL region having the sequence shown in SEQ ID NO: 51

[226] .

[0138] In particular, the present invention provides an antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), wherein the antibody is capable of blocking the binding of an antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region

[059] to 5T4, the heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 5 and the light chain variable (VL) region having the sequence shown in SEQ ID NO: 9.

[0139] In particular, the antibody may be capable of blocking the binding of an antibody selected from the group consisting of:

[0140] a) An antibody comprising a heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 40 and a light chain variable (VL) region having the sequence shown in SEQ ID NO: 44

[207] ,

[0141] b) An antibody comprising a heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 47 and a light chain variable (VL) region having the sequence shown in SEQ ID NO: 51

[226] ; and

[0142] An antibody comprising a heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 5 and a light chain variable (VL) region having the sequence shown in SEQ ID NO: 9

[059] .

[0143] In certain embodiments of the invention, the antibody is capable of blocking the binding of an antibody selected from the group consisting of:

[0144] a) An antibody comprising a heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 40 and a light chain variable (VL) region having the sequence shown in SEQ ID NO: 44

[207] ; and

[0145] b) An antibody comprising a heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 47 and a light chain variable (VL) region having the sequence shown in SEQ ID NO: 51

[226] .

[0146] The antibody according to the invention is characterized by being specific for human (Homo sapiens) 5T4 or having the ability to bind human (Homo sapiens) 5T4. Thus, 5T4 as referred to herein may in particular be human 5T4, such as the mature polypeptide of SEQ ID NO: 1.

[0147] In a further embodiment, the antibody of the invention is characterized by being specific for cynomolgus monkey (Macaca fascicularis) 5T4 or having the ability to bind cynomolgus monkey (Macaca fascicularis) 5T4, for example being specific for or binding both human and cynomolgus monkey 5T4. Cynomolgus monkey 5T4 can particularly be the mature polypeptide of SEQ ID NO:2.

[0148] In a further embodiment, the antibody of the invention is specific for chicken (Gallus gallus) 5T4 or has the ability to bind chicken (Gallus gallus) 5T4, for example being specific for or binding human 5T4 and chicken 5T4, or being specific for or binding human, cynomolgus monkey and chicken 5T4, wherein particularly, chicken 5T4 can have the amino acid sequence of the mature polypeptide of SEQ ID NO:3.

[0149] Thus, the antibody of the invention can be specific for human 5T4 such as the mature polypeptide of SEQ ID NO:1 and cynomolgus monkey 5T4 such as the mature polypeptide of SEQ ID NO:2 or can be capable of binding human 5T4 such as the mature polypeptide of SEQ ID NO:1 and cynomolgus monkey 5T4 such as the mature polypeptide of SEQ ID NO:2.

[0150] Furthermore, the antibody according to the invention can be specific for human 5T4, such as the mature polypeptide of SEQ ID NO:1, cynomolgus monkey 5T4, such as the mature polypeptide of SEQ ID NO:2, and chicken 5T4, such as the mature polypeptide of SEQ ID NO:3, or can be capable of binding human 5T4, such as the mature polypeptide of SEQ ID NO:1, cynomolgus monkey 5T4, such as the mature polypeptide of SEQ ID NO:2, and chicken 5T4, such as the mature polypeptide of SEQ ID NO:3.

[0151] The antibody according to the invention can be capable of binding with a K D value corresponding to 1E-7 M or less, a K DValues, a binding affinity of 5E-8 M or less, about 5E-8 M or less, 1E-8 M or less, about 1E-8 M or less, 5E-9 M or less, about 5E-9 M or less, such as 1E-9 M or less or about 1E-9 M or less, such as in the range of 1E-7 to 5E-10 M, such as about 1E-7 to about 5E-10 M, such as 1E-7 to 1E-9 M, such as about 1E-7 to about 1E-9 M, such as 5E-8 to 5E-10 M, such as about 5E-8 to about 5E-10 M, such as 5E-8 to 1E-9 M, such as about 5E-8 to about 1E-9 M, such as 1E-8 to 5E-10 M, such as about 1E-8 to about 5E-10 M, such as 1E-8 to 1E-9 M, such as about 1E-8 to about 1E-9 M, such as 1E-8 to 5E-9 M or about 1E-8 to about 5E-9 M of K D The binding affinity of the value binds to human 5T4, cynomolgus monkey and / or chicken 5T4.

[0152] Although it is within the ability of those skilled in the art to determine the affinity of an antibody for its target, the binding affinity of the antibodies according to the present invention for 5T4 can in particular be determined by biolayer interferometry, optionally as described in Example 2 herein.

[0153] More specifically, procedures such as biolayer interferometry procedures can be used to determine the binding affinity of the antibodies according to the present invention, and the procedures include the following steps:

[0154] I) Immobilize 1 μg / mL of the antibody on an anti-human IgG Fc capture biosensor for 600 seconds;

[0155] II) Using a two-fold dilution series ranging from 100 nM to 1.56 nM, determine the association for 200 seconds and the dissociation for 1000 seconds of 5T4ECDHis (the mature protein of SEQ ID NO: 99) or cynomolgus monkey 5T4 (the mature protein of SEQ ID NO: 2) or recombinant cynomolgus monkey 5T4 protein (Cusabio; catalog number CSB-MP024093MOV),

[0156] III) Refer to the buffer control (0 nM) reference data.

[0157] In particular, the binding affinity of the antibodies according to the present invention can be determined using the antibodies defined in any one of the preceding claims, which are monospecific bivalent antibodies, such as antibodies of full-length IgG1.

[0158] In a further embodiment of the present invention, the antibody recognizes or binds to an epitope or antibody-binding region or binding site on 5T4, and the binding site or epitope or antibody-binding region is recognized by any one of the antibodies selected from the following group:

[0159] a) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:5 and a VL region containing the sequence shown in SEQ ID NO:9

[059] ,

[0160] b) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:12 and a VL region containing the sequence shown in SEQ ID NO:16

[076] ,

[0161] c) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:19 and a VL region containing the sequence shown in SEQ ID NO:23

[085] ,

[0162] d) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:26 and a VL region containing the sequence shown in SEQ ID NO:30

[106] ,

[0163] e) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:33 and a VL region containing the sequence shown in SEQ ID NO:37

[127] ,

[0164] f) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:40 and a VL region containing the sequence shown in SEQ ID NO:44

[207] ; and

[0165] g) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:47 and a VL region containing the sequence shown in SEQ ID NO:51

[226] .

[0166] In a further embodiment, an antibody according to the invention recognizes or binds to an antibody-binding region, binding site or epitope on 5T4 which is not the antibody-binding region or binding site or epitope bound by an antibody selected from the group consisting of or is different from the antibody-binding region, binding site or epitope bound by an antibody selected from the group consisting of:

[0167] a) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:87 and a VL region containing the sequence shown in SEQ ID NO:88 [H8],

[0168] b) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:83 and a VL region containing the sequence shown in SEQ ID NO:84 [A1]; and

[0169] c) An antibody comprising a VH region containing the sequence shown in SEQ ID NO:85 and a VL region having the sequence shown in SEQ ID NO:86 [A3].

[0170] In other embodiments, the binding of an antibody according to the invention to 5T4 is blocked by the binding of an antibody [A3] comprising a heavy chain variable (VH) region and a light chain variable (VL) region to 5T4, wherein the heavy chain variable (VH) region comprises the sequence shown in SEQ ID NO:85 and the light chain variable (VL) region comprises the sequence shown in SEQ ID NO:86. The antibody comprising the VH and VL sequences shown in SEQ ID NOs 85 and 86 respectively is antibody A3, one of the three murine 5T4 antibodies disclosed in WO2007106744. Rewrite: Antibody A3 with a single amino acid substitution. In the CDR sequences?

[0171] In other embodiments, the antibody according to the invention shows displacement of an antibody that binds to 5T4 or to the His-tagged extracellular domain of 5T4 (e.g., the mature protein of 5T4ECDHis / SEQ ID NO:99), wherein the antibody that binds to 5T4 contains a heavy chain variable (VH) region comprising the sequence shown in SEQ ID NO:85 and a light chain variable (VL) region comprising the sequence shown in SEQ ID NO:86 [A3]. Such displacement indicates that the antibody of the invention binds to an epitope that is different from the epitope bound by antibody A3, but may be adjacent to or even overlap with the epitope bound by A3.

[0172] The ability of a "displacing" or displacement-binding antibody can be measured in a biolayer interferometry assay, such as in the assay conducted as described in Example 4 of the present application.

[0173] The "cross-blocking" or the ability of an antibody according to the invention to block the binding of another antibody to 5T4 can be measured by using a fluorescence-activated cell sorting (FACS) assay, such as in the assay conducted as described in Example 5.

[0174] In particular, the "cross-blocking" or the ability of an antibody according to the invention to block the binding of another antibody to 5T4 is measured by the ability of an unconjugated antibody to block a conjugated antibody, and is optionally measured in a procedure comprising the following steps:

[0175] i) Providing a set of samples, each sample comprising a mixture of human ovarian adenocarcinoma SK-OV-3 cells, an antibody that binds to 5T4 and is conjugated to fluorescein isothiocyanate (FITC), and an excess of an unconjugated antibody targeting 5T4,

[0176] ii) Incubating the samples at 4°C for 30 minutes and then centrifuging the samples,

[0177] iii) The supernatant was removed from each sample, and the cells were resuspended in buffer, and the mean fluorescence intensity (MFI) of FITC was measured using a flow cytometer; and

[0178] iv) Calculate the percentage of binding as follows:

[0179] Multiply the difference in MFI between cells incubated with a mixture of the antibody conjugated with FITC and the unconjugated antibody and cells not incubated with the antibody conjugated with FITC or the unconjugated antibody by 100, and then divide by the difference in MFI between cells incubated with a mixture of the antibody conjugated with FITC and the IgG-b12 antibody and cells not incubated with the antibody conjugated with FITC or the unconjugated antibody.

[0180] While those skilled in the art will be familiar with suitable techniques for determining the ability of an antibody to block the binding of another antibody to its target or displace another antibody bound to its target, the present application discloses procedures suitable for assaying blocking binding and displacement. Thus, in some embodiments, biolayer interferometry can be used to determine the ability of an antibody according to the invention to block the binding of another antibody to 5T4 or displace another antibody bound to 5T4, for example, in a biolayer interferometry performed as described in Example 3.

[0181] In particular, biolayer interferometry can be used to determine the ability of an antibody according to the invention to block the binding of another antibody to 5T4 or displace another antibody bound to 5T4, and can be determined in a procedure comprising the following steps:

[0182] i) Immobilize the antibody according to the invention at a quantity of 20 μg / mL in 10 mM sodium acetate buffer onto an activated amine-reactive second-generation biosensor,

[0183] ii) Quench the biosensor with the immobilized antibody in ethanolamine at pH 8.5,

[0184] iii) Immerse the biosensor with the immobilized antibody in a composition comprising 3.6 μg / mL (100 nM) human 5T4ECDHis (mature protein of SEQ ID NO: 99) for a period of 500 seconds, and then

[0185] iv) Immerse the biosensor with the immobilized antibody and 5T4ECDHis in a composition comprising 10 μg / mL of another antibody targeting 5T4, and measure the association response within a period of 500 seconds;

[0186] wherein steps i)-iv) are carried out at a temperature of 30 °C and with shaking at 1000 rpm.

[0187] The antibodies provided herein can bind to an epitope or antibody-binding region on human 5T4 that includes amino acid residues R73, Y92, and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0188] The present invention also provides antibodies that bind to an epitope or antibody-binding region on human 5T4 that includes amino acid residues S69, R73, Y92, and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0189] The present invention also provides antibodies that bind to an epitope or antibody-binding region on human 5T4 that includes amino acid residues R73, T74, Y92, R94, and N95; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0190] Based on the results provided in Example 16 herein, and without wishing to be bound by theory, it is hypothesized that any one or more of these amino acid residues (i.e., S69, R73, T74, Y92, R94, and N95) are directly involved in antibody binding, e.g., by non-covalent interactions; e.g., having amino acid residues within the CDR sequences of the antibody. This hypothesis is supported by the fact that these residues are identified as surface-exposed on the structure of 5T4 (4cnm; provided by the RCSB PDB Protein Data Bank; DOI: 10.2210 / pdb4CNM / pdb); as published in Zhao, Y., Malinauskas, T., Harlos, K., & Jones, E.Y. (2014). Structural insights into the inhibition of Wnt signaling by cancer antigen 5T4 / Wnt-activated inhibitory factor 1. Structure, 22(4), 612–620.

[0191] One or more of the following additional amino acid residues may be involved in antibody binding, e.g., indirectly involved in binding, e.g., by affecting protein folding and / or the positioning of one or more amino acid residues directly involved in antibody binding: L89, F111, L117, F138, L144, D148, N152; the numbering of each amino acid residue refers to its position in SEQ ID NO:1. In particular, as described in Zhao et al., Structure, 22(4), 612–620, L89, F111, L117, F138, L144 have been identified as part of the hydrophobic core within 5T4.

[0192] In addition, the antibodies disclosed herein can be directed against an epitope or antibody-binding region on human 5T4, wherein the amino acid residues R73, Y92, and R94 within the epitope or antibody-binding region are directly involved in binding the antibody, and wherein one or more of the amino acid residues F111, F138, L144, and D148 are indirectly involved in said binding; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0193] The antibodies provided herein can bind to an epitope or antibody-binding region on human 5T4, wherein the amino acid residues S69, R73, Y92, and R94 within the epitope or antibody-binding region are directly involved in binding the antibody, and wherein one or more of the amino acid residues F111, F138, and D148 are indirectly involved in said binding; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0194] In addition, the present disclosure provides antibodies that bind to an epitope or antibody-binding region on human 5T4, wherein the amino acid residues R73, T74, Y92, R94, and N95 within the epitope or antibody-binding region are directly involved in binding the antibody, and wherein the amino acid residue F138 is indirectly involved in said binding; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0195] The amino acid residues contained within the epitope or antibody-binding region and optionally one or more additional amino acid residues that are indirectly involved in binding can be identified by alanine scanning of human 5T4 having the amino acid sequence shown in SEQ ID NO:1 or the mature polypeptide sequence of SEQ ID NO:1, or by alanine scanning of a polypeptide comprising amino acid residues 32-355 of SEQ ID NO:1.

[0196] Alanine scanning can be carried out specifically as described in Example 16 herein or substantially as described in Example 16 herein.

[0197] In addition, alanine scanning can be carried out by a procedure comprising the following steps:

[0198] i) Individually express mutant human 5T4 polypeptides and wild-type 5T4 polypeptides (amino acid residues 32-355 of SEQ ID NO:1) in human embryonic kidney cells, such as HEK 293 cells, so as to provide a sample containing 70-90,000 cells, such as 80,000 cells, for each mutant or wild-type 5T4, wherein all amino acid residues in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO:1), except for cysteine and alanine, are individually replaced with alanine in the mutant human 5T4 polypeptides.

[0199] ii) At room temperature, incubate the cells in each sample with 20 μL of the antibody for 40 minutes. The antibody is conjugated to an antibody conjugated with fluorescein isothiocyanate (FITC) (3 μg / mL; in FACS buffer), and then wash each sample twice in 150 - 180 μL of FACS buffer (phosphate - buffered saline [PBS; Lonza, catalog number BE17 - 517] + 0.1% [w / v] BSA [Roche, catalog number 10735086001] + 0.02% [w / v] sodium azide [NaN3; EMELCA Bioscience, catalog number 41920044 - 3]), and resuspend the cells in each sample in 30 μL of FACS buffer.

[0200] iii) For each sample, determine the average amount of antibody bound per cell as the geometric mean fluorescence intensity (gMFI) of the live single - cell population in the sample, and normalize the data for each test antibody relative to the binding strength of a non - cross - blocking 5T4 - specific control antibody using the following equation:

[0201]

[0202] where "aa position" refers to the position mutated to alanine.

[0203] where the Z - score is calculated according to the following to express loss or gain of antibody binding:

[0204]

[0205] where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all mutants, respectively.

[0206] where if the gMFI of the control antibody for a particular 5T4 mutant is below the mean gMFI 对照抗体 - 2.5x mean gMFI 对照抗体 of the SD (from all mutants), the data are excluded from the analysis; and optionally

[0207] where if a residue binds with a Z - score just below - 1.5 (e.g., between - 1.5 and - 1.8, such as between - 1.5 and - 1.7 or such as between - 1.5 and - 1.6), and the residue is predicted to be buried and spatially separated from most residues, the data are excluded from the analysis. The most residues are predicted to be surface - exposed and loss of binding or reduced binding is determined for the most residues.

[0208] A suitable non - cross - blocking 5T4 - specific control antibody to be used in step iii) is a bispecific antibody that contains

[0209] - An antigen-binding region comprising a VH sequence as shown in SEQ ID NO:83 and a VL sequence as shown in SEQ ID NO:84 [A1]; and

[0210] - An antigen-binding region comprising a VH sequence as shown in SEQ ID NO:97 and a VL sequence as shown in SEQ ID NO:98 [B12].

[0211] The present invention provides an antibody that binds 5T4 such that if any one or more of the amino acid residues R73, Y92, and R94 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0212] In particular, if any one or more of the amino acid residues S69, R73, Y92, and R94 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0213] In addition, if any one or more of the amino acid residues R73, T74, Y92, R94, and N95 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0214] Similarly, the antibodies disclosed herein can bind 5T4 such that if any one or more of the amino acid residues L89, F111, L117, F138, L144, D148, N152 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0215] In addition, the antibody can bind 5T4 such that if any one or more of the amino acid residues R73, Y92, R94, F111, F138, L144, and D148 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0216] The antibody can bind 5T4 such that if any one or more of the amino acid residues S69, R73, Y92, R94, F111, F138, and D148 are replaced with alanine, there is a loss of binding or the binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0217] In other embodiments, the antibodies of the invention can bind 5T4 such that if any one or more of the amino acid residues R73, T74, Y92, R94, N95, and F138 are replaced with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO:1.

[0218] The effect of any of the alanine substitutions provided above can be determined by alanine scanning a polypeptide comprising amino acid residues 32 - 355 of SEQ ID NO:1.

[0219] In particular, the effect of the alanine substitution can be determined by the procedure described in Example 16 herein or substantially as described in Example 16 herein.

[0220] Loss of binding can be defined as a Z - score of binding less than 1.5; the Z - score is optionally calculated as described in Example 16 herein or substantially as described in Example 16 herein.

[0221] The effect of the alanine substitution can be determined by a procedure comprising the following steps:

[0222] i) Individually express mutant human 5T4 polypeptide and wild - type 5T4 polypeptide in human embryonic kidney cells, such as HEK 293 cells, to provide a sample comprising 70 - 90,000 cells, such as 80,000 cells, for each mutant or wild - type 5T4, wherein all amino acid residues in the extracellular domain of human 5T4 (corresponding to amino acid residues 32 - 355 of SEQ ID NO:1), except cysteine and alanine, in the mutant human 5T4 polypeptide are individually replaced with alanine.

[0223] ii) Incubate the cells in each sample with 20 μL of the antibody conjugated to an antibody conjugated with fluorescein isothiocyanate (FITC) (3 μg / mL; in FACS buffer) for 40 minutes at room temperature, then wash each sample twice in 150 - 180 μL of FACS buffer (phosphate - buffered saline [PBS; Lonza, catalog number BE17 - 517]+0.1% [w / v] BSA [Roche, catalog number 10735086001]+0.02% [w / v] sodium azide [NaN3; EMELCA Bioscience, catalog number 41920044 - 3]), and resuspend the cells in each sample in 30 μL of FACS buffer.

[0224] iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean fluorescence intensity (gMFI) of the live single-cell population in the sample, and the data for each test antibody was normalized relative to the binding strength of a non-cross-blocking 5T4-specific control antibody using the following equation:

[0225]

[0226] where "aa position" refers to the position mutated to alanine,

[0227] where the Z-score was calculated to represent loss or gain of antibody binding according to the following calculation:

[0228]

[0229] where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all mutants, respectively,

[0230] where if the gMFI of the control antibody for a particular 5T4 mutant is below the mean gMFI 对照抗体 - 2.5 x mean gMFI 对照抗体 SD (from all mutants), the data was excluded from the analysis; and optionally

[0231] where if a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, such as between -1.5 and -1.7 or such as between -1.5 and -1.6), and the residue is predicted to be buried and spatially separated from most residues, the data was excluded from the analysis, where the most residues are predicted to be surface-exposed and loss of binding or reduced binding was determined for the most residues.

[0232] A suitable non-cross-blocking 5T4-specific control antibody in step iii) of the foregoing procedure is a bispecific antibody that comprises

[0233] - an antigen-binding region that comprises a VH sequence shown in SEQ ID NO:83 and a VL sequence shown in SEQ ID NO:84 [A1]; and

[0234] - an antigen-binding region that comprises a VH sequence shown in SEQ ID NO:97 and a VL sequence shown in SEQ ID NO:98 [B12].

[0235] The antibody according to the present invention may be characterized by having a reduced internalization ability, as shown by a reduced cytotoxicity when conjugated to a cytotoxic moiety compared to the same conjugated antibody [H8] containing a heavy chain variable (VH) region comprising the sequence shown in SEQ.ID.No.: 87 and a light chain variable (VL) region comprising the sequence shown in SEQ ID NO: 88. The antibody comprising the VH and VL sequences shown in SEQ ID NO: 87 and 88 respectively may be the murine 5T4 antibody mAb5T4, also known as the H8 antibody (Shaw et al. (2002), Biochem.J. 363:137 - 45, WO98 / 55607). Various chimeric or humanized forms of the antibody H8 are disclosed in WO06 / 031653.

[0236] The cytotoxicity or internalization of a 5T4 antibody that binds 5T4 monovalently can be determined using the procedures described in Example 7 of this application. In particular, cytotoxicity can be determined in an assay comprising the following steps:

[0237] i) Provide a bispecific antibody that binds 5T4 monovalently and is conjugated to a toxin, which comprises a first Fab arm of an antibody as defined in any one of the preceding claims and a second Fab arm capable of binding to the HIV viral protein gp120 (HIV-1 gp120), wherein the HIV-1 gp120-specific Fab arm is conjugated to Duostatin-3,

[0238] ii) Incubate 5T4-positive breast cancer cells MDA-MB-468 (ATCC clone HTB-132) or HCC1954 (ATCC clone CRL-1338) with the bispecific antibody that binds 5T4 monovalently at 37 °C for 5 days; and

[0239] iii) Determine the viability of the cells.

[0240] IgG-b12 is an HIV-1 gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812 - 23). The sequences of the heavy chain (VH) and light chain variable (VL) regions are listed in SEQ ID NO: 97 and 98 respectively.

[0241] In certain embodiments, the antibody of the present invention is an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region selected from the group consisting of:

[0242] a) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 6, 7, and 8

[059] ,

[0243] b) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 13, 14, and 15

[076] ,

[0244] c) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 20, 21, and 22

[085] ,

[0245] d) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 27, 28, and 29

[106] ,

[0246] e) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, 35, and 36

[127] ,

[0247] f) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 41, 42, and 43

[207] ,

[0248] g) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 48, 49, and 50

[226] , and

[0249] h) A heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 sequences which, when compared to the CDR1, CDR2, and CDR3 sequences defined in any one of a) to g), contain in total up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions.

[0250] In other embodiments, the antibody according to the invention is an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region selected from the group consisting of:

[0251] a) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 6, 7, and 8

[059] ,

[0252] b) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 41, 42, and 43

[207] ,

[0253] c) A heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 48, 49, and 50

[226] ; and

[0254] d) A heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 sequences, wherein the CDR1, CDR2, and CDR3 sequences in total contain at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions when compared to the CDR1, CDR2, and CDR3 sequences defined in any one of a) to c).

[0255] In particular, an antibody according to the present invention may be an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 6, 7, and 8

[059] .

[0256] Alternatively, an antibody according to the present invention may be an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region selected from the group consisting of: a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 41, 42, and 43

[207] .

[0257] An antibody according to the present invention may also be an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region selected from the group consisting of: a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 48, 49, and 50

[226] .

[0258] In other embodiments, an antibody according to the present invention is an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of:

[0259] a) A heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.: 6, 7, and 8 respectively and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, AAS, and SEQ ID NO: 11 respectively

[059] ,

[0260] b) A heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 13, 14, and 15 respectively and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17, DAS, and SEQ ID NO: 18 respectively

[076] ,

[0261] c) The heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:20, 21, and 22 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:24, DAS, and SEQ ID NO:25 respectively

[085] ,

[0262] d) The heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.:27, 28, and 29 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:31, DVS, and SEQ ID NO:32 respectively

[106] ,

[0263] e) The heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.:34, 35, and 36 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:38, DAS, and SEQ ID NO:39 respectively

[127] ,

[0264] f) The heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.:41, 42, and 43 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:45, DAS, and SEQ ID NO:46 respectively

[207] ,

[0265] g) The heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO.:48, 49, and 50 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:52, DAS, and SEQ ID NO:53 respectively

[226] ; and

[0266] h) The heavy chain variable region (VH) and the light chain variable region (VL), each region comprising CDR1, CDR2, and CDR3 sequences, which, when compared to the CDR1, CDR2, and CDR3 sequences defined in any one of a) to g), the CDR1, CDR2, and CDR3 sequences in total comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions.

[0267] The antibody according to the invention may be an antibody, wherein when compared to the following, the six complementarity determining regions (CDRs) of the antigen-binding region capable of binding 5T4 in total comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions;

[0268] iv) CDR sequences of SEQ ID NO:6, 7, 8, 10, AAS and SEQ ID NO:11

[059]

[0269] v) CDR sequences of SEQ ID NO.:41, 42, 43, 45, DAS and SEQ ID NO:46

[207] ; or

[0270] vi) CDR sequences of SEQ ID NO.:48, 49, 50, 52, DAS and SEQ ID NO:53

[226] .

[0271] Preferably, 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acid substitutions are conservative amino acid substitutions.

[0272] The antibody may particularly comprise one or two heavy chain variable regions, wherein Complementary Determining Region 3 (CDR3) comprises six consecutive amino acid residues of the sequence shown as SEQ ID NO:102 (YYGMDV) [059, 207, 226]. These six consecutive amino acid residues may be the most C-terminal amino acid residues within CDR3.

[0273] The antibody according to the invention may be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises one or two heavy chain variable regions (VH) and a light chain variable region (VL), the heavy chain variable region comprises the CDR1 sequence of SEQ ID NO:41 (GGSFSGYY), the CDR2 sequence of SEQ ID NO:103 (IDHSX1ST) and the CDR3 sequence of SEQ ID NO:104 (AX2WFGELX3X4YYYGMDV), the light chain variable region comprises the CDR1 sequence of SEQ ID NO:105 (QSVSSX5), the CDR2 sequence DAS and the CDR3 sequence of SEQ ID NO:46 (QQRSNWPLT), wherein X1 is G or E, X2 is A or G, X3 is W or Y, X4 is D or H, and X5 is Y or F [207, 226].

[0274] The antibody according to the invention may be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL)

[059] , the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6, 7 and 8 respectively, and the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:10, AAS and SEQ ID NO:11 respectively.

[0275] Alternatively, an antibody according to the invention can be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region

[207] , the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NOs: 41, 42 and 43 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO: 45, DAS and SEQ ID NO: 46 respectively.

[0276] In addition, an antibody according to the invention can be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL)

[226] , the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NOs: 48, 49 and 50 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO: 52, DAS and SEQ ID NO: 53 respectively.

[0277] In some embodiments, an antibody according to the invention is an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of:

[0278] a) a heavy chain variable region (VH) which comprises the sequence of SEQ ID NO: 5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 5

[059] ,

[0279] b) a heavy chain variable region (VH) which comprises the sequence of SEQ ID NO: 12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 12

[076] ,

[0280] c) a heavy chain variable region (VH) which comprises the sequence of SEQ ID NO: 19 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 19

[085] ,

[0281] d) a heavy chain variable region (VH) which comprises the sequence of SEQ ID NO: 26 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 26

[106] ,

[0282] e) a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:33 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:33

[127] ,

[0283] f) a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:40

[207] ; and

[0284] g) a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:47

[226] .

[0285] An antibody according to the invention can in particular be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:5

[059] .

[0286] Likewise, an antibody according to the invention can be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:40

[207] .

[0287] In addition, an antibody according to the invention can be an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a variable heavy chain region (VH) that comprises the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:47

[226] .

[0288] In other embodiments, an antibody according to the invention is an antibody, wherein the antigen-binding region capable of binding 5T4 comprises a variable heavy chain region (VH) and a variable light chain region (VL) selected from the group consisting of:

[0289] a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 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:5 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:9 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:9,

[0290] b) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:12 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:12 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:16 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:16,

[0291] c) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:19 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:19 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:23 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:23,

[0292] d) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:26 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:26 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:30 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:30,

[0293] e) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:33 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:33 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:37 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:37,

[0294] f) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 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:40 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:44 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:44

[207] .

[0295] g) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 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:47 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:51 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:51

[226] .

[0296] In one embodiment, at least one binding region comprises a heavy chain variable (VH) region and a light chain variable (VL) region that have at most 10 mutations or substitutions, at most 5 mutations or substitutions, such as at most 4 mutations or substitutions, such as at most 3 mutations or substitutions, such as at most 2 mutations or substitutions, such as at most 1 mutation or substitution across the heavy chain variable region (VH) and light chain variable region (VL) selected from the group consisting of:

[0297] a) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or consisting of this sequence and a light chain variable region (VL) comprising the sequence of SEQ ID NO:9 or consisting of this sequence

[059] .

[0298] b) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:12 or consisting of this sequence and a light chain variable region (VL) comprising the sequence of SEQ ID NO:16 or consisting of this sequence

[076] .

[0299] c) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:19 or consisting of this sequence and a light chain variable region (VL) comprising the sequence of SEQ ID NO:23 or consisting of this sequence

[085] .

[0300] d) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:26 or consisting of this sequence and a light chain variable region (VL) comprising the sequence of SEQ ID NO:30 or consisting of this sequence

[106] .

[0301] e) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:33 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:37

[127] ,

[0302] f) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:40 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:44

[207] ; and

[0303] g) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:47 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:51

[226] .

[0304] In some embodiments of the present disclosure, up to 10 mutations or substitutions are allowed across the full length of the variable heavy chain and the entire variable light chain, up to 5 mutations or substitutions, such as up to 4 mutations or substitutions, such as up to 3 mutations or substitutions, such as up to 2 mutations or substitutions, such as 1 mutation or substitution. In other embodiments, up to 10 mutations or substitutions, up to 5 mutations or substitutions, such as up to 4 mutations or substitutions, such as up to 3 mutations or substitutions, such as up to 2 mutations or substitutions, such as up to 1 mutation or substitution may not be within any of the 6 CDR sequences of the variable heavy chain and variable light chain.

[0305] Up to 10 mutations or substitutions can be distributed across the full length of the variable heavy chain and variable light chain of each binding region. Some or all of the mutations or substitutions can be conservative substitutions, where one amino acid residue is replaced by an amino acid residue of the same class as indicated under "Amino Acids" defined above; for example, replacing one acidic amino acid residue with another acidic amino acid residue, and an aromatic residue can be replaced by another aromatic residue. It may be preferred that 35% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 92% or more, 93% or more or 94% or more of the substitutions in the variant are conservative amino acid residue replacements.

[0306] In particular, some or all of the mutations or substitutions can be made with one or more amino acid residues, each of which has the same physical or functional properties as the corresponding amino acid residue it replaces. Amino acid residues sharing physical and functional properties are provided under "Amino Acids" defined above; for example, under the definition of "Amino Acids" above; for example, one hydrophobic amino acid residue can be replaced by another hydrophobic amino acid residue, or one cycloalkenyl-related residue can be replaced by another cycloalkenyl-related residue.

[0307] In particular, an antibody comprising a substituted or mutated antibody as disclosed above can be a functional variant of the VL region, VH region or one or more CDRs as defined by the reference sequence identifier above. The functional variants of VL, VH or CDR used in the context of the antibodies of the present invention still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or higher) of the affinity and / or specificity / selectivity of the parental antibody, and in some cases, such 5T4 antibodies can even be associated with higher affinity, selectivity and / or specificity than the parental antibody.

[0308] In other embodiments of the invention, the antibody is an antibody wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

[0309] a) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:5 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:9

[059] ,

[0310] b) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:12 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:16

[076] ,

[0311] c) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:19 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:23

[085] ,

[0312] d) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:26 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:30

[106] ,

[0313] e) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:33 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:37

[127] ,

[0314] f) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:40 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:44

[207] ; and

[0315] g) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:47 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:51

[226] .

[0316] The antibodies of the present invention can be full-length antibodies, such as full-length IgG1 antibodies.

[0317] In addition, the antibodies of the present invention can be monovalent antibodies. Alternatively, the antibodies according to the present invention can be bivalent antibodies.

[0318] In other embodiments, the antibodies provided according to the present invention are monospecific antibodies.

[0319] Alternatively, the antibodies according to the present invention can be bispecific antibodies.

[0320] Also within the scope of the present disclosure are antibodies as defined above, which comprise an antigen-binding region of an antibody that binds CD3, such as human CD3ε (epsilon), such as human CD3ε (epsilon) as specified in SEQ ID NO:4.

[0321] In particular, the present disclosure provides bispecific antibodies that comprise a first antigen-binding region of an antibody as disclosed above and a second binding region that binds CD3, such as human CD3, as defined above.

[0322] Examples of bispecific antibody molecules useful in the present invention include, but are not limited to, (i) a single antibody having two arms comprising different antigen-binding regions, (ii) a single-chain antibody specific for two different epitopes, such as two scFvs tandemly linked via an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig TM ) in which each light and heavy chain contains two variable domains tandemly linked via a short peptide linker, Wu et al., Generation and Characterization ofa Dual Variable Domain Immunoglobulin (DVD-Ig TM ) Molecule, in: AntibodyEngineering, Springer Berlin Heidelberg (2010); (iv) chemically linked bispecific (Fab’)2 fragments; (v) It is a fusion of two single-chain diabodies, generating a tetravalent bispecific antibody that has two binding sites for each target antigen; (vi) flexibody, which is a combination of scFv and diabody, generating a multivalent molecule; (vii) the so-called "dock-and-lock" molecule based on the "dimerization and docking domain" in protein kinase A When applied to Fab, the dimerization and docking domain can generate a trivalent bispecific binding protein composed of two identical Fab fragments linked to different Fab fragments; (viii) the so-called Scorpion molecule, which contains, for example, two scFvs fused to the two ends of a human Fab arm; and (ix) diabody.

[0323] In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body such as CrossMabs, or a bispecific antibody obtained by controlled Fab arm exchange (e.g., as described in WO 2011 / 131746).

[0324] Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual-targeting molecules, where each side of the molecule contains at least two different antibody Fab fragments or parts of Fab fragments; (iii) IgG fusion molecules, where a full-length IgG antibody is fused to an additional Fab fragment or part of a Fab fragment; (iv) Fc fusion molecules, where a single-chain Fv molecule or a stabilized diabody is fused to a heavy-chain constant domain, Fc region, or part thereof; (v) Fab fusion molecules, where different Fab fragments are fused together and fused to a heavy-chain constant domain, Fc region, or part thereof; (vi) ScFv-based and diabody-based heavy-chain antibodies (e.g., domain antibodies, ), where different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, ) are fused to each other or to another protein or carrier molecule that is fused to a heavy-chain constant domain, Fc region, or part thereof.

[0325] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to (TrionPharma / Fresenius Biotech, WO / 2002 / 020039), Knobs-into-Holes (Genentech, WO9850431;), CrossMAbs (Roche, WO2011117329) and electrostatically paired (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y (Genentech), DIG-body and PIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), Biclonics (Merus), FcΔAdp (Regeneron, WO 2010 / 015792), bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), Azymetric scaffold (Zymeworks / Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), bivalent bispecific antibody (Roche WO 2009 / 080254) and molecules (GenmabA / S, WO 2011 / 131746).

[0326] Examples of recombinant IgG class dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig (GSK / Domantis), tandem diabody (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybodies TM (Zyngenia), the method using a common light chain (Crucell / Merus, US 7,262,028), κλBodies (NovImmune) and CovX-body (CovX / Pfizer).

[0327] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig TM(Abbott, US7,612,181), dual-domain, dual-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecifics (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec, US007951918), scFv fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc, CN 102250246) and TvAb (Roche, WO2012025525, WO2012025530).

[0328] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART TM )(MacroGenics, WO2008157379, WO2010 / 080538) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine–China).

[0329] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), (DNL) (ImmunoMedics), bivalent bispecifics (Biotecnol) and Fab-Fv (UCB-Celltech).

[0330] Examples of scFv-based antibodies, diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engagers (Engager) (Micromet, Tandem Diabody (Tandab TM)(Affimed), Dual Affinity ReTargeting (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODIES (Epigen Biotech), dual-targeting nanobodies (Ablynx), dual-targeting heavy-chain-only domain antibodies.

[0331] The antibodies according to the present disclosure can in particular be antibodies, wherein the antigen-binding region that binds CD3 comprises

[0332] a heavy-chain variable region (VH) that comprises CDR1, CDR2, and CDR3 sequences that are SEQ ID NOs: 54, 55, and 56, respectively; [huCD3-H1L1] (WO2015001085 (Genmab A / S));

[0333] and optionally

[0334] a light-chain variable region (VL) that comprises CDR1, CDR2, and CDR3 sequences that are SEQ ID NO: 58, GTN, and 59, respectively [huCD3-H1L1].

[0335] Also disclosed are antibodies wherein the antigen-binding region that binds CD3 comprises

[0336] a heavy-chain variable region (VH) that comprises the sequence of SEQ ID NO: 57 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 57 [huCD3-H1L1];

[0337] and optionally

[0338] a light-chain variable region (VL) that comprises the sequence of SEQ ID NO: 60 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with the sequence of SEQ ID NO: 60 [huCD3-H1L1].

[0339] The present disclosure further provides antibodies, wherein

[0340] The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO.:6, 7 and 8 respectively, and the light-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:10, AAS and SEQ ID NO:11 respectively [

[059] ].

[0341] and

[0342] The antigen-binding region capable of binding CD3 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively, and the light-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [huCD3-H1L1].

[0343] In addition, the present disclosure provides an antibody, wherein

[0344] The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO.:41, 42 and 43 respectively, and the light-chain variable region (VL) comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO.:45, DAS and 46 respectively

[207] ;

[0345] and

[0346] The antigen-binding region capable of binding CD3 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 56 respectively, and the light-chain variable region (VL) comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [huCD3-H1L1].

[0347] In addition, the disclosure provides an antibody, wherein

[0348] An antigen-binding region capable of binding to 5T4 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences that are SEQ ID NO:48, 49, and 50, respectively. The light-chain variable region (VL) comprises CDR1, CDR2, and CDR3 sequences that are SEQ ID NO:52, DAS, and SEQ ID NO:53, respectively [226-VH+VL CDR1, -2, and -3 sequences];

[0349] and

[0350] An antigen-binding region capable of binding to CD3 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 having sequences shown as SEQ ID NO:54, 55, and 56, respectively. The light-chain variable region (VL) comprises CDR1, CDR2, and CDR3 having the sequences shown as SEQ ID NO:58, CDR GTN, and SEQ ID NO:59, respectively [huCD3-H1L1].

[0351] The antigen-binding region that binds to CD3 can have a dissociation constant Kd in the range of 200–1000 nM, such as in the range of 300–1000 nM, 400–1000 nM, 500–1000 nM, 300–900 nM, 400–900 nM, 400–700 nM, 500–900 nM, 500–800 nM, 500–700 nM, 600–1000 nM, 600–900 nM, 600–800 nM, or such as in the range of 600–700 nM for binding. D binding.

[0352] In a further embodiment, the antibodies disclosed herein have a lower human CD3ε binding affinity than an antibody having an antigen-binding region comprising a VH sequence shown as SEQ ID NO:57 and a VL sequence shown as SEQ ID NO:60 [huCD3-H1L1], preferably wherein the affinity is at least 2-fold lower, such as at least 5-fold lower, such as at least 10-fold lower, for example at least 20-fold lower, at least 30-fold lower, at least 40-fold lower, at least 45-fold lower, at least 50-fold lower, at least 55-fold lower, or such as at least 50-fold lower.

[0353] In particular, the antigen-binding region that binds CD3 can have a dissociation constant K in the range of 1–100 nM, such as in the range of 5–100 nM, in the range of 10–100 nM, in the range of 1–80 nM, in the range of 1–60 nM, in the range of 1–40 nM, in the range of 1–20 nM, in the range of 5–80 nM, in the range of 5–60 nM, in the range of 5–40 nM, in the range of 5–20 nM, in the range of 10–80 nM, in the range of 10–60 nM, in the range of 10–40 nM, or in the range of 10–20 nM. D Binding.

[0354] The affinity of the antibodies according to the invention for binding CD3 can be determined by biolayer interferometry, using a modification of the above procedure or as described in Example 2 herein, in which the antibody is immobilized on a human IgG Fc capture biosensor and the association and dissociation of CD3E27-GSKa (the mature protein of SEQ ID NO: 101) with the immobilized antibody are determined. Additionally, the affinity of the antibodies according to the invention for binding CD3 can be determined by biolayer interferometry as provided in Example 9 herein.

[0355] WO 2017 / 009442 provides antibodies that bind CD3, particularly human CD3, with reduced affinity, and it should be understood that any of these antibodies can serve as a basis for generating the antibodies according to the invention, which have the ability to bind CD3 with reduced affinity in addition to the ability to bind 5T4. Thus, in other embodiments, the antibodies according to the invention are antibodies in which

[0356] the antigen-binding region that binds CD3 comprises a heavy-chain variable (VH) region that comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence,

[0357] when compared to a heavy-chain variable (VH) region comprising the sequence shown in SEQ ID NO: 57, the heavy-chain variable (VH) region has an amino acid substitution in one of the CDR sequences at a position selected from the group consisting of: T31, N57, H101, G105, S110, and Y114, the positions being numbered according to the sequence of SEQ ID NO: 57; and

[0358] the wild-type light-chain variable (VL) region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, GTN, and SEQ ID NO: 59, respectively.

[0359] Preferably, when compared to the sequence shown in SEQ ID NO:57, the CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds CD3 together contain at most 1, 2, 3, 4 or 5 amino acid substitutions.

[0360] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds CD3 may have at least 95% sequence identity with the amino acid sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain variable (VH) region, such as at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity. The sequence identity is calculated based on aligning the amino acid sequence composed of the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds CD3 with the amino acid sequence containing the sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain variable (VH) region.

[0361] In particular, the antigen-binding region that binds CD3 may contain mutations selected from the group consisting of: T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, Y114V, and the positions are numbered according to the reference sequence of SEQ ID NO:57.

[0362] In certain embodiments, the antibody according to the present invention is an antibody, wherein when the antibody is a bispecific antibody lacking Fc-mediated effector function or having reduced Fc-mediated effector function ("inert" antibody) and contains the antigen-binding region of an antibody that binds CD3, then the antibody:

[0363] a) When using purified peripheral blood mononuclear cells (PBMCs) or T cells as effector cells, it is capable of mediating concentration-dependent cytotoxicity of SK-OV-3 cells. For example, when measured as described in Example 14 herein.

[0364] b) When using purified T cells as effector cells, it is capable of mediating concentration-dependent cytotoxicity of MDA-MB-231 cells. For example, when measured as described in Example 13 herein.

[0365] c) It is capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells; for example, when measured as described in Example 13(II) herein.

[0366] d) It is capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells; for example, when measured as described in Example 17 herein.

[0367] e) When using purified T cells as effector cells, it is capable of inducing cytotoxicity in BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, for example when measured as described in Example 17 herein; and / or

[0368] f) It shows anti-tumor activity in a humanized immune hematopoietic stem cell reconstituted mouse xenograft model, such as NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ inoculated with human MDA-MB-231 tumor cells, such as delayed tumor growth; for example when measured as described in Example 15; and

[0369] In addition, the antibody according to the present invention is an antibody that does not bind to leukocyte FcγR when evaluated by flow cytometry or ELISA, and does not induce CD3 antibody-dependent FcγR-mediated CD3 cross-linking by binding C1q in the absence of target (5T4)-specific tumor cells.

[0370] A more detailed disclosure of antibodies with reduced Fc-mediated effector function or without Fc-mediated effector function ("inert" antibodies) can be found hereinafter.

[0371] The ability of the antibody to mediate concentration-dependent cytotoxicity of SK-OV-3 cells is determined in an in vitro cytotoxicity assay, which comprises the following steps:

[0372] i) Isolate PBMC or T cells from the buffy coat of healthy human donors,

[0373] ii) Provide

[0374] A first set of samples, each of which contains PBMC and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of PBMC:SK-OV-3 cells in the sample is 1:2, 1:1, 2:1, 4:1, 8:1 and 12:1; and

[0375] A second set of samples, each of which contains T cells and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of T cells:SK-OV-3 cells in the sample is 1:2, 1:1, 2:1, 4:1 and 8:1

[0376] iii) Add the antibody to each set of samples at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubate the samples at 37 °C for 72 hours; then

[0377] iv) The viability of the SK-OV-3 cells was evaluated using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide).

[0378] The ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells can be determined in an assay comprising the following steps:

[0379] i) T cells were isolated from the buffy coat of healthy human donors,

[0380] ii) A set of samples was provided, each sample containing T cells and human breast cancer MDA-MB-231 cells, and wherein the ratio of T cells:MDA-MB-231 cells in the sample was 8:1,

[0381] iii) The antibody was added to the set of samples at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL, and the samples were incubated at 37 °C for 72 hours,

[0382] iv) The T cells were stained with a fluorescently labeled antibody against a T cell activation marker, such as CD69-APC, CD25-PE-Cy7, and CD279 / PD1-BV604 antibodies, by incubating with the antibody at 4 °C for 30 minutes; and

[0383] v) The samples were analyzed by flow cytometry.

[0384] APC anti-human CD69 (CD69-APC) antibody is available from, for example, BioLegend (catalog numbers 310909 and 310910). CD25 monoclonal antibody PE-Cyanine7 (CD25-PE-Cy7) can also be purchased from, for example, ThermoFisher Scientific (catalog number 25-0259-42) and BD Biosciences (catalog number 557741). Finally, CD279 / PD1-BV604 antibody can be purchased from Genscript (catalog number A01828).

[0385] The activation of T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski, and / or SiHa tumor cells can be determined by a procedure comprising the following steps:

[0386] i) T cells isolated from the buffy coat of healthy human donors were provided,

[0387] ii) A set of samples was provided, each sample containing the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells, and wherein the ratio of T cells:tumor cells in the sample was 4:1,

[0388] iii) Add the antibody to the set of samples at a concentration ranging from 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilution), and incubate the samples at 37 °C for 72 hours.

[0389] iv) Collect 110 μL of the T cell-containing supernatant from each sample, and stain the T cells with fluorescently labeled antibodies against T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and with antibodies against T cell markers, such as 69-APC, CD25-PE-Cy7, and CD279 / PD1-BV604 antibodies, by incubating with the antibodies at 4 °C for 30 minutes; and

[0390] v) Analyze the samples by flow cytometry.

[0391] The ability to induce cytotoxicity in BxPC-3, PANC-1, Ca Ski, and / or SiHa tumor cells can be determined by a procedure comprising the following steps:

[0392] i) Provide T cells isolated from the buffy coat of healthy human donors.

[0393] ii) Provide a set of test samples and control samples, where each sample contains BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells and the T cells that have been allowed to adhere to the bottom of a 96-well tissue culture plate, and where the ratio of T cells:tumor cells in the samples is 4:1.

[0394] iii) Add the antibody to the set of test samples at a concentration ranging from 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilution), while the control samples remain untreated or are incubated with 5 μM staurosporine, and then incubate all samples at 37 °C for 72 hours.

[0395] iv) Incubate the adherent cells at 37 °C for 4 hours in RPMI-1640 medium supplemented with 10% (w / w) iron-supplemented donor bovine serum and penicillin / streptomycin in 10% (w / w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin).

[0396] v) Measure the absorbance of the cells; set the absorbance of the cells incubated with staurosporine to 0% viability, and set the absorbance of the untreated cells to 100% viability, and calculate the percentage of viable cells as follows:

[0397]

[0398] The antibody of the present invention can particularly be an antibody, wherein the antigen-binding region capable of binding to CD3 comprises:

[0399] a) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 61, 55, and 56 respectively [VHCDR1-T31P + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 58, the sequence GTN, and the sequence shown in SEQ ID NO: 59 respectively [wild-type VL CDR1, 2, 3], or

[0400] b) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 63, 55, and 56 respectively [VHCDR1-T31M + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 58, the sequence GTN, and the sequence shown in SEQ ID NO: 59 respectively [wild-type VL CDR1, 2, 3], or

[0401] c) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 54, 65, and 56 respectively [VHCDR-N57E + wild-type VH CDR1, 3] and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 58, the sequence GTN, and the sequence shown in SEQ ID NO: 59 respectively [wild-type VL CDR1, 2, 3], or

[0402] d) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 54, 55, and 67 respectively [wild-type VH CDR1, 2 + VH CDR3-H101G] and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 58, the sequence GTN, and the sequence shown in SEQ ID NO: 59 respectively [wild-type VL CDR1, 2, 3], or

[0403] e) A heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 69 respectively [wild-type VH CDR 1, 2+VH CDR3-H101N] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0404] f) A heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 71 respectively [wild-type VH CDR 1, 2+VH CDR3-G105P] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0405] g) A heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 73 respectively [wild-type VH CDR 1, 2+VH CDR3-S110A] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0406] h) A heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 75 respectively [wild-type VH CDR 1, 2+VH CDR3-S110G] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0407] i) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 77 respectively [wild-type VH CDR 1, 2+VH CDR3-Y114V], and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0408] j) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 79 respectively [wild-type VH CDR 1, 2+VH CDR3-Y114M], and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3], or

[0409] k) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 81 respectively [wild-type VH CDR 1, 2+VH CDR3-Y114R], and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3].

[0410] In certain embodiments, the antigen-binding region capable of binding CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively [wild-type VH CDR 1, 2+VH CDR3-H101G], and the light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1, 2, 3].

[0411] In addition, the present invention provides an antibody as defined above, wherein

[0412] The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO.:6, 7 and 8 respectively. The light-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:10, AAS and SEQ ID NO:11 respectively

[059] .

[0413] and

[0414] The antigen-binding region capable of binding CD3 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively [wild-type VH CDR 1,2+VH CDR3-H101G]. The light-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1,2,3].

[0415] The present invention also provides an antibody as defined above, wherein

[0416] The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO.:41, 42 and 43 respectively. The light-chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:45, DAS and SEQ ID NO:46 respectively

[207] .

[0417] and

[0418] The antigen-binding region capable of binding CD3 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively [wild-type VH CDR 1,2+VH CDR3-H101G]. The light-chain variable region comprises CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1,2,3].

[0419] In addition, the present invention provides an antibody as defined above, wherein

[0420] The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences which are SEQ ID NO.:48, 49, and 50 respectively. The light-chain variable region comprises CDR1, CDR2, and CDR3 sequences which are SEQ ID NO:52, DAS, and SEQ ID NO:53 respectively

[226] .

[0421] and

[0422] The antigen-binding region capable of binding CD3 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 which have the sequences shown in SEQ ID NO:54, 55, and 67 respectively [wild-type VHCDR 1,2+VH CDR3-H101G]. The light-chain variable region comprises CDR1, CDR2, and CDR3 which have the sequences shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59 respectively [wild-type VL CDR 1,2,3].

[0423] In the antibodies of the present invention, the antigen-binding region capable of binding human CD3 may comprise a VH sequence and a VL sequence selected from the group consisting of:

[0424] a) a VH sequence shown in SEQ ID NO:62 [VH T31P] and a VL sequence shown in SEQ ID NO:60,

[0425] b) a VH sequence shown in SEQ ID NO:64 [VH T31M] and a VL sequence shown in SEQ ID NO:60,

[0426] c) a VH sequence shown in SEQ ID NO:66 [VH N57E] and a VL sequence shown in SEQ ID NO:60,

[0427] d) a VH sequence shown in SEQ ID NO:68 [VH H101G] and a VL sequence shown in SEQ ID NO:60,

[0428] e) a VH sequence shown in SEQ ID NO:70 [VH H101N] and a VL sequence shown in SEQ ID NO:60,

[0429] f) a VH sequence shown in SEQ ID NO:72 [VH G105P] and a VL sequence shown in SEQ ID NO:60,

[0430] g) The VH sequence shown as SEQ ID NO:74 [VH S110A] and the VL sequence shown as SEQ ID NO:60,

[0431] h) The VH sequence shown as SEQ ID NO:76 [VH S110G] and the VL sequence shown as SEQ ID NO:60,

[0432] i) The VH sequence shown as SEQ ID NO:78 [VH Y114V] and the VL sequence shown as SEQ ID NO:60,

[0433] j) The VH sequence shown as SEQ ID NO:80 [VH Y114M] and the VL sequence shown as SEQ ID NO:60; and

[0434] k) The VH sequence shown as SEQ ID NO:82 [VH Y114R] and the VL sequence shown as SEQ ID NO:60.

[0435] In particular, the antibody according to the present invention can be an antibody as follows, wherein the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown as SEQ ID NO:68 [VH H101G] and the VL sequence shown as SEQ ID NO:60.

[0436] In some embodiments, the antibody according to the present invention is an antibody as follows, wherein

[0437] the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:5

[059] ;

[0438] and

[0439] the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown as SEQ ID NO:68 [VH H101G] and the VL sequence shown as SEQ ID NO:60.

[0440] In other embodiments, the antibody according to the present invention is an antibody as follows, wherein

[0441] the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:40

[207] ;

[0442] and

[0443] The antigen-binding region capable of binding to human CD3 comprises a VH sequence [VH H101G] shown as SEQ ID NO:68 and a VL sequence shown as SEQ ID NO:60.

[0444] In other embodiments, the antibody according to the invention is an antibody wherein

[0445] the antigen-binding region capable of binding to 5T4 comprises a heavy-chain variable region (VH) which comprises the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:47

[226] ;

[0446] and

[0447] the antigen-binding region capable of binding to human CD3 comprises a VH sequence [VH H101G] shown as SEQ ID NO:68 and a VL sequence shown as SEQ ID NO:60.

[0448] As is known to the person skilled in the art, each antigen-binding region of an antibody typically comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), and wherein each variable region comprises three CDR sequences, namely CDR1, CDR2 and CDR3, and four framework sequences, namely FR1, FR2, FR3 and FR4. This structure can also be found in the antibodies according to the invention. In addition, the antibodies according to the invention can comprise two heavy-chain constant regions (CH) and two light-chain constant regions (CL).

[0449] In certain embodiments, an antibody according to the invention comprises a first and a second heavy chain, such as a first and a second heavy chain, each of which comprises at least a hinge region, CH2 and CH3 regions. Stable heterodimeric antibodies can be obtained in high yield based on two homologous dimeric starting proteins which contain only a few asymmetric mutations in the CH3 region, for example by so-called Fab-arm exchange as provided in WO 2008 / 119353 and WO 2011 / 131746. Thus, in some embodiments of the invention, an antibody, a first heavy chain, wherein at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted, and a second heavy chain, wherein at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted, wherein the substitutions in the first and the second heavy chain are not in the same position, and wherein the amino acid positions are numbered according to EU numbering.

[0450] In certain embodiments, the invention provides an antibody, wherein the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain, and vice versa.

[0451] In some embodiments, an antibody according to the invention further comprises an Fc region composed of the Fc sequences of two heavy chains in addition to the antigen-binding region. The first and second Fc sequences can each have any isotype, including any human isotype, such as IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM or IgA isotype or a mixed isotype. Preferably, the Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or a mixed isotype, such as the human IgG1 isotype.

[0452] Antibodies according to the invention may comprise modifications in the Fc region to render the antibody an inert or non-activated antibody. Thus, in the antibodies disclosed herein, one or both heavy chains may be modified such that the antibody induces Fc-mediated effector functions to a lesser extent relative to an otherwise identical antibody that comprises unmodified first and second heavy chains. Fc-mediated effector functions may be measured by determining Fc-mediated CD69 expression on T cells (i.e., CD69 expression resulting from CD3 antibody-mediated, Fcγ receptor-dependent CD3 crosslinking), by binding to Fcγ receptors, by binding to C1q, or by inducing Fc-mediated FcγR crosslinking. In particular, the heavy chain constant sequence may be modified such 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 a wild-type (unmodified) antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay as described in Example 3 of WO2015001085. Modifications of the heavy and light chain constant sequences may also result in reduced binding of C1q to the antibody. The binding of C1q may be reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to an unmodified antibody, and C1q binding may be determined by ELISA. In addition, the Fc region may be modified such that the antibody mediates at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% reduced Fc-mediated T cell proliferation compared to an unmodified antibody, wherein the T cell proliferation is measured in a PBMC-based functional assay.

[0453] Examples of amino acid positions that may be modified (e.g., in an IgG1 isotype antibody) include positions L234 and L235. Thus, an antibody according to the invention may comprise a first and a second heavy chain, and wherein in both the first and second heavy chains, the amino acid residues at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively.

[0454] In addition, 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). Accordingly, an antibody according to the invention can comprise a first and a second heavy chain, wherein in both the first and the second heavy chain, the amino acid residue at the position corresponding to position D265 in the human IgG1 heavy chain according to EU numbering is A. Further embodiments of the invention provide an antibody, wherein in at least one of, e.g., both, the first and the second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively. In the present application, antibodies having the combination of the three amino acid substitutions L234F, L235E and D265A and the additionally above-disclosed K409R or F405L mutations are designated with the suffix "FEAR" or "FEAL", respectively.

[0455] The amino acid sequence of the wild-type IgG1 heavy chain constant region is herein identified as SEQ ID NO:89. Consistent with the above-disclosed embodiments, an antibody according to the invention can comprise an IgG1 heavy chain constant region carrying an F405L substitution and having the amino acid sequence shown in SEQ ID NO:90 and / or an IgG1 heavy chain constant region carrying a K409R substitution and having the amino acid sequence shown in SEQ ID NO:94.

[0456] The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E and D265A substitutions is herein identified as SEQ ID NO:91. The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E, D265A and F405L substitutions is herein identified as SEQ ID NO:92. The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E, D265A and K409R substitutions is herein identified as SEQ ID NO:93.

[0457] The invention further provides an antibody, wherein

[0458] a) the antigen-binding region capable of binding 5T4 is humanized, and / or

[0459] b) if present, the antigen-binding region capable of binding CD3 is humanized.

[0460] In addition, the invention provides an antibody, wherein

[0461] a) the antigen-binding region capable of binding 5T4 is human, and / or

[0462] b) If present, the antigen-binding region capable of binding CD3 is human.

[0463] In addition, the present invention provides antibodies, wherein

[0464] a) the antigen-binding region capable of binding 5T4 is chimeric, and / or

[0465] b) If present, the antigen-binding region capable of binding CD3 is chimeric.

[0466] In some embodiments of the present invention, the antibody comprises a kappa (κ) light chain. In certain embodiments of the present invention regarding the sequences of bispecific antibodies, the kappa light chain comprises the CDR1, -2, and -3 sequences of the 5T4 antibody light chain as disclosed above.

[0467] In a further embodiment of the present invention, the antibody according to any one of the preceding claims, wherein the antibody comprises a lambda (λ) light chain. In certain embodiments of the present invention regarding bispecific antibodies, the lambda light chain comprises the CDR1, -2, and -3 sequences of the CD3 antibody light chain as disclosed above, particularly the CDR1, -2, and -3 sequences of the CD3 antibody with reduced affinity for CD3 as disclosed above. The amino acid sequence of the kappa light chain constant region is included herein as SEQ ID NO:95, and the lambda light chain constant region is included herein as SEQ ID NO:96.

[0468] In certain embodiments, the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain; for example, an antibody having a heavy chain comprising a binding region capable of binding CD3 and a lambda light chain; and a heavy chain comprising a binding region capable of binding 5T4 and a kappa light chain.

[0469] Nucleic acid construct

[0470] On the other hand, the present invention provides an immunoconjugate or an antibody-drug conjugate (ADC) comprising the antibody defined above, and a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic drug, a cytokine, an immunosuppressant, an antibiotic, or a radioisotope. In general, those skilled in the art will dispose of many cytotoxic agents, chemotherapeutic drugs, cytokines, immunosuppressants, antibiotics, and radioisotopes according to the desired application of the immunoconjugate, the optimal choice of the therapeutic moiety. For certain applications, a preferred cytotoxic agent may be a microtubule-disrupting agent, such as duostatin, for example Duostatin-3.

[0471] Expression vector

[0472] Another aspect of the present invention provides a nucleic acid construct, which comprises

[0473] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above, and / or

[0474] b) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above.

[0475] The nucleic acid construct may further comprise

[0476] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above, and / or

[0477] b) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above.

[0478] Cells and host cells

[0479] Another aspect of the present invention provides an expression vector, which comprises a nucleic acid sequence encoding a heavy chain and / or a light chain sequence of an antibody according to the present invention. In particular, the expression vector may comprise:

[0480] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above, and / or

[0481] b) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above.

[0482] The expression vector may further comprise:

[0483] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above, and / or

[0484] b) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above.

[0485] In another embodiment, the expression vector further comprises a nucleic acid sequence encoding a constant region of a light chain, a heavy chain, or both a light chain and a heavy chain of an antibody such as a human IgG1, κ monoclonal antibody.

[0486] In the context of the present invention, an expression vector can be any suitable vector, including chromosomal, episomal and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-5T4 antibody is contained in a naked DNA or RNA vector, such as a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17 , 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in US 6,077,835 and / or WO00 / 70087), a plasmid vector, such as pBR322, pUC 19 / 18 or pUC 118 / 119, a "midge" minimal-size nucleic acid vector (as described in Schakowski et al., Mol Ther 3 , 793-800 (2001)), or a nucleic acid vector construct as a precipitate, such as a CaP04-precipitated construct (as described in, for example, WO 00 / 46147, Benvenisty and Reshef, PNAS USA 83 , 9551-55 (1986), Wigler et al., Cell 14 , 725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7 , 603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0487] In one embodiment, the vector is suitable for expressing the anti-5T4 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison WI), etc.).

[0488] In addition / or, the expression vector can be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516 - 544 (1987)).

[0489] The nucleic acid construct and / or vector can also contain a nucleic acid sequence encoding a secretion / localization sequence, which can target a polypeptide such as a nascent polypeptide chain to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), etc.

[0490] In the expression vector of the present invention, the nucleic acid encoding the anti - 5T4 antibody can contain any suitable promoter, enhancer, and other expression - promoting elements or be associated therewith. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3 - 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in Escherichia coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., multiple cloning sites). Contrary to a constitutive promoter such as CMV IE, the nucleic acid can also contain an inducible promoter (those skilled in the art will recognize that these terms are actually descriptors of the degree of gene expression under certain conditions).

[0491] In one embodiment, the expression vector encoding the anti - 5T4 antibody can be localized in and / or delivered to a host cell or host animal by a viral vector.

[0492] Composition

[0493] In a further aspect, the present invention provides a cell comprising the nucleic acid construct as defined above or the expression vector as defined above. It should be understood that the cell can be obtained by transfecting a host cell with the nucleic acid construct or the expression vector, such as a recombinant host cell.

[0494] The host cell can be of human origin, such as human embryonic kidney (HEK) cells, like HEK / Expi cells. Alternatively, it can be of rodent origin, such as Chinese hamster ovary cells, like CHO / N50 cells. In addition, the host cell can be of bacterial origin.

[0495] The cell can contain a nucleic acid sequence encoding the antibody of the present invention or a portion thereof that is stably integrated into the cell genome. Alternatively, the cell can contain non-integrated nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, which contain a sequence encoding the anti-5T4 antibody of the present invention or the expression of a portion thereof. In particular, the host cell can contain non-integrated nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, which contain a sequence encoding the anti-5T4 antibody or the expression of a portion thereof.

[0496] Use and therapeutic application

[0497] A further aspect of the present invention provides a composition comprising an antibody; such as a bispecific antibody or an immunoconjugate as defined above. The composition can be a pharmaceutical composition comprising an antibody, bispecific antibody, or immunoconjugate and a pharmaceutically acceptable carrier.

[0498] The pharmaceutical composition can be formulated with carriers, excipients, and / or diluents, and any other components suitable for a pharmaceutical composition, including known adjuvants, according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutically acceptable carrier or diluent and any known adjuvants and excipients should be suitable for the antibody or antibody conjugate of the present invention and the chosen mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on having no significant negative impact on the desired biological properties of the selected compound or pharmaceutical composition of the present invention (e.g., less than a substantial effect on antigen binding [10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0499] The pharmaceutical composition of the present invention can include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0500] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition or its amide employed in the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0501] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents and the like that are physiologically compatible with the compounds of the present invention.

[0502] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethylcellulose colloidal solutions, acacia, and injectable organic esters such as ethyl oleate and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0503] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for active pharmaceutical substances is known in the art. Except insofar as any conventional media or reagent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated.

[0504] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants such as (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid.

[0505] The pharmaceutical compositions of the present invention may also contain in the composition isotonic agents such as sugars, polyols such as mannitol, sorbitol, glycerol or sodium chloride.

[0506] The pharmaceutical composition of the present invention may further comprise one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffering agents, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention can be prepared with carriers that protect the compounds from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate used alone or with waxes, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, or other materials known in the art. Methods for preparing such formulations are generally known to those skilled in the art, see, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0507] In one embodiment, the compounds of the present invention can be formulated to ensure proper distribution in the body. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for active pharmaceutical substances is known in the art. Unless any conventional media or reagent is incompatible with the active compound, it may be considered for use in the pharmaceutical compositions of the present invention. Other active or therapeutic compounds may also be incorporated into the composition.

[0508] Injectable pharmaceutical compositions generally must be sterile and stable under production and storage conditions. The compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium that includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. In many cases, it is preferred to include in the composition isotonic agents such as sugars, polyols such as glycerol, mannitol, sorbitol or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption such as monostearates and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent having, as required, a combination of one or more of the ingredients (e.g., as listed above), followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients (e.g., from those listed above). In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation methods are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient and any other desired ingredients from its previously sterile-filtered solution.

[0509] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent having, as required, a combination of one or more of the ingredients listed above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation methods are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient and any other desired ingredients from its previously sterile-filtered solution.

[0510] The pharmaceutical compositions of the present invention can comprise an antibody, bispecific antibody or antibody-drug conjugate (ADC) of the present invention, a combination of an antibody, bispecific antibody or ADC according to the present invention with another therapeutic compound or a combination of compounds of the present invention.

[0511] The pharmaceutical compositions can be administered by any suitable route and manner. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by a person of ordinary skill in the art.

[0512] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally; that is, by a mode of administration other than enteral and topical administration; typically by injection, including epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion. In particular, the pharmaceutical composition of the present invention can be administered by intravenous or subcutaneous injection or infusion.

[0513] Antibody production

[0514] The present invention further provides an antibody as defined herein for use as a medicament, such as a bispecific antibody, or an immunoconjugate or an antibody-drug conjugate (ADC). The anti-5T4 antibody or immunoconjugate of the present invention can be used to treat or prevent diseases or disorders involving cells expressing 5T4. In particular, the bispecific antibodies according to the present invention; that is, antibodies comprising antigen-binding regions capable of binding 5T4 and CD3 can be useful in a therapeutic setting where specific targeting of cells expressing 5T4 and T cell-mediated killing are required, and they can be more effective than conventional anti-5T4 in certain such indications and settings.

[0515] In one embodiment, the antibodies of the present invention for treating cancer are disclosed herein, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADC). Antibodies, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADC) can be particularly useful for treating cancers, wherein the cancers are characterized by 5T4 expression in at least some tumor cells.

[0516] The cancers can in particular be selected from kidney / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia.

[0517] In addition, the present invention relates to the use of an antibody according to the present invention in the preparation of a medicament, such as a medicament for treating cancer, such as cancers selected from the group consisting of: kidney / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia.

[0518] In another aspect, the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof an antibody, immunoconjugate, composition such as a pharmaceutical composition or an antibody-drug conjugate (ADC) of the present invention.

[0519] In certain embodiments of the present invention, the method is for treating cancer. The method of the present invention particularly comprises the following steps:

[0520] a) selecting a subject having cancer comprising tumor cells expressing 5T4 and / or cancer known to express 5T4; and

[0521] b) administering to the subject an antibody, such as a bispecific antibody of the present invention, a pharmaceutical composition or an antibody-drug conjugate (ADC).

[0522] The cancer may particularly be selected from kidney / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia..

[0523] Adjust the dosage regimen in the above treatment methods and uses to provide an optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be indicated to be proportionally reduced or increased depending on the urgency of the treatment situation. The parenteral composition may be formulated in unit dosage form for ease of administration and uniformity of dosage.

[0524] The effective dose and dosage regimen of the antibody depend on the disease or condition to be treated and can be determined by those skilled in the art. Exemplary non-limiting ranges of the therapeutically effective amount of the compounds of the present invention are about 0.001 - 10 mg / kg, such as about 0.001 - 5 mg / kg, for example about 0.001 - 2 mg / kg, such as about 0.001 - 1 mg / kg, for example about 0.001, about 0.01, about 0.1, about 1 or about 10 mg / kg. Another exemplary non-limiting range of the therapeutically effective amount of the antibody of the present invention is about 0.1 - 100 mg / kg, such as about 0.1 - 50 mg / kg, for example about 0.1 - 20 mg / kg, for example about 0.1 - 10 mg / kg, for example about 0.5, for example about 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0525] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may start with a dose of the antibody used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the appropriate daily dose of the antibody of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Administration may be, for example, parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, it may be by mg / m 2The calculated weekly dose administers the antibody by infusion. Such doses can be, for example, based on the mg / kg doses provided above according to: dose (mg / kg) x 70:1.8. Such administration can be repeated, for example, 1 to 8 times, such as 3 to 5 times. Administration can be carried out by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours. In one embodiment, the antibody can be administered by slow continuous infusion over a long period (e.g., more than 24 hours) to reduce toxic side effects.

[0526] In one embodiment, when administered once a week, the antibody can be administered at a weekly dose calculated as a fixed dose up to 8 times, such as 4 to 6 times. Such a regimen can be repeated one or more times as needed, for example, after 6 months or 12 months. Such a fixed dose can be, for example, based on the mg / kg doses provided above, with a body weight estimated at 70 kg. The dose can be determined or adjusted by, for example, taking a biological sample and measuring the amount of the antibody of the invention in the blood after administration using an anti-idiotypic antibody targeting the 5T4 antigen antigen-binding region of the antibody of the invention.

[0527] In one embodiment, the antibody can be administered as maintenance therapy, such as, for example, once a week for 6 months or longer.

[0528] The antibody can also be administered prophylactically to reduce the risk of developing cancer, delay the occurrence of events in cancer progression, and / or reduce the risk of recurrence upon cancer remission.

[0529] The antibody of the invention can also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition to be treated. Thus, in one embodiment, a medicament containing the antibody is used in combination with one or more other therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents.

[0530] Kit

[0531] Also provided herein are methods for generating antibodies, such as bispecific antibodies of the invention. In particular, methods for generating the antibodies of the invention are provided, including the following steps:

[0532] a) culturing a host cell containing an expression vector as defined herein; and

[0533] b) purifying the antibody from the culture medium.

[0534] In an embodiment of the invention, where the antibody comprises a binding region capable of binding 5T4 and a binding region capable of binding CD3, a method comprising the following steps can be used to generate the antibody:

[0535] a) providing an antibody capable of binding 5T4, by culturing a host cell comprising an expression vector as defined herein under conditions permitting expression of the antibody capable of binding 5T4, and purifying the antibody capable of binding 5T4 from the culture medium;

[0536] b) providing an antibody capable of binding CD3, by culturing a host cell comprising an expression vector under conditions permitting expression of the antibody capable of binding CD3, and purifying the antibody capable of binding CD3 from the culture medium, the expression vector comprising

[0537] I) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding CD3 as defined herein, and

[0538] II) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding CD3 as defined herein;

[0539] c) incubating the antibody capable of binding 5T4 with the antibody capable of binding CD3 under reducing conditions sufficient to permit cysteine in the hinge region to undergo disulfide bond isomerization, and

[0540] d) obtaining the antibody.

[0541] Anti-idiotypic antibody

[0542] The present invention further provides a kit comprising the antibody disclosed above, such as a kit for use as a companion diagnostic / to identify within a patient population those patients having a propensity to respond to treatment with an antibody or an immunoconjugate or an antibody-drug conjugate (ADC) as defined herein, or for predicting the efficacy of the antibody or immunoconjugate or ADC when used to treat a patient, the kit comprising an antibody as defined herein; and instructions for use of the kit.

[0543] Example 1 - Generation of 5T4 antibody and screening materials

[0544] In another aspect, the present invention relates to an anti-idiotype antibody that binds an antibody comprising at least one antigen-binding region capable of binding 5T4, namely the antibody according to the present invention described herein. In a particular embodiment, the anti-idiotype antibody binds an antigen-binding region capable of binding 5T4.

[0545] An anti-idiotype (Id) antibody is an antibody that recognizes unique determinants usually associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genetic type as the source of the anti-5T4 monoclonal antibody with the monoclonal antibody against which the anti-Id is to be prepared. By generating antibodies (anti-Id antibodies) against these idiotype determinants, the immunized animal can generally recognize and respond to the idiotype determinants of the immunizing antibody. Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0546] Anti-Id antibodies can also be used as an "immunogen" to induce an immune response in another animal, thereby generating so-called anti-anti-Id antibodies. The anti-anti-Id antibodies can be identical to the original monoclonal antibody that induced the anti-Id antibody in terms of epitope. Thus, by using antibodies against the idiotype determinants of a monoclonal antibody, other clones expressing antibodies of the same specificity can be identified. Anti-Id antibodies can be varied (thereby generating anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for the 5T4-specific antibodies of the present invention. For example, a monoclonal anti-Id antibody can be conjugated to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will generally contain anti-anti-Id antibodies that have similar (if not identical) binding properties to the original / parental-5T4 antibody.

[0547] Sequence

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558] The present invention is further illustrated by the following examples, which should not be construed as further limitations. Examples

[0559] Example 2 – Determination of binding affinity of 5T4-specific antibody using biolayer interferometry

[0560] Expression constructs of 5T4

[0561] The following codon-optimized constructs were generated for the expression of various full-length 5T4 variants: human (Homo sapiens) 5T4 (Uniprot accession number Q13641), cynomolgus monkey (Macaca fascicularis) 5T4 (Uniprot accession number Q4R8Y9), and chicken (Gallus gallus) 5T4 (Uniprot accession number R4GM46). Additionally, the following codon-optimized constructs were generated for various 5T4 extracellular domain (ECD) variants: the ECD of human 5T4 with a C-terminal His tag (aa 1-355 from Uniprot accession number Q13641) (5T4ECDHis) (SEQ ID NO:99), and the ECD of human 5T4 (aa 1-91) fused to a rabbit Fc domain and a C-terminal His-tag (5T4ECD91-FcRbHis). In SEQ ID NO:99, amino acid residues 1-31 are the signal peptide; thus, the mature 5T4ECDHis protein corresponds to amino acid residues 32-363 of SEQ ID NO:99. Similarly, amino acid residues 1-31 of SEQ ID NO:100 are the signal peptide, and the mature 5T4ECD91-FcRbHis protein corresponds to amino acid residues 32-327 of SEQ ID NO:100.

[0562] The constructs contain restriction sites suitable for cloning and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999; 234(2):187-208). The codon-optimized constructs of full-length human 5T4 and cynomolgus monkey 5T4 were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen). The codon-optimized construct of full-length chicken 5T4 was cloned into pSB (a mammalian expression vector that contains Sleeping Beauty inverted terminal repeats flanking an expression cassette consisting of a CMV promoter and an HSV-TK polyA signal).

[0563] Generation of HEK-293F cell lines transiently expressing full-length human, cynomolgus monkey, or chicken 5T4

[0564] Freestyle was obtained from Invitrogen (catalog number R790-07) TMHEK-293 subclone 293-F (HEK-293F) cells, which are suitable for suspension growth and chemically defined Freestyle medium, were transfected with the above codon-optimized construct using 293fectin (Invitrogen, catalog number 12347-019) according to the manufacturer's instructions.

[0565] Purification of His-tagged 5T4

[0566] As described above, 5T4ECDHis (the mature protein of SEQ ID NO:99) was expressed in HEK-293F cells. The 5T4ECD91-FcRbHis was expressed using the Expi293F expression platform (Thermo Fisher Scientific, Waltham, MA, USA, catalog number A14527) essentially as described by the manufacturer.

[0567] The His-tag enables purification by immobilized metal affinity chromatography. In this process, the chelating agent immobilized on the chromatography resin is filled with Co 2+ cations. The supernatant containing the His-tagged protein was incubated with the resin in a batch manner (i.e., in solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant do not bind or bind weakly compared to the His-tagged protein. After incubation, the beads were recovered from the supernatant and packed into a column. The column was washed to remove weakly bound proteins. Then, the strongly bound His-tagged protein was eluted with a buffer containing imidazole, which competes for the binding of His to Co 2+ . The eluate was desalted by exchanging the buffer on a desalting column.

[0568] Immunization

[0569] To generate the antibodies IgG1-5T4-207 and IgG1-5T4-226, HCo17-BalbC transgenic mice (Bristol-Myers Squibb, New York, NY, USA) were immunized intraperitoneally (IP) and subcutaneously (SC) alternately with 20 μg of 5T4ECDHis protein in Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA, catalog number S6322) at 14-day intervals. A total of 8 immunizations were performed: 4 IP and 4 SC.

[0570] To generate antibodies IgG1-5T4-076 and IgG1-5T4-059, transgenic mice (Bristol-Myers Squibb) of HCo12-BalbC (IgG1-5T4-076) and HCo20-BalbC (IgG1-5T4-059) were immunized alternately by IP and SC with 20 μg of 5T4ECDHis protein in Sigma adjuvant system at 14-day intervals. A total of 8 immunizations were performed: 4 IP and 4 SC.

[0571] To generate antibody IgG1-5T4-085, transgenic mice of HCo17-BalbC were immunized alternately by IP and SC with 20 μg of 5T4ECDHis protein and 20 μg of 5T4ECD91-FcRbHis mature protein in Sigma adjuvant system at 14-day intervals. A total of 8 immunizations were performed: 4 IP and 4 SC.

[0572] To generate antibodies IgG1-5T4-106 and IgG1-5T4-127, transgenic mice of HCo12-BalbC (IgG1-5T4-106) and HCo17-BalbC (IgG1-5T4-127) were immunized alternately by IP and SC with 20 μg of 5T4ECD91-FcRbHis mature protein in Sigma adjuvant system at 14-day intervals. A total of 8 immunizations were performed: 4 IP and 4 SC.

[0573] Mice having at least two consecutive 5T4-specific antibody titers in the antigen-specific screening fluorescence measurement microvolume assay (FMAT) as described below were boosted with 10 μg of 5T4ECDHis or 10 μg of 5T4ECD91-FcRbHis (in PBS by intravenous injection), and the spleen cells and lymph node cells of these mice were fused 3 - 4 days later.

[0574] Homogeneous antigen-specific screening assay

[0575] The presence of 5T4 antibodies in the sera of immunized mice or in the HuMAb (human monoclonal antibody) hybridoma or transfectoma culture supernatants was determined by homogeneous antigen-specific screening assay using FMAT (Applied Biosystems, Foster City, CA, USA). For this purpose, a combination of 4 cell-based assays was used.

[0576] Analysis of human antibodies in serum samples from immunized mice or supernatants of hybridomas or transfected cell lines by binding to HEK-293F cells transiently expressing human 5T4, HEK-293F cells transiently expressing cynomolgus monkey 5T4, streptavidin-coated polystyrene particles (0.5% w / v; 6.7 μm; Spherotech, Lake Forest, IL, USA, catalog number SVP-60-5) coated with 5T4ECD91-FcRBHis, and HEK-293 wild-type cells (negative control).

[0577] Add the sample to the cells to allow binding to 5T4. Subsequently, binding of HuMAb was detected using a fluorescent conjugate (AffiniPure Goat anti-Human IgG Fc 647; Jackson ImmunoResearch, catalog number 109-605-098). IgG1-5T4-H8-F405L was used as a positive control, and ChromPure human IgG whole molecule (Jackson ImmunoResearch, catalog number 009-000-003) was used as a negative control. Samples were scanned using ImageXpress Velos (Molecular Devices, LLC, Sunnyvale, CA, USA), and total fluorescence was used as the readout. Samples were described as positive when the count was higher than 50 and the count x fluorescence was at least three-fold higher than the negative control.

[0578] Generation of HuMAb hybridomas

[0579] HuMAb mice with sufficient antigen - specific titers developed (as described above) were sacrificed, and spleens and lymph nodes flanking the abdominal aorta and vena cava were harvested. Spleen cells and lymph node cells were fused with a murine myeloma cell line (SP2.0 cells) by electrofusion using a CytoPulse CEEF 50 electrofusion system (Cellectis, Paris, France) essentially according to the manufacturer's instructions. Next, antigen - positive primary wells were subcloned using a ClonePix system (Genetix, Hampshire, UK). For this purpose, specific primary well hybridomas were inoculated into a semi - solid medium made of 40% Clone Media (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA). As described above, the subclones were retested for 5T4 binding according to the antigen - specific binding assay and scanned using an IsoCyte system (Molecular Devices). IgG levels were measured using an Octet system (Fortebio, Menlo Park, USA) in order to select the best - producing clone for each primary well for further amplification. The resulting HuMAb hybridomas were further amplified and cultured based on standard protocols (e.g., as described in Coligan J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M. and Strober, W. eds Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).

[0580] Sequence analysis of the variable domains of the 5T4 antibody and cloning into an expression vector

[0581] From 2 to 5x10 6Total RNA was prepared from hybridoma cells 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 cloned directly in-frame into the p33G1f and p33Kappa expression vectors (pcDNA3.3-based vectors with codon-optimized human IgG1m(f) and Kappa constant domains, respectively) by ligation-independent cloning (Aslanidis, C. and P. J. 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 the antigen. The leader sequences were ordered as codon-optimized sequences (GeneArt, Thermo Fisher Scientific) and produced using the Expi293 expression system according to the manufacturer's instructions (Thermo Fisher Scientific). Antibodies in these supernatants were purified and used for functional characterization. The sequences of the resulting lead clones are shown in the table above.

[0582] 5T4 control antibody

[0583] In some embodiments, comparative antibodies against 5T4 (IgG1-5T4-H8, IgG1-5T4-A3 and IgG1-5T4-A1) previously described in WO2007 / 106744 were used. The codon-optimized antibody coding sequences were synthesized and cloned into the pCDNA3.3 expression vector (Thermo Fisher Scientific).

[0584] IgG1-b12 antibody

[0585] In some embodiments, the antibody b12, an HIV-1 gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3): 812-23) is used as a negative control. A codon-optimized antibody-encoding sequence for this control antibody is synthesized and cloned into the pCDNA3.3 expression vector (Thermo Fisher Scientific). The sequences of the heavy chain variable (VH) region and the light chain variable (VL) region are included herein as SEQ ID NO: 97 and 98, respectively.

[0586] Example 3 - Cross-blocking of 5T4 antibody determined by biolayer interferometry

[0587] The affinity of the 5T4 antibody for the recombinant 5T4 protein was determined using label-free biolayer interferometry on an Octet HTX instrument (ForteBio, Portsmouth, UK). The 5T4 antibody (1 μg / mL) was immobilized on an anti-human IgG Fc capture biosensor (ForteBio) for 600 s. After baseline measurement (100 s), a 2-fold dilution series (ranging from 100 nM to 1.56 nM) starting from 3.58 μg / mL (100 nM) human 5T4ECDHis or 3.99 μg / mL (100 nM) cynomolgus monkey 5T4 was used to determine the association (200 s) and dissociation (1000 s) of human 5T4ECDHis (the mature protein of SEQ ID NO: 99) or recombinant cynomolgus monkey 5T4 protein (Cusabio; catalog number CSB-MP024093MOV) in the sample diluent (ForteBio) while shaking at 1000 rpm at 30 °C. The data was analyzed using data analysis software v9.0.0.12 (ForteBio). For each antibody separately, the values of the reference wells containing only the sample diluent during the association and dissociation steps were subtracted from the values of the wells containing the antigen. The Y-axis was aligned with the last 10 s of the baseline, and step-to-step correction alignment with dissociation and Savitzky-Golay filtering were applied. Responses < 0.05 nm were excluded from the analysis. The data was fit using a 1:1 model and a global full fit with an association time of 200 s and a dissociation time of 1000 s or 50 s as the window of interest. A fit with a full dissociation time (1000 s) as the window of interest was used by default. Based on the R 2 value and visual inspection of the fit, a dissociation time of 50 s was used as the window of interest for IgG1-5T4-127-FEAR.

[0588] Table 1 shows the association rate constant k a (1 / Ms), dissociation rate constant kd (1 / s) and equilibrium dissociation constant K D (M). The affinity range of the antibody for human 5T4 was measured, ranging from 1.3x10 -9 –2.7x10 -8 M. The response of IgG1-5T4-085-FEAR was below 0.05 nm, which prevented the correct fitting of the data (for these fits, lower R 2 values). In addition, the response of IgG1-5T4-076-FEAR could not be correctly fitted. These data are shown in italics.

[0589] Table 2 shows the association rate constant k a (1 / Ms), dissociation rate constant k d (1 / s) and equilibrium dissociation constant K D (M) of cynomolgus monkey 5T4 measured by biolayer interferometry. The affinity range of the antibody for cynomolgus monkey 5T4 was measured, ranging from 1.1x10 -9 –4.1x10 -8 M. The responses of IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR were below 0.05 nm, which prevented the correct fitting of the data (for these fits, lower R 2 values). In addition, the response of IgG1-5T4-076-FEAR could not be correctly fitted. These data are shown in italics.

[0590] Table 1: Binding affinities of monospecific bivalent 5T4 antibodies for the extracellular domain of human 5T4, determined by label-free biolayer interferometry.

[0591]

[0592]

[0593] Table 2: Binding affinities of monospecific bivalent 5T4 antibodies for the extracellular domain of cynomolgus monkey 5T4, determined by label-free biolayer interferometry.

[0594]

[0595] Example 4 - Antibody replacement of IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR

[0596] Antibody cross-blocking analysis (epitope binning) was performed using biolayer interferometry on an Octet HTX instrument (ForteBio). According to the manufacturer's instructions, 5T4 antibody (20 μg / mL in 10 mM sodium acetate buffer, pH 6.0, ForteBio) was immobilized on an amine-reactive second-generation (AR2G) biosensor (ForteBio). After baseline measurements (100 s) in sample diluent (ForteBio), the biosensor containing the immobilized antibody was loaded with 100 nM (3.6 μg / mL) of human 5T4ECDHis (mature protein of SEQ ID NO:99) for 500 s. Next, the association response of a second 5T4 antibody (10 μg / mL) was measured for 500 s. The biosensor was regenerated by three 5-s exposures to 10 mM glycine pH 2.5 followed by sample diluent, and measurements were repeated with a new set of the second 5T4 antibody starting from the baseline step. Each biosensor was used up to four times. Measurements were performed at 30 °C using a shaker speed of 1000 rpm. Data were analyzed using data analysis software v9.0.0.12 (ForteBio). The Y-axis was aligned with the association step and Savitzky-Golay filtering was applied. The response of the sample diluent in the association step was subtracted from the association response of the second antibody to correct for dissociation of 5T4ECDHis from the immobilized antibody. The corrected association responses were plotted in matrix form. Typically, responses >0.1 nm were considered non-blocking antibody pairs (white), while responses between -0.1 and 0.1 nm were considered blocking antibody pairs (dark gray). For some antibody pairs, the second antibody showed an initial positive response followed by a decrease in signal. This was considered antibody displacement (light gray), i.e., the second antibody displaced the interaction between the first antibody and the antigen (Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morishige W, et al. (2017) Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another. PLoS ONE 12(1):e0169535. doi:10.1371 / journal.pone.0169535). In some cases, the data curves required visual inspection by an expert to assign blocking, non-blocking, or displacement characteristics to the antibody pairs.

[0597] Cross-blocking experiments were performed on the antibodies IgG1-5T4-059-FEAR, IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-127-FEAR, IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, and the prior art antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L. The results are summarized in Table 3.

[0598] None of the antibodies (except IgG1-5T4-A1-F405L itself) blocked the binding of IgG1-5T4-A1-F405L to 5T4ECDHis. The antibodies IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, IgG1-5T4-127-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR (as well as IgG1-5T4-H8-FEAR itself) blocked the binding of IgG1-5T4-H8-FEAR to 5T4ECDHis. The antibodies IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR and IgG1-5T4-127-FEAR (as well as IgG1-5T4-A3-F405L itself) also blocked the binding of IgG1-5T4-A3-F405L to 5T4ECDHis, while the antibodies IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR did not block the binding of IgG1-5T4-A3-F405L to 5T4ECDHis. The antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR in combination with IgG1-5T4-A3-F405L (which is described in more detail in Example 4) showed antibody displacement.

[0599] Table 3: Antibody cross-blocking as determined by biolayer interferometry.

[0600] The first column shows the immobilized antibody, and the first row shows the antibody in solution. The corrected association responses of the antibodies in solution are shown. Antibody cross-blocking is indicated by dark gray, and displacing antibody combinations are indicated by light gray and asterisks. Non-blocking antibody combinations are unlabeled (transparent background).

[0601]

[0602] and IgG1-5T4-226-FEAR in combination with IgG1-5T4-A3-F405L Figure 1

[0603] Antibody displacement was demonstrated using a biolayer interferometer on an Octet HTX instrument (ForteBio). IgG1-5T4-A3-F405L (20 μg / mL in 10 mM sodium acetate buffer pH 6.0, ForteBio) was immobilized on an amine-reactive second generation (AR2G) biosensor (ForteBio) according to the manufacturer's instructions. After baseline measurements (100 s) in sample diluent (ForteBio), human 5T4ECDHis (mature protein of SEQ ID NO:99) at 100 nM (3.6 μg / mL) was loaded onto the biosensor containing the immobilized IgG1-5T4-A3-F405L antibody for 500 s. Next, the association response of a second 5T4 antibody (IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR; 10 μg / mL) or sample diluent (buffer control) was measured for 500 s. Experiments were performed at 30 °C using an oscillator speed of 1000 rpm. Data were analyzed using data analysis software v9.0.0.12 (ForteBio). The buffer control response was subtracted from the response of the second antibody to correct for dissociation of human 5T4ECDHis from the immobilized IgG1-5T4-A3-F405L, the Y-axis was aligned with the association step, and Savitzky-Golay filtering was applied.

[0604] As Example 5 - Simultaneous binding of 5T4 antibody to membrane-bound 5T4 measured by flow cytometry As shown, IgG1-5T4-A3-F405L did not show binding, indicating cross-blocking with IgG1-5T4-A3-F405L (self-blocking). IgG1-5T4-H8-FEAR showed binding to 5T4ECDHis and thus no cross-blocking with IgG1-5T4-A3-F405L. IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR initially showed a positive response (indicating binding to the IgG1-5T4-A3-F405L-5T4ECDHis complex rather than cross-blocking with IgG1-5T4-A3-F405L), then the response decreased, dropping to the self-blocking response of IgG1-5T4-A3-F405L. This demonstrated a mass loss from the IgG1-5T4-A3-F405L-5T4ECDHis complex, indicating dissociation of 5T4ECDHis from IgG1-5T4-A3-F405L after binding of the complex by IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR. This phenomenon was described as antibody displacement and indicates that the epitopes are closely adjacent or minimally overlapping (Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morishige W, et al. (2017) Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another. PLoS ONE 12(1):e0169535. doi:10.1371 / journal.pone.0169535). This indicates that the antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR bind to different epitopes on 5T4 compared to IgG1-5T4-A3-F405L.

[0605] ([MFI of cells with Ab-FITC and unconjugated antibody – MFI of cells without Ab-FITC or unconjugated antibody] * 100)

[0606] The binding of IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR antibodies to membrane-bound 5T4 in the presence of IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L was evaluated by flow cytometry. According to the manufacturer's instructions, IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR were conjugated to fluorescein isothiocyanate (FITC, Thermo Fisher Scientific). SK-OV-3 cells expressing approximately 20,000 5T4 molecules / cell (50,000 cells per condition) were incubated with a mixture of 10 μg / mL unconjugated 5T4 antibodies (IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12, IgG1-5T4-207-FEAR, or IgG1-5T4-226-FEAR) and 2 μg / mL 5T4 antibodies conjugated with FITC (IgG1-5T4-H8-FEAR-FITC, IgG1-5T4-207-FEAR-FITC, and IgG1-5T4-226-FEAR-FITC). Table 4 shows an overview of the combinations tested. After incubation at 4 °C for 30 minutes, the cells were centrifuged at 1200 RPM for 5 minutes and the supernatant was discarded. The cells were resuspended in 100 μL of FACS buffer supplemented with 1:4000 Topro-3-iodine (Molecular Probes). The mean fluorescence intensity (MFI) of the FITC signal was measured using a flow cytometer (FACS Fortessa, BD Biosciences). The percentage of binding was calculated using the following formula:

[0607] Figure 2 FITC-labeled antibody (2 μg / mL) (MFI of cells with Ab-FITC and isotype control – MFI of cells without Ab-FITC or unconjugated antibody)

[0608] Unconjugated antibody (10 μg / mL) Binding of IgG1-5T4-H8-FEAR-FITC, IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC was blocked in the presence of their unconjugated counterparts. However, binding of IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC to membrane-bound 5T4 was still observed in the presence of unconjugated IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L or IgG1-b12, and was comparable to the binding of IgG1-5T4-H8-FEAR-FITC to membrane-bound 5T4 in the presence of unconjugated IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L or IgG1-b12. Antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind to different epitopes on 5T4 compared to antibodies IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L.

[0609] Table 4: Overview of antibody combinations used in flow cytometry experiments.

[0610] IgG1-5T4-H8-FEAR-FITC IgG1-5T4-H8-FEAR 1 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A3-F405L 2 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-207-FEAR 3 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-226-FEAR 4 ​ ​ 5 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A1-F405L 6 IgG1-5T4-H8-FEAR-FITC IgG1-b12 7 IgG1-5T4-207-FEAR-FITC IgG1-5T4-H8-FEAR 8 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A3-F405L 9 IgG1-5T4-207-FEAR-FITC IgG1-5T4-207-FEAR 10 IgG1-5T4-207-FEAR-FITC IgG1-5T4-226-FEAR 11 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A1-F405L 12 IgG1-5T4-207-FEAR-FITC IgG1-b12 13 IgG1-5T4-226-FEAR-FITC IgG1-5T4-H8-FEAR 14 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A3-F405L 15 IgG1-5T4-226-FEAR-FITC IgG1-5T4-207-FEAR 16 IgG1-5T4-226-FEAR-FITC IgG1-5T4-226-FEAR 17 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A1-F405L 18 IgG1-5T4-226-FEAR-FITC IgG1-b12

[0611] Example 6 - Binding of 5T4 Antibodies to HEK-293 Cells Transfected with Human or Chicken 5T4

[0612] Binding of 5T4 antibodies to HEK-293 cells transiently transfected with full-length human or chicken 5T4 (produced as described in Example 1) was analyzed by flow cytometry. Cells (5x10 4Cells ( cells / well) were incubated for 30 minutes in 50 μL of 5T4 antibody in PBS / 0.1% BSA / 0.02% azide (staining buffer) serially diluted (range 0.01 to 10 μg / mL, in 3-fold dilution steps) in a polystyrene 96-well round bottom plate (Greiner bio-one, catalog number 650180). After washing twice in staining buffer, the cells were incubated for 30 minutes at 4 °C in 50 μL of goat anti-human IgG F(ab')2 conjugated to R-phycoerythrin (PE) (1:500 in staining buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, catalog number 109-116-098). The cells were washed twice in staining buffer, resuspended in 20 μL of staining buffer, and analyzed on an iQue screener (Intellicyt Corporation, USA). Binding curves were analyzed by non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0613] Figure 3 A shows the dose-dependent binding of IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, and IgG1-5T4-A3-F405L to HEK-293 cells transfected with full-length human 5T4. Figure 3 B shows that while dose-dependent binding of IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, and IgG1-5T4-059-FEAR to HEK-293 cells transfected with full-length chicken 5T4 was observed, IgG1-5T4-A3-F405L showed minimal binding to HEK-293 cells transfected with full-length chicken 5T4. The negative control antibody IgG1-b12-K409R did not show binding to HEK-293 cells transfected with full-length human or chicken 5T4 at a concentration of 10 μg / mL.

[0614] Example 7 - Internalization Ability of 5T4 Antibodies in Tumor Cells

[0615] Experiments were conducted to characterize the internalization ability of the monovalent 5T4 antibody. Intracellular payload delivery and the resulting cytotoxicity were used as readouts of 5T4 antibody internalization after target binding. A toxin-conjugated bispecific antibody was generated by controlled Fab-arm exchange of an unconjugated 5T4 antibody with an (HIV-1 gp120-specific) IgG1-b12 antibody that had been conjugated to the microtubule-disrupting agent Duostatin-3. This antibody recognized 5T4 with one Fab arm and an irrelevant antigen (HIV-1 gp120, which is not expressed on tumor cells) with the second Fab arm. The resulting bispecific Duostatin-3-conjugated antibody carried 1 toxin molecule per antibody (drug-antibody ratio 1). Serial dilutions (0.00152 - 10 μg / mL, 3-fold) of the monovalent 5T4-binding Duostatin-3-conjugated bispecific antibody were added to MDA-MB-468 (breast cancer cell line, ATCC, clone HTB-132) or HCC1954 (breast cancer cell line ATCC, clone CRL-2338) cells seeded in flat-bottom 96-well tissue culture plates (5,000 cells / well; Greiner-bio-one, The Netherlands, catalog number 655180). The cells were incubated at 37 °C for 5 days, and then cell viability was assessed using the CellTiter-Glo luminescent cell viability assay (Promega, USA, catalog number G7570) according to the manufacturer's instructions. Cytotoxicity curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) with GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0616] Figure 4Shows the cytotoxic ability of the monovalent binding 5T4 Duostatin-3 conjugated bispecific antibody in MDA-MB-468 (A) or HCC1954 cells (B). BsIgG1-5T4-H8-FEARxb12-vcDuo3 was highly capable of inducing cytotoxicity, indicating the effective internalization ability of the antibody. In contrast, bsIgG1-5T4-076-FEARxb12-vcDuo3, bsIgG1-5T4-085-FEARxb12-vcDuo3, and bsIgG1-5T4-127-FEARxb12-vcDuo3 did not induce any cytotoxicity; the dose-response curves were similar to those of the non-binding IgG1-b12-vcDuo3 control antibody. This indicates poor internalization of those antibodies after binding to membrane-bound 5T4. BsIgG1-5T4-059-FEARxb12-vcDuo3, bsIgG1-5T4-106-FEARxb12-vcDuo3, bsIgG1-5T4-207-FEARxb12-vcDuo3, and bsIgG1-5T4-226-FEARxb12-vcDuo3 induced moderate cytotoxicity in both tested cell lines, indicating that these monovalent 5T4 antibodies induced internalization, but to a lesser extent than bsIgG1-5T4-H8-FEARxb12-vcDuo3.

[0617] Example 8 - Humanized CD3 Antibody for the Generation of CD3x5T4 Bispecific Antibodies

[0618] The generation of the humanized antibody IgG1-huCD3-H1L1 is described in Example 1 of WO2015 / 001085. IgG1-huCD3-H1L1 is referred to herein as "IgG1-huCD3". The antibody IgG1-huCD3-H1L1-FEAL is a variant thereof that has amino acid substitutions in the Fc domain that prevent interaction with IgG Fc receptors (Fc gamma receptors [FcγR]) and complement, in addition to mutations that allow the generation of bispecific antibodies by controlled Fab arm exchange: L234F, L235E, D265A, and F405L, as described above. It has previously been demonstrated that these mutations have no effect on the binding of the antibody into which they are introduced to the target (see, for example, US2015 / 0337049).

[0619] The generation of the humanized antibody IgG1-huCD3-H1L1-H101G is described in Example 2 of WO2017 / 009442. IgG1-huCD3-H1L1-H101G is referred to as "IgG1-huCD3-H101G". As described above, the antibody IgG1-huCD3-H101G-FEAL is a variant having amino acid substitutions L234F, L235E, D265A and F405L.

[0620] Example 9 - Determination of CD3 Binding Affinity Using Biolayer Interferometry

[0621] As described in Example 7 of WO2017 / 009442, the binding affinities of selected CD3 antibodies, including IgG1-huCD3 and IgG1-huCD3-H101G, were determined.

[0622] Briefly, the binding affinity of the selected CD3 antibodies in the form of IgG1-huCD3-FEAL for recombinant soluble CD3ε (CD3E27-GSKa) (mature protein of SEQ ID NO: 101) was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio). An anti-human Fc capture biosensor (ForteBio, catalog number 18-5060) was loaded with hlgG (1 mg / mL) for 600 s. After baseline measurement (200 s), the association (1000 s) and dissociation (2000 s) of CD3E27-GSKa were determined using a CD3E27-GSKa concentration range of 27.11 μg / mL - 0.04 μg / mL (1000 nM - 1.4 nM) with a three-fold dilution step (sample diluent, ForteBio, catalog number 18-5028). For calculations, the theoretical molecular weight of CD3E27-GSKa based on the amino acid sequence, i.e., 27.11 kDa, was used. The experiment was performed with shaking at 1000 rpm at 30 °C. Each antibody was tested in at least two independent experiments. Using ForteBio Data Analysis Software v8.1, the data were analyzed using a 1:1 model and a global complete fit with an association time of 1000 s and a dissociation time of 100 s. The data traces were corrected by subtracting the reference curve (antibody on the biosensor, measured using only the sample diluent), aligning the Y-axis with the last 10 s of the baseline, and applying between-step correction and Savitzky-Golay filtering. Data traces with a response < 0.05 nm were excluded from the analysis.

[0623] Table 5 shows the association rate constant k a (1 / Ms), dissociation rate constant k d (1 / s) and equilibrium dissociation constant KD (M). Compared with IgG1-huCD3-H101G-FEAL (K D : 638 nM), IgG1-huCD3-FEAL shows a relatively high binding affinity for recombinant CD3ε (K D : 15 nM).

[0624] Table 5: Binding affinities of monospecific bivalent CD3 antibodies for recombinant CD3ε, determined by label-free biolayer interferometry

[0625]

[0626] Example 10 - Generation of Bispecific Antibodies by 2-MEA-Induced Fab Arm Exchange

[0627] 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), using a platform technology, namely 2-MEA-induced Fab-arm exchange, bispecific antibodies were generated in vitro. To generate bispecific antibodies by this method, IgG1 molecules carrying a single mutation in the CH3 domain were generated: the F405L mutation (i.e., the CD3 antibody) in one parental IgG1 antibody and the K409R mutation (i.e., 5T4 or control, an HIV-1 gp120-specific antibody) in the other parental IgG1 antibody. In addition to these mutations, the parental IgG1 antibodies contain substitutions in the Fc domain that result in the inability to interact with IgG Fc receptors (Fcgamma receptors) and complement: L234F, L235E, D265A (FEA).

[0628] To generate bispecific antibodies, the two parental antibodies were mixed in equal mass amounts in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4 2- , 1.8 mM H2PO4 - , 163.9 mM Na + , 140.3 mM Cl -, in pH 7.4), and 2-mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM, and the reaction mixture was incubated at 31 °C for 5 hours. According to the manufacturer's protocol, 2-MEA was removed by dialysis into PBS buffer using Slide-A-Lyzer carriages with a 10 kDa molecular weight cut-off (Thermo Fisher Scientific) to allow reoxidation of interchain disulfide bonds and formation of intact bispecific antibodies.

[0629] The following antibodies were used in the examples:

[0630] CD3 antibody

[0631] IgG1-huCD3-FEAL (having VH and VL sequences shown in SEQ ID NO:57 and SEQ ID NO:60).

[0632] IgG1-huCD3-H101G-FEAL (having VH and VL sequences shown in SEQ ID NO:68 and SEQ ID NO:60)

[0633] 5T4 antibody

[0634] IgG1-5T4-207-FEAR (having VH and VL sequences shown in SEQ ID NO:40 and SEQ ID NO:44)

[0635] IgG1-5T4-226-FEAR (having VH and VL sequences shown in SEQ ID NO:47 and SEQ ID NO:51)

[0636] IgG1-5T4-059-FEAR (having VH and VL sequences shown in SEQ ID NO:5 and SEQ ID NO:9)

[0637] IgG1-5T4-076-FEAR (having VH and VL sequences shown in SEQ ID NO:12 and SEQ ID NO:16)

[0638] IgG1-5T4-085-FEAR (having VH and VL sequences shown in SEQ ID NO:19 and SEQ ID NO:23)

[0639] IgG1-5T4-106-FEAR (having VH and VL sequences shown in SEQ ID NO:26 and SEQ ID NO:30)

[0640] IgG1-5T4-127-FEAR (having VH and VL sequences shown in SEQ ID NO:33 and SEQ ID NO:37)

[0641] IgG1-5T4-H8-FEAR (based on 5T4 antibody H8 from Wyeth (WO 2007 / 106744 and US2010 / 0173382); having VH and VL sequences shown in SEQ ID NO:87 and SEQ ID NO:88)

[0642] IgG1-5T4-A1-F405L (based on 5T4 antibody A1 from Wyeth (WO 2007 / 106744 and US8044178); having VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO:84)

[0643] IgG1-5T4-A1-FEAR (based on 5T4 antibody A1 from Wyeth (WO 2007 / 106744 and US8044178); having VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO:84)

[0644] IgG1-5T4-A3-F405L (based on 5T4 antibody A3 from Wyeth (WO 2007 / 106744 and US8759495); having VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO:86)

[0645] IgG1-5T4-A3-FEAR (based on 5T4 antibody A3 from Wyeth (WO 2007 / 106744 and US8759495); having VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO:86)

[0646] Bispecific antibody

[0647] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR

[0648] bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR

[0649] bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR

[0650] bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR

[0651] bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR

[0652] bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR

[0653] bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR

[0654] bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR

[0655] bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR

[0656] bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR

[0657] bsIgG1-huCD3-H101G-FEALxb12-FEAR

[0658] bsIgG1-huCD3-FEALx5T4-207-FEAR

[0659] bsIgG1-huCD3-FEALx5T4-226-FEAR

[0660] bsIgG1-huCD3-FEALx5T4-059-FEAR

[0661] bsIgG1-huCD3-FEALx5T4-106-FEAR

[0662] bsIgG1-huCD3-FEALx5T4-H8-FEAR

[0663] bsIgG1-huCD3-FEALx5T4-A1-FEAR

[0664] bsIgG1-huCD3-FEALx5T4-A3-FEAR

[0665] bsIgG1-b12-FEALx5T4-207-FEAR

[0666] Fluorescein isothiocyanate (FITC)-labeled bispecific antibody

[0667] bsIgG1-b12-FEALx5T4-059-FEAR-FITC

[0668] bsIgG1-b12-FEALx5T4-207-FEAR-FITC

[0669] bsIgG1-b12-FEALx5T4-226-FEAR-FITC

[0670] bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC

[0671] bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC

[0672] Bispecific antibody conjugated with Duostatin-3

[0673] BsIgG1-5T4-H8-FEARxb12-vcDuo3

[0674] bsIgG1-5T4-076-FEARxb12-vcDuo3

[0675] bsIgG1-5T4-085-FEARxb12-vcDuo3

[0676] bsIgG1-5T4-127-FEARxb12-vcDuo3

[0677] BsIgG1-5T4-059-FEARxb12-vcDuo3

[0678] bsIgG1-5T4-106-FEARxb12-vcDuo3

[0679] bsIgG1-5T4-207-FEARxb12-vcDuo3

[0680] bsIgG1-5T4-226-FEARxb12-vcDuo3.

[0681] Unconjugated control antibody

[0682] IgG-b12 is an HIV-1 gp120 specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23), which is used as the negative, non-binding, control second arm of the bispecific antibody in some examples.

[0683] IgG1-b12-F405L is a variant thereof with substitution of F405L.

[0684] IgG1-b12-FEAL is a variant having the following substitutions in addition to the mutations L234F, L235E, D265A, and F405L that allow for the generation of bispecific antibodies by controlled Fab-arm exchange, said substitutions resulting in an Fc domain that is unable to interact with IgG Fc receptors (Fc gamma receptors) and complement.

[0685] IgG1-b12-K409R is a variant having the substitution K409R.

[0686] IgG1-b12-FEAR is a variant having the following substitutions in addition to the mutations L234F, L235E, D265A, and K409R that allow for the generation of bispecific antibodies by controlled Fab-arm exchange, said substitutions resulting in an Fc domain that is unable to interact with IgG Fc receptors (Fc gamma receptors) and complement.

[0687] Example 11 - Binding of CD3x5T4 Bispecific Antibodies to Cynomolgus Monkey and Human 5T4 Expressed in HEK-293 Cells

[0688] The binding of bispecific monovalent CD3x5T4 antibodies and monospecific bivalent 5T4 antibodies to the plasma membrane of HEK-293 cells transiently transfected with human 5T4 or cynomolgus monkey (cynomolgus macaque) 5T4 (produced as described in Example 1) was analyzed by flow cytometry.

[0689] Cells (3x10 4 cells / well) were incubated for 30 minutes at 4 °C in a polystyrene 96-well round bottom plate (Greiner bio-one, catalog number 650180) with serial dilutions of the antibodies (ranging from 0.0137 to 10 μg / mL in 3-fold dilution steps) in 100 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer). The experiment was performed in technical replicates. After washing twice in staining buffer, the cells were incubated for 30 minutes at 4 °C in 50 μL of secondary antibody. As the secondary antibody, goat anti-human IgG F(ab’)2 conjugated with FITC (SouthernBiotech, USA, catalog number 2043-02) diluted 1:200 in staining buffer was used in all experiments. The cells were washed twice in staining buffer, resuspended in 30 μL of staining buffer, and analyzed on an iQue Screener (Intellicyt Corporation, USA). Binding curves were analyzed using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA) using nonlinear regression (sigmoidal dose response with variable slope).

[0690] Figure 5(I) (left panel) shows the bispecific antibody

[0691] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR(A),

[0692] bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR(B),

[0693] bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR(C) and

[0694] bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR(D) (which bind 5T4 monovalently)

[0695] showed dose-dependent binding to HEK-293 cells transfected with human 5T4, comparable to that of the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR and IgG1-5T4-H8-FEAR.

[0696] Figure 5(I) (right panel) shows the bispecific antibodies

[0697] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR(A),

[0698] bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR(B) and

[0699] bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR(C) (which bind 5T4 monovalently)

[0700] showed dose-dependent binding to HEK-293 cells transfected with cynomolgus monkey 5T4, comparable to that of the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR.

[0701] BsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T4-H8-FEAR showed poor binding to cynomolgus monkey 5T4, consistent with the experiments described in Example 2 and WO2007 / 106744. As a negative control, IgG1-b12-K409R (3 μg / mL) was included in these experiments, which did not show binding to HEK-293 cells transfected with human or cynomolgus monkey 5T4.

[0702] In the second experiment, staining was performed with slight adjustments as described above. The cells were incubated with serial dilutions of the antibody in the range of 0.000128 to 10 μg / mL in 5-fold dilution steps. As the secondary antibody, goat anti-human IgG F(ab’)2 conjugated with phycoerythrin (PE) (Jackson Immunoresearch, UK, catalog number 109-116-098) diluted 1:200 in staining buffer was used.

[0703] Figure 5 (II) shows that the antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR (A), bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR (B), bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR (C), bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR (D), bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR and IgG1-5T4-076-FEAR (E), bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR (F), bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR (G), bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR (H), bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR (I) showed dose-dependent binding to HEK-293 cells transfected with human 5T4 (left panel) and HEK-293 cells transfected with cynomolgus monkey 5T4 (right panel). Similarly, the binding curves of the bivalent monospecific and bispecific monovalent antibodies showed similar trends between human and macaque 5T4.

[0704] Example 12 - Binding of CD3x5T4 Bispecific Antibodies to 5T4-Positive Human Tumor Cells

[0705] The binding of the CD3x5T4 bispecific antibody to human tumor cell lines HeLa (cervical adenocarcinoma; ATCC, catalog number CCL-2) and MDA-MB-231 (breast cancer; ATCC, catalog number HTB-26) expressing 5T4 was analyzed by flow cytometry. Neither HeLa nor MDA-MB-231 cells express CD3.

[0706] The cells (3x10 4 cells / well) were incubated at 4 °C for 30 minutes with serial dilutions of the antibody (range 0.000152 to 3 μg / mL, in 3-fold dilution steps) in 100 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer) in a polystyrene 96-well round bottom plate (Greiner bio-one, catalog number 650180). After washing twice in the staining buffer, the cells were incubated at 4 °C for 30 minutes in 50 μL of the secondary antibody. As the secondary antibody, goat anti-human IgG F(ab’)2 conjugated with fluorescein isothiocyanate (FITC) diluted 1:400 in the staining buffer (Southern Biotech, USA, catalog number 2043-02) was used for the first experiment. Next, the cells were washed twice in the staining buffer, resuspended in 120 μL of the staining buffer, and analyzed on a BD LSRFortessa FACS (BD Biosciences, USA). The binding curves were analyzed using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA) using non-linear regression (sigmoidal dose response with variable slope).

[0707] Figure 6(I) (left panel) shows that the CD3x5T4 bispecific antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (A) and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (B) exhibit dose-dependent binding to HeLa cells, with higher maximum binding than the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR and IgG1-5T4-059-FEAR. For bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (C), the maximum binding on HeLa cells is similar to that of the monospecific bivalent 5T4 antibody IgG1-5T4-226-FEAR.

[0708] Figure 6(I) (right panel) shows that the CD3x5T4 bispecific antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (A), bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (B) and bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (C) exhibit dose-dependent binding to MDA-MB-231 cells with higher maximal binding than the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR. The negative control antibody IgG1-b12-K409R (3 μg / mL) included in these experiments did not show binding to HeLa and MDA-MB-231 cells.

[0709] In a second experiment, staining was performed with minor modifications as described above. Cells were incubated with serial dilutions of the antibodies ranging from 0.000128 to 10 μg / mL in 5-fold dilution steps. As the secondary antibody, goat anti-human IgG F(ab’)2 conjugated to phycoerythrin (PE) (Jackson Immunoresearch, UK, catalogue number 109-116-098) diluted 1:200 in staining buffer was used.

[0710] Figure 6 (II) and 6 (III)The antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR showed dose-dependent binding to HeLa and MDA-MB-231 tumor cells. Generally, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-085-FEAR and IgG1-5T4-127-FEAR showed binding at lower antibody concentrations compared to bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR.

[0711] Example 13 - Induction of T Cell Activation, Cytokine Release, and Cytotoxicity In Vitro by CD3x5T4 Bispecific Antibodies Using Purified T Cells as Effector Cells Figure 11

[0712] The CD3x5T4 bispecific antibody was tested in an in vitro cytotoxicity assay using 5T4-positive tumor cell lines as target cells and purified T cells as effector cells. The T cells were derived from buffy coats of healthy human donors (Sanquin, Amsterdam, The Netherlands) and were isolated using the RosetteSep Human T Cell Enrichment Cocktail (Stemcell Technologies, France, catalog number 15061) according to the manufacturer's instructions. To determine the percentage of viable T cells after isolation (total T cells, CD4 + T cells or CD8 + T cells), the isolated T cell samples (2.5x10 5 cells per condition) were stained for 30 minutes at 4°C in a U-bottom 96-well plate (Cellstar, catalog number 650180) with the following antibodies: Pacific Blue CD3 (eBiosciences, clone OKT3) in 100 μL PBS / 0.1% BSA / 0.02% azide (staining buffer), APC-Cy anti-CD4 (eBiosciences, clone OKT4), AF700-anti-CD8 (Biolegend, clone RPA-T8), and the viability marker FVS 510 (BD Biosciences). Next, the cells were washed twice in staining buffer, resuspended in 120 μL staining buffer, and analyzed on a BD LSRFortessa FACS (BD Biosciences, USA). The percentages of CD3 + , CD3 + CD4 + and CD3 + CD8 + T cells for each donor used in the cytotoxicity experiments are described in Table 6.

[0713] Table 6: CD3 + , CD4 + and CD8 + T cell ratios

[0714]

[0715]

[0716] MDA-MB-231 cells (16,000 cells / well) were seeded into flat-bottom 96-well plates (Greiner-bio-one, The Netherlands, catalog number 655180) and allowed to adhere at 37 °C for 4 hours. T cells were added to the tumor cells at an E:T ratio = 8:1. Serial dilutions of bispecific CD3x5T4 antibody or monospecific bivalent 5T4 antibody (final concentration range from 1000 to 0.0128 ng / mL; 5-fold dilutions) were added, and the plates were incubated at 37 °C for 72 hours. Next, 110 μL of the supernatant containing T cells was transferred to a U-shaped 96-well culture plate (CellStar, catalog number 650180). The plates were centrifuged at 4 °C (300 xg) for 3 minutes, then 75 μL of the supernatant was transferred to a new plate for cytokine production measurement, and the T cells were retained to assess T cell activation markers (as described below). Cytokine production induced by 0.2 μg / mL CD3x5T4 bispecific antibody was analyzed by multiplex U-plex assay (MeSo Scale Discovery, USA, catalog number K15049K) according to the manufacturer's instructions.

[0717] 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). Single-stained samples with Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42) were used for compensation adjustment of the flow cytometer. After incubation at 4 °C for 30 minutes, the plates were washed 3 times with PBS / 0.1% BSA / 0.02% azide (staining buffer). The cells were resuspended in 120 μL of staining buffer and analyzed using a FACS Fortessa (BD Biosciences). Data were processed using FlowJo (BD Biosciences).

[0718] In parallel, the viability of tumor cells was evaluated using resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide). The adherent tumor cells were washed twice with PBS and incubated for 4 hours at 37 °C with 10% resazurin (150 μL; Life Technologies, The Netherlands, catalog number DAL1100) in RPMI-1640 (Lonza, Switzerland, catalog number BE12-115F) medium containing 10% iron-supplemented donor bovine serum (Life Technologies, The Netherlands, catalog number 10371-029) and penicillin / streptomycin (Lonza, catalog number DE17-603E). Absorbance was measured using an Envision multimode plate reader (PerkinElmer, US). The absorbance of the staurosporine-treated (Sigma-Aldrich, US, catalog number S6942) tumor cell samples was set as 0% viability, and the absorbance of the untreated tumor cell samples was set as 100% viability. The "percentage of live cells" was calculated as follows:

[0719] % live cells = [sample absorbance - absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells - absorbance of staurosporine-treated target cells] x 100

[0720] The dose-response curves, EC50, and IC50 values were analyzed using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA) with non-linear regression (sigmoidal dose-response with variable slope).

[0721] Figure 7(I) shows that bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR induce dose-dependent cytotoxicity (shown as reduced % live cells) in the 5T4-positive tumor cell line MDA-MB-231. Differences between donors were observed, but T cells from both donors induced maximal killing in the presence of 1 μg / mL of the CD3x5T4 bispecific antibody. Monospecific bivalent antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not induce cytotoxicity. The IC 50 values calculated from the figure are presented in Figure 7(II).

[0722] The IC 50 values of bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR are lower compared to bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively.

[0723] In contrast, the IC 50 value of bsIgG1-huCD3-FEALx5T4-226-FEAR is comparable to bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR.

[0724] Figure 8(I) shows that bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, bsIgG1-huCD3-FEALx5T4-A3-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induce T cell-mediated cytotoxicity (shown as a reduction in tumor cell survival) in the MDA-MB-231 cell line. The bivalent monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR do not induce T cell-mediated cytotoxicity. The IC50 values calculated from the figure are presented in Figure 8(II). The IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-FEALx5T4-106-FEAR are lower than those of bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR.Similarly, the IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, and bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR were lower than those of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.

[0725] T cell activation was determined by staining for activation markers PD1, CD25, and CD69 by flow cytometry (Figure 9(I)). Monospecific bivalent antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, and IgG1-5T4-059-FEAR did not induce upregulation of these T cell activation markers, while bispecific antibodies bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR induced dose-dependent upregulation of PD1, CD25, and CD69. The EC 50 values calculated from the figure are presented in Figure 9(II). Compared with bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively, bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR had lower EC 50 values for upregulating PD1, CD25, and CD69. Compared with bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR had lower EC 50 values for upregulating CD25 and CD69, while the EC 50The values are comparable between bsIgG1-huCD3-FEALx5T4-226-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR.

[0726] Figure 10(I) shows that bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, bsIgG1-huCD3-FEALx5T4-A3-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induce T cell activation (exemplified by the increase in % CD69+ T cells within the CD4 + and CD8 + T cell populations) when incubated with the MDA-MB-231 cell line, while the bivalent monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR do not induce T cell activation. The EC50 values for the three T cell activation markers are shown in Figure 10(II). Generally, the EC50 values for T cell activation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-FEALx5T4-106-FEAR (% CD69 + and CD8 + within the T cell populations), CD25 + and PD1 + + ​The increase in (e.g., CD4 and CD8 T cell populations within the % CD69, CD25, and PD1 T cells) induced by bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR was lower than the EC50 values of bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR. Similarly, the EC50 values of T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, and bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR (as the increase in CD4 + and CD8 + T cell populations within the % CD69 + 、CD25 + and PD1 + T cells) were lower than the EC50 values of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.

[0727] After exposing the co-culture of T cells and MDA-MB-231 cells to 0.2 μg / mL of the CD3x5T4 bispecific antibody, the production of cytokines IL-10, IL-13, and TNF was measured in the culture supernatant by multiplex U-plex assay. Figure 11 The cytokine levels in the supernatant of the T cell-tumor cell co-culture after incubation with the bispecific antibody are shown. Experiments were performed using T cells from two different healthy donors; Figure 11 Panel A shows the results from co-culture with T cells derived from donor A, Figure 11Panel B shows the results from co - cultures with T cells derived from donor B. Although the cytokine levels in the co - cultures of T cell - tumor cell with the CD3x5T4 bispecific antibody containing the IgG1 - huCD3 - H101G - FEAL - derived CD3 - specific Fab - arms were lower than those in the co - cultures with the bispecific antibody containing the IgG1 - huCD3 - FEAL - derived CD3 - specific Fab - arms, the bispecific antibodies bsIgG1 - huCD3 - FEALx5T4 - 207 - FEAR, bsIgG1 - huCD3 - H101G - FEALx5T4 - 207 - FEAR, bsIgG1 - huCD3 - FEALx5T4 - 226 - FEAR, bsIgG1 - huCD3 - H101G - FEALx5T4 - 226 - FEAR, bsIgG1 - huCD3 - FEALx5T4 - 059 - FEAR and bsIgG1 - huCD3 - H101G - FEALx5T4 - 059 - FEAR all induced cytokine release. The monospecific antibodies IgG1 - 5T4 - 207 - FEAR, IgG1 - 5T4 - 226 - FEAR and IgG1 - 5T4 - 059 - FEAR did not induce any cytokine release.

[0728] Example 14 - Induction of Cytotoxicity In Vitro by CD3x5T4 Bispecific Antibodies at Different Effector-to-Target Ratios Using PBMCs or Purified T Cells as Effector Cells Figure 12

[0729] To more precisely determine the efficiency of T - cell - mediated killing by the bispecific antibodies bsIgG1 - huCD3 - H101G - FEALx5T4 - 207 - FEAR and bsIgG1 - huCD3 - H101G - FEALx5T4 - 207 - FEAR, cytotoxicity assays were performed at different effector - to - target (E:T) ratios as described in Example 13. Additionally, peripheral blood mononuclear cells (PBMCs) or isolated T cells were used as effector cells. The ovarian cancer cell line SK - OV - 3 (9,000 cells / well, ATCC, catalog number HTB - 77) was used as the target cell line. PBMCs were isolated from 40 mL of human blood buffy coat (Sanquin) using a Ficoll gradient (Lonza; Lymphocyte Separation Medium, catalog number 17 - 829E) according to the manufacturer's instructions. T cells were isolated as described in Example 13. For PBMCs, the following E:T ratios were used: 1:2, 1:1, 2:1, 4:1, 8:1 and 12:1. For isolated T cells, the following E:T ratios were used: 1:2, 1:1, 2:1, 4:1 and 8:1. In each experiment, effector cells from two separate donors were used. Table 7 provides the CD3 in the PMBC or T - cell isolates from each donor + ,CD3 + CD4+ and CD3 + CD8 + Overview of the percentage of CD3, CD4, and CD8 T cells (determined as described in Example 13).

[0730] Table 7: CD3, CD4, and CD8 T cell ratios for each donor + , CD4 + and CD8 + T cell ratios.

[0731] Donor % CD3 within Live Cell Population <![CDATA[CD3 + % CD4 within the cell + > <![CDATA[CD3 + % CD8 within the cell + > C (PBMC) 75 56.8 28.9 D (PBMCs) 60 63.2 32 E (T Cells) 98.3 59.6 31.6 F (T Cells) 97.2 70 26.4

[0732] As Figure 13 shown, the use of effector cells from two different donors at an E:T ratio of 4:1 to 12:1 in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR resulted in effective PBMC-mediated killing of SK-OV-3 cells. At an E:T ratio of 2:1 and lower, maximum killing of SK-OV-3 cells was not achieved at the highest antibody concentration (1000 ng / mL) used. Similar results were observed when isolated T cells were used as effector cells ( Figure 13 ). Using effector cells from two different donors, at the highest antibody concentration (1000 ng / mL) used, E:T ratios of 4:1 and 8:1 in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR resulted in maximum T cell-mediated killing of SK-OV-3 cells, while lower E:T ratios were not sufficient to induce maximum killing. Thus, the T cell-mediated killing efficacy induced by bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR depends on a sufficiently high E:T ratio.

[0733] Example 15 - Antitumor Activity of CD3x5T4 Bispecific Antibodies in a Humanized Immune System Mouse Xenograft Model Figure 14

[0734] In humanized (CD34+ hematopoietic stem cells [HSC] injected into the tail vein at 3 - 4 weeks of age) NOD.Cg-Prkdc mice subcutaneously inoculated with human MDA-MB-231 tumor cells scid Il2rg tm1WjlThe in vivo antitumor efficacy of the CD3x5T4 bispecific antibodies bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR was evaluated in / SzJ(NSG-HIS) mice (obtained from The Jackson Laboratory). Sixteen weeks after implantation, humanization of the NSG-HIS mouse immune system was confirmed by flow cytometry. Subsequently, according to the HSC donor (#5239 or #2328) and the percentage of human CD3 + in the human CD45 + population and human CD3 + T cells (mean % hCD45 + and % hCD3 + cells, respectively; 42% hCD45 + and 39% hCD3 + for the PBS group, 34% hCD45 + and 25% hCD3 + for the bsIgG1-huCD3-FEALx5T4-207-FEAR group, and 36% hCD45 + and 29% hCD3 6 ), the NSG-HIS mice were randomly divided into three groups (8 mice per group). 5 x 10 2 MDA-MB-231 cells (in 100 μL PBS) were injected subcutaneously (SC) into the flanks of the mice; this was designated as day 0 in the study. On days 14, 18, 21, and 25, the mice were injected intravenously (IV) with 0.5 mg / kg of the antibody or PBS. The treatment groups are shown in Table 8. Tumor growth was evaluated twice a week (starting from day 14) using calipers. Tumor volume (mm 3 ) was calculated from caliper measurements as 0.52 x (length) x (width).

[0735] The results are shown in Figure 14 . Figure 14 Figure A shows that both bsIgG1-huCD3-FEALx5T4-207-FEAR (p < 0.01) and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p < 0.05) effectively inhibited tumor growth compared to the control group on day 43 based on Mann-Whitney statistical analysis. In addition, statistical analysis of the tumor-free survival curves using the Mantel Cox test (Kaplan Meier plot, using less than 500 mm 3The tumors as retention) demonstrated that the differences in tumor-free survival were statistically different, showing increased tumor-free survival in animals treated with bsIgG1-huCD3-FEALx5T4-207-FEAR (p<0.001) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p<0.001) compared to untreated animals. Figure 14 B).

[0736] Table 8 Treatment groups

[0737]

[0738] Example 16. Determination of the contribution of 5T4 amino acid residues to antibody binding using alanine scanning.

[0739] Library design

[0740] A human 5T4 (Uniprot ID Q13641) single-residue alanine library (GeneArt, Thermo Fisher Scientific) was synthesized, in which all amino acid residues in the extracellular domain of human 5T4 were individually mutated to alanine except for positions that already contained alanine or cysteine. To minimize the chance of disrupting the structure of the antigen, cysteines were not mutated. The library was cloned into the pMAC expression vector containing the CMV / TK-polyA expression cassette, the ampicillin resistance gene, and the pBR322 origin of replication.

[0741] Library Generation and Screening

[0742] Wild-type 5T4 and alanine mutants were individually expressed in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Fisher Scientific, catalog number 12347-019). One day after transfection, the cells were harvested. Approximately 80,000 cells were incubated with 20 μL of FITC-conjugated antibody (3 μg / mL; in FACS buffer (PBS [Lonza, catalog number BE17-517] + 0.1% [w / v] BSA [Roche, catalog number 10735086001] + 0.02% [w / v] sodium azide [NaN3; EMELCABioscience, catalog number 41920044-3]; Table 9) for 40 minutes at room temperature. Subsequently, the cells were washed twice by centrifugation with 150 - 180 μL of FACS buffer. The cells were resuspended in 30 μL of FACS buffer and stored at 4°C until analysis by flow cytometry using an iQue screener (Intellicyt Corporation).

[0743] The entire experiment was performed twice, generating duplicate measurements.

[0744] Table 9: Antibodies used to determine the contribution of 5T4 amino acid residues to antibody binding using alanine scanning. Prior to performing the experiment, antibodies that bind monovalently to 5T4 were labeled with FITC (Thermo Fisher Scientific, catalog number 46425). IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L are substitution A1 and A3 antibodies, respectively, which were cloned into a human IgG1 backbone containing the F405L mutation. Thus, the substitution A1 antibody has the same variable region as the A1 antibody disclosed in WO2007106744. Similarly, the A3 substitution antibody has the same variable region as the A3 antibody disclosed in WO2007106744. In both antibodies, the Fc domain carries the F405L substitution.

[0745] Antibody Test or Control Antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC Test antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC Test antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC Test antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC Test antibody bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC Control antibody for normalization

[0746] Data analysis

[0747] For each sample, the average amount of antibody bound per cell was determined as the geometric mean fluorescence intensity (gMFI) of the live single-cell population. The gMFI is affected by the affinity of the antibody for the 5T4 mutant and the level of 5T4 mutant expression per cell. Since specific alanine mutations can affect the surface expression level of the mutant 5T4 and the expression differences of each 5T4 mutant are typically corrected, the data for each test antibody was normalized relative to the binding strength of a non-cross-blocking 5T4-specific control antibody using the following equation:

[0748]

[0749] where "aa position" refers to the position mutated to alanine, and a Z-score was calculated to express the loss or gain of antibody binding according to the following equation:

[0750]

[0751] where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all mutants, respectively,

[0752] where data was excluded from the analysis if the gMFI of the control antibody for a particular 5T4 mutant was below the mean gMFI of the control antibody - 2.5x the SD of the mean gMFI of the control antibody (from all mutants) (assuming that the expression level of those 5T4 mutants was not sufficient to draw conclusions). This was the case for the amino acid W at position 296 (SEQ ID NO:1).

[0753] Results

[0754] Figure 15 Shows the binding results of the tested antibodies to human 5T4 variants with single alanine mutations in the ECD: positions 32 to 355 (according to SEQ ID NO:1). The results indicate that when aa R at position 73, T at position 74, Y at position 92, R at position 94, N at position 95, or F at position 138 of human 5T4 is mutated to alanine, the antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC shows loss of binding. This suggests that the binding of antibody IgG1-5T4-059-04-FEAR depends at least on R73, T74, Y92, R94, N95, F138 of human 5T4 (SEQ ID NO:1). When aa S at position 69, R at position 73, Y at position 92, R at position 94, F at position 111, F at position 138, D at position 148 of human 5T4 is mutated to alanine, the antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC shows loss of binding. This suggests that the binding of antibody IgG1-5T4-207-FEAR depends at least on aa S69, R73, Y92, R94, F111, F138, and D148 of human 5T4 (SEQ ID NO:1). When aa R at position 73, Y at position 92, R at position 94, F at position 111, F at position 138, L at position 144, or D at position 148 of human 5T4 is mutated to alanine, the antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC shows loss of binding. This suggests that the binding of antibody IgG1-5T4-226-FEAR depends at least on aa R73, Y92, R94, F111, F138, L144, and D148 of human 5T4 (SEQ ID NO:1). When aa D at position 60, Q at position 61, D at position 88, L at position 89, Y at position 92, F at position 111, P at position 115, L at position 117, F at position 138, D at position 148, or N at position 152 of human 5T4 is mutated to alanine, the antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC shows loss of binding. This suggests that the binding of antibody IgG1-5T4-A3-FEAR depends at least on aa D60, Q61, D88, L89, Y92, F111, P115, L117, F138, D148, and N152 of human 5T4 (SEQ ID NO:1).

[0755] Some amino acids can participate in binding indirectly. For example, mutating hydrophobic residues to alanine can affect local folding and the positioning of residues that directly interact (Zhao et al., 2014 Structure 22, 612 - 620). Based on structural data (human 5T4 crystal structure 4cnm; RCSB Protein Data Bank), the following residues are buried and thus are expected to indirectly contribute to binding to the following:

[0756] · Antibody bsIgG1 - b12 - FEALx5T4 - 059 - 04 - FEAR - FITC: F138,

[0757] · Antibody bsIgG1 - b12 - FEALx5T4 - 207 - FEAR - FITC: F111, F138, D148,

[0758] · Antibody bsIgG1 - b12 - FEALx5T4 - 226 - FEAR - FITC: F111, F138, L144, D148,

[0759] · Antibody bsIgG1 - 5T4 - A3 - F405Lxb12 - FEAR - FITC: L89, F111, L117, F138, D148, N152.

[0760] Since only surface - exposed residues can directly interact with the antibody, the following residues are expected to directly interact with the following antibodies:

[0761] · Antibody bsIgG1 - b12 - FEALx5T4 - 059 - FEAR - FITC: R73, T74, Y92, R94 and N95,

[0762] · Antibody bsIgG1 - b12 - FEALx5T4 - 207 - FEAR - FITC: S69, R73, Y92 and R94,

[0763] · Antibody bsIgG1 - b12 - FEALx5T4 - 226 - FEAR - FITC: R73, Y92 and R94,

[0764] · Antibody bsIgG1 - 5T4 - A3 - F405Lxb12 - FEAR - FITC: D60, Q61, D88, Y92 and P115.

[0765] These results together suggest that antibodies IgG1-5T4-059, IgG1-5T4-207, and IgG1-5T4-226 all bind by direct interaction with amino acid residues R73, Y92, and R94. The results also indicate that antibodies IgG1-5T4-059, IgG1-5T4-207, and IgG1-5T4-226 each bind an epitope that is different from but partially overlapping with the epitope bound by IgG1-5T4-A3. This is consistent with the substitution behavior described in Examples 3 and 4.

[0766] Example 17: CD3x5T4 Bispecific Antibody Induces T Cell Activation and Cytotoxicity In Vitro in Cell Lines of Different Indications

[0767] The tumor cell lines of pancreatic cancer and cervical cancer were used as target cells, and purified T cells were used as effector cells to test the CD3x5T4 bispecific antibody in an in vitro cytotoxicity assay. For each indication (pancreatic cancer and cervical cancer), two representative cell lines were selected. Table 10 summarizes the tumor cell lines used in the in vitro cytotoxicity assay. T cells were derived from human donor buffy coats (Sanquin, Amsterdam, The Netherlands) and were isolated using the RosetteSep Human T Cell Enrichment Cocktail (Stemcell Technologies, France, catalog number 15061) according to the manufacturer's instructions. For each cell line, at least three different donors were tested in the in vitro cytotoxicity assay and T cell activation assays, as summarized in Table 10.

[0768] Table 10: Tumor Cell Lines Used in the In Vitro Cytotoxicity Assay

[0769]

[0770] Tumor cells (16,000 cells / well) were seeded into flat-bottom 96-well plates (Greiner Bio-One, The Netherlands, catalog number 655180) and allowed to adhere at 37 °C for 4 hours. T cells were added to the tumor cells at an E:T ratio = 4:1. Serial dilutions of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) were added (final concentration range from 5000 to 0.0128 ng / mL; 5-fold dilutions), and the plates were incubated at 37 °C for 72 hours. Next, 110 μL of the T cell-containing supernatant was transferred to round-bottom 96-well culture plates (CellStar, catalog number 650180) and centrifuged at 4 °C (300 xg) for 3 minutes. T cells were stained for T cell markers by incubation with CD3-eFluor450 (1:200; eBioscience, clone OKT3), CD4-APC-eFluor780 (1:50; eBioscience, clone OKT4), CD8-AF700 (1:100; Biolegend, clone RPA-T8), and T cell activation markers CD69-APC (1:50; BD Biosciences, clone AB2439), CD25-PE-Cy7 (1:50; eBioscience, clone BC96), and CD279 / PD1-BV605 (1:50; Biolegend, clone EH12.2H7) diluted in 50 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer). Single-stained samples with Ultracomp beads (5 μL; Invitrogen, catalog number 01-2222-42) were used for compensation adjustment of the flow cytometer. After incubation at 4 °C for 30 minutes, the plates were washed 3 times with the staining buffer. The cells were resuspended in 120 μL of the staining buffer and analyzed using a FACSFortessa (BD Biosciences). Data were processed using FlowJo (version 10, BD Biosciences).

[0771] In parallel, the viability of tumor cells was evaluated using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide). Adherent tumor cells were washed twice with PBS and incubated at 37 °C for 4 h with 10% resazurin (150 μL; Life Technologies, The Netherlands, catalog number DAL1100) in RPMI-1640 medium (Lonza, Switzerland, catalog number BE12-115F) supplemented with 10% iron-supplemented donor bovine serum (Life Technologies, The Netherlands, catalog number 10371-029) and penicillin / streptomycin (Lonza, catalog number DE17-603E). Absorbance was measured using an Envision multimode plate reader (PerkinElmer, US). The absorbance of staurosporine-treated (Sigma-Aldrich, US, catalog number S6942) cells was set as 0% viability, and the absorbance of untreated cells was set as 100% viability. The "percentage of live cells" was calculated as follows:

[0772] % live cells = [absorbance of sample – absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells – absorbance of staurosporine-treated target cells] x 100

[0773] Using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA), dose-cytotoxicity curves, T cell activation curves, IC50 (cytotoxicity) and EC50 (T cell activation) values were analyzed using non-linear regression (sigmoidal dose response with variable slope).

[0774] Figure 16(I) shows that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR induces cytotoxicity in a panel of cell lines with different indications, while control bispecific antibodies targeting only tumor cells or T cells (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) do not show any cytotoxicity. Figure 16(II) shows the mean IC 50 values for each cell line tested with different donors (at least n = 3). Figure 17(I) shows the T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in a panel of cell lines with different indications, as measured by CD4 + and CD8 + CD69 on T cells (CD4 + or CD8+ CD69 within the population + measurement of upregulation of cells (%)). Control bispecific antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) that target only tumor cells or T cells do not induce any T cell activation. Figure 17 (II) shows the average EC of each cell line tested using different donors (at least n = 3). 50 value.

[0775] These data indicate that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR can specifically induce T cell-mediated cytotoxicity and T cell activation in pancreatic cancer and cervical cancer, while the control bispecific antibodies bsIgG1-huCD3-H101G-FEALxb12-FEAR and bsIgG1-b12-FEALx5T4-207-FEAR do not induce T cell activation and T cell-mediated cytotoxicity.

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US 2015 / 0337049 Sequence Listing <110> Genmab Satijn, David Parren, Paul Horbach, Sjeng Verzijl, Dennis Rademakers, Rik van den Brink, Edward Engelberts, Patrick Kemper, Kristel de Goeij, Bart Breij, Esther <120> Antibody <130> P / 0131-WO-PCT <150> EP18161293.8 <151> 2018-03-12 <150> EP 18175347.6 <151> 2018-05-31 <160> 105 <170> PatentIn version 3.5 <210> 1 <211> 420 <212> PRT <213> Homo sapiens <400> 1 Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu 1 5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser 20 25 30 Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu 35 40 45 Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala 50 55 60 Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg 65 70 75 80 Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu 85 90 95 Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala 100 105 110 Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser 115 120 125 Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu 130 135 140 Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe 145 150 155 160 Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val 165 170 175 Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg Gln Asn 180 185 190 Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala 195 200 205 Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220 Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp 225 230 235 240 Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn 245 250 255 Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val 260 265 270 Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg 275 280 285 Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp 290 295 300 Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Asp Arg 305 310 315 320 Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu 325 330 335 Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu 340 345 350 Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala 355 360 365 Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp 370 375 380 Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His 385 390 395 400 Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser 405 410 415 Asn Ser Asp Val 420 <210> 2 <211> 420 <212> PRT <213> Clustered monkeys <400> 2 Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu 1 5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser 20 25 30 Ser Ser Thr Ser Ser Ala Ser Ser Ser Ser Ser Ser Ala Pro Phe Leu 35 40 45 Ala Ser Ala Ala Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala 50 55 60 Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg 65 70 75 80 Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Leu Tyr Val Arg Asn Leu 85 90 95 Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala 100 105 110 Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser 115 120 125 Arg Leu Asp Glu Val Arg Gly Gly Ala Phe Glu His Leu Pro Ser Leu 130 135 140 Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Tyr Leu Ser Pro Phe 145 150 155 160 Ala Phe Ser Gly Ser Asn Ala Ser Ile Ser Ala Pro Ser Pro Leu Val 165 170 175 Glu Leu Ile Leu Asn His Ile Val Pro Pro Asp Asp Lys Arg Gln Asn 180 185 190 Arg Ser Phe Glu Gly Met Val Ala Ala Ala Leu Val Ala Gly Arg Ala 195 200 205 Leu Gln Gly Leu His Leu Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220 Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg Tyr Leu Asp 225 230 235 240 Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn 245 250 255 Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val 260 265 270 Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Val Arg 275 280 285 Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp 290 295 300 Met Val Thr Trp Leu Lys Gln Thr Gly Val Val Gln Gly Lys Asp Arg 305 310 315 320 Leu Thr Cys Ala Phe Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu 325 330 335 Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu 340 345 350 Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala 355 360 365 Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp 370 375 380 Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His 385 390 395 400 Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser 405 410 415 Asn Ser Asp Val 420 <210> 3 <211> 379 <212> PRT <213> Red junglefowl <400> 3 Met Pro Gly Arg Glu Ala Glu Arg Arg Gly Ala Leu Cys Leu Gly Leu 1 5 10 15 Leu Leu His Ala Leu Leu Gly Cys Gly Ser Ala Gln Pro Pro Ala Ala 20 25 30 Cys Pro Ala Pro Cys Glu Cys Ser Glu Ala Ala Lys Thr Val Lys Cys 35 40 45 Val Asn Lys Asn Leu Thr Glu Val Pro Pro Asp Leu Pro Pro Tyr Val 50 55 60 Arg Asn Leu Phe Ile Thr Gly Asn Arg Leu Gly Arg Leu Pro Ala Gly 65 70 75 80 Ala Leu Ser Ala Pro Arg Leu Ala Glu Leu Gly Ser Leu Asn Leu Ser 85 90 95 Gly Asn His Leu Arg Ala Val Glu Ala Gly Ala Leu Ala Ala Leu Pro 100 105 110 Ala Leu Arg Gln Leu Asp Leu Gly Gly Asn Pro Leu Ala Glu Leu Ser 115 120 125 Pro Leu Ala Phe Gly Arg Ala Ser Pro Leu Glu Glu Leu Ala Leu Arg 130 135 140 Gly Ala Leu Arg Glu Gln Gly Ala Leu Leu Gly Leu Ala Asp Leu Leu 145 150 155 160 Gln Ala Gly Ala Leu Arg Asn Leu Ser Arg Leu Glu Leu Ala Asp Asn 165 170 175 Gly Leu Leu Leu Leu Pro Thr Gly Met Leu Gly Ala Leu Pro Ala Leu 180 185 190 Arg His Leu Asp Leu Ser Asn Asn Ser Leu Val Gly Leu Arg Asn Val 195 200 205 Ser Phe Gln Gly Leu Val Arg Leu Gln Ser Leu Asn Leu Ser Asp Asn 210 215 220 Ser Leu Gly Val Leu Arg Asn Gly Thr Leu Ala Gln Trp Arg Gly Leu 225 230 235 240 Pro Ala Leu Arg Arg Ile Ser Leu Ser His Asn Thr Trp Val Cys Asp 245 250 255 Cys Ala Ile Glu Asp Met Val Ala Trp Leu Lys Glu Ser Asp Gln Val 260 265 270 Glu Gly Lys Glu Ala Leu Ser Cys Ala Phe Pro Glu Lys Met Ala Gly 275 280 285 Arg Ala Leu Leu Lys Leu Asn Thr Ser Glu Leu Asn Cys Ser Ala Pro 290 295 300 Val Asp Val Pro Ser Gln Leu Gln Thr Ser Tyr Val Phe Leu Gly Ile 305 310 315 320 Val Leu Ala Leu Ile Gly Ala Ile Phe Leu Leu Val Leu Tyr Leu Asn 325 330 335 Arg Lys Gly Ile Lys Lys Trp Met His Asn Ile Arg Asp Ala Cys Arg 340 345 350 Asp His Met Glu Gly Tyr His Tyr Arg Tyr Glu Ile Asn Ala Asp Pro 355 360 365 Arg Leu Thr Asn Leu Ser Ser Asn Ser Asp Val 370 375 <210> 4 <211> 186 <212> PRT <213> Homo sapiens <400> 4 Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile 100 105 110 Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr 115 120 125 Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly 130 135 140 Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro 145 150 155 160 Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175 Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 180 185 <210> 5 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 6 Gly Phe Thr Phe Ser Ser Tyr Asp 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 7 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 8 <211> 18 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 8 Ala Arg Asp Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met 1 5 10 15 Asp Val <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 10 Gln Gly Ile Ser Ser Trp 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 11 Gln Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 <210> 12 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Gly Tyr Phe Asp Trp Leu Tyr Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 13 Gly Tyr Thr Phe Thr Ser Tyr Gly 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 14 Ile Ser Ala Tyr Asn Gly Asn Thr 1 5 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 15 Ala Arg Asp Pro Gly Tyr Phe Asp Trp Leu Tyr Gly Asp Tyr 1 5 10 <210> 16 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 16 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 17 Gln Gly Ile Ser Ser Ala 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 18 Gln Gln Phe Asn Ser Tyr Pro Arg Thr 1 5 <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Asn Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu His Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Gly Tyr Asn Asn Val Glu Tyr Leu Asp His Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 20 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 21 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 22 Ala Arg Asp Pro Gly Tyr Asn Asn Val Glu Tyr Leu Asp His 1 5 10 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 23 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 24 Gln Gly Ile Ser Ser Ala 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 25 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 26 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 26 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Arg Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ala Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 27 Gly Tyr Arg Phe Thr Ser Tyr Trp 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 28 Ile Tyr Pro Gly Asp Ser Asp Ala 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 29 Ala Arg Ser Val Leu Phe Asp Tyr 1 5 <210> 30 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 30 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Val Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 31 Gln Gly Ile Ser Ser Ala 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 32 Gln Gln Phe Asn Ser Tyr Pro His Thr 1 5 <210> 33 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 33 Glu Val Gln Leu Leu Glu Ser Arg Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 34 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 35 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 36 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 36 Ala Lys Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His 1 5 10 15 <210> 37 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 37 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Leu Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 38 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 38 Gln Ser Val Ser Ser Tyr 1 5 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 39 Gln Gln Arg Ser Asn Trp Leu Met Tyr Thr 1 5 10 <210> 40 <211> 122 <212> PRT <213> Artificial Sequence <400> 40 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Glu Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Gly Trp Phe Gly Glu Leu Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 41 Gly Gly Ser Phe Ser Gly Tyr Tyr 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 42 Ile Asp His Ser Glu Ser Thr 1 5 <210> 43 <211> 16 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 43 Ala Gly Trp Phe Gly Glu Leu Tyr His Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 44 <211> 107 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 44 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 45 Gln Ser Val Ser Ser Tyr 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR Sequence <400> 46 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 47 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 47 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Trp Phe Gly Glu Leu Trp Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 48 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 48 Gly Gly Ser Phe Ser Gly Tyr Tyr 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 49 Ile Asp His Ser Gly Ser Thr 1 5 <210> 50 <211> 16 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 50 Ala Ala Trp Phe Gly Glu Leu Trp Asp Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 51 <211> 107 <212> PRT <213> Synthetic sequence <220> <223> Antibody variable region <400> 51 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 52 <211> 6 <212> PRT <213> Synthetic sequence <220> <223> N / A <400> 52 Gln Ser Val Ser Ser Phe 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 53 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 54 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDr sequence <400> 55 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 1 5 10 <210> 56 <211> 16 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 56 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 57 <211> 125 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 57 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 58 Thr Gly Ala Val Thr Thr Ser Asn Tyr 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR sequence <400> 59 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 60 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Antibody variable region <400> 60 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 61 Gly Phe Thr Phe Asn Pro Tyr Ala 1 5 <210> 62 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 62 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Pro Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 63 Gly Phe Thr Phe Asn Met Tyr Ala 1 5 <210> 64 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 64 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 65 Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr 1 5 10 <210> 66 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Antibody Variable Region <400> 66 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 67 Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 68 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 68 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 69 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 69 Val Arg Asn Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 70 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 70 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Asn Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 71 Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 72 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 72 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly...

Claims

1. An antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of: a) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 6, 7 and 8 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 10, AAS and SEQ ID NO: 11 respectively, b) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 13, 14 and 15 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 17, DAS and SEQ ID NO: 18 respectively, c) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 20, 21 and 22 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 24, DAS and SEQ ID NO: 25 respectively, d) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 27, 28 and 29 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 31, DVS and SEQ ID NO: 32 respectively, e) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 34, 35 and 36 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 38, DAS and SEQ ID NO: 39 respectively, f) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO.: 41, 42 and 43 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 45, DAS and SEQ ID NO: 46 respectively, and g) A heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO.: 48, 49 and 50 respectively, and a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 52, DAS and SEQ ID NO: 53 respectively.

2. The antibody according to claim 1, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:6, 7 and 8 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:10, AAS and SEQ ID NO:11 respectively.

3. The antibody according to claim 1, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:41, 42 and 43 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:45, DAS and SEQ ID NO:46 respectively.

4. The antibody according to claim 1, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:48, 49 and 50 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:52, DAS and SEQ ID NO:53 respectively.

5. The antibody according to claim 1, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of: a) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:5 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:5 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:9 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:9; b) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:12 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:16 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:

16. c) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:19 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:19, and a light chain variable (VL) region comprising the sequence of SEQ ID NO:23 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:23, d) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:26 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:26, and a light chain variable (VL) region comprising the sequence of SEQ ID NO:30 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:30, e) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:33 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:33, and a light chain variable (VL) region comprising the sequence of SEQ ID NO:37 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:37, f) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:40 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:40, and a light chain variable (VL) region comprising the sequence of SEQ ID NO:44 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:44, and g) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:47 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:47, and a light chain variable (VL) region comprising the sequence of SEQ ID NO:51 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:

51.

6. The antibody according to claim 1, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of: a) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:5 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:9, b) A heavy chain variable (VH) region comprising the sequence of SEQ ID NO:12 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:16, c) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:19 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:23, d) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:26 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:30, e) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:33 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:37, f) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:40 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:44; and g) a heavy chain variable (VH) region comprising the sequence of SEQ ID NO:47 and a light chain variable (VL) region comprising the sequence of SEQ ID NO:

51.

7. The antibody according to claim 1, wherein the antibody is a full-length antibody.

8. The antibody according to claim 1, wherein the antibody is a full-length IgG1 antibody.

9. The antibody according to claim 1, which is a monovalent antibody.

10. The antibody according to claim 1, which is a bivalent antibody.

11. The antibody according to claim 1, which is a monospecific antibody.

12. The antibody according to claim 1, which is a bispecific antibody.

13. The antibody according to claim 1, which is a bispecific antibody comprising: an antigen-binding region capable of binding CD3.

14. The antibody according to claim 1, which is a bispecific antibody comprising: an antigen-binding region capable of binding CD3, wherein the CD3 is human CD3ε (epsilon).

15. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 sequences that are SEQ ID NO:54, 55, and 56, respectively; and, a light chain variable (VL) region comprising CDR1, CDR2, and CDR3 sequences that are SEQ ID NO:58, GTN, and 59, respectively.

16. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises A heavy chain variable (VH) region that comprises the sequence of SEQ ID NO:57 or a sequence having at least 90% amino acid sequence identity to the sequence of SEQ ID NO:57; and, A light chain variable (VL) region that comprises the sequence of SEQ ID NO:60 or a sequence having at least 90% amino acid sequence identity to the sequence of SEQ ID NO:

60.

17. The antibody according to claim 13 or 14, wherein the antibody has a lower human CD3ε binding affinity than an antibody having an antigen-binding region that comprises a VH sequence shown in SEQ ID NO:57 and a VL sequence shown in SEQ ID NO:

60.

18. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 has an equilibrium dissociation constant K in the range of 200 - 1000 nM D .

19. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 has an equilibrium dissociation constant K in the range of 1 - 100 nM D .

20. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises: a) A heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:61, 55, and 56 respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59 respectively, or b) A heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:63, 55, and 56 respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59 respectively, or c) A heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 65, and 56 respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59 respectively, or d) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or e) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 69, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or f) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 71, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or g) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 73, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or h) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 75, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or i) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 77, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or j) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:54, 55, and 79, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 having the sequence shown in SEQ ID NO:58, the sequence GTN, and the sequence shown in SEQ ID NO:59, respectively, or k) A heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 81 respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively.

21. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively.

22. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:6, 7 and 8 respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:10, AAS and SEQ ID NO:11 respectively, and the antigen-binding region capable of binding CD3 comprises a heavy chain variable (VH) region and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:54, 55 and 67 respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, the sequence GTN and the sequence shown in SEQ ID NO:59 respectively.

23. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:41, 42 and 43 respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3 sequences which are SEQ ID NO:45, DAS and SEQ ID NO:46 respectively, and The antigen-binding region capable of binding CD3 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 having the sequences shown as SEQ ID NO:54, 55, and 67, respectively. The light-chain variable region comprises CDR1, CDR2, and CDR3 having the sequence shown as SEQ ID NO:58, the sequence GTN, and the sequence shown as SEQ ID NO:59, respectively.

24. The antibody according to claim 13 or 14, wherein The antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences which are SEQ ID NO:48, 49, and 50, respectively. The light-chain variable region comprises CDR1, CDR2, and CDR3 sequences which are SEQ ID NO:52, DAS, and SEQ ID NO:53, respectively. and The antigen-binding region capable of binding CD3 comprises a heavy-chain variable (VH) region and a light-chain variable region (VL). The heavy-chain variable region comprises CDR1, CDR2, and CDR3 having the sequences shown as SEQ ID NO:54, 55, and 67, respectively. The light-chain variable region comprises CDR1, CDR2, and CDR3 having the sequence shown as SEQ ID NO:58, the sequence GTN, and the sequence shown as SEQ ID NO:59, respectively.

25. The antibody of claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises a VH sequence and a VL sequence selected from the group consisting of: a) a VH sequence shown as SEQ ID NO:62 and a VL sequence shown as SEQ ID NO:60, b) a VH sequence shown as SEQ ID NO:64 and a VL sequence shown as SEQ ID NO:60, c) a VH sequence shown as SEQ ID NO:66 and a VL sequence shown as SEQ ID NO:60, d) a VH sequence shown as SEQ ID NO:68 and a VL sequence shown as SEQ ID NO:60, e) a VH sequence shown as SEQ ID NO:70 and a VL sequence shown as SEQ ID NO:60, f) a VH sequence shown as SEQ ID NO:72 and a VL sequence shown as SEQ ID NO:60, g) a VH sequence shown as SEQ ID NO:74 and a VL sequence shown as SEQ ID NO:60, h) the VH sequence shown in SEQ ID NO:76 and the VL sequence shown in SEQ ID NO:60, i) the VH sequence shown in SEQ ID NO:78 and the VL sequence shown in SEQ ID NO:60, j) the VH sequence shown in SEQ ID NO:80 and the VL sequence shown in SEQ ID NO:60; and k) the VH sequence shown in SEQ ID NO:82 and the VL sequence shown in SEQ ID NO:

60.

26. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

27. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:5 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:5; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

28. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:40 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:40 over its entire length; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

29. The antibody according to claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:47 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:47 over its entire length; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

30. The antibody of claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprises the sequence of SEQ ID NO:5 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:5, and the light-chain variable region comprises the sequence of SEQ ID NO:9 or a sequence having at least 90% amino acid sequence identity with the sequence of SEQ ID NO:9; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

31. The antibody of claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprises the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:40, and the light-chain variable region comprises the sequence of SEQ ID NO:44 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:44; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

32. The antibody of claim 13 or 14, wherein the antigen-binding region capable of binding 5T4 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprises the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:47, and the light-chain variable region comprises the sequence of SEQ ID NO:51 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the sequence of SEQ ID NO:51; and the antigen-binding region capable of binding CD3 comprises the VH sequence shown in SEQ ID NO:68 and the VL sequence shown in SEQ ID NO:

60.

33. The antibody according to claim 1 or 13, wherein each antigen-binding region comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), and wherein each of said variable regions comprises three CDR sequences, namely CDR1, CDR2 and CDR3, and four framework sequences, namely FR1, FR2, FR3 and FR4.

34. The antibody according to claim 1 or 12, wherein the antibody comprises two heavy-chain constant regions (CH) and two light-chain constant regions (CL).

35. The antibody according to claim 1 or 12, wherein the antibody comprises a first and a second heavy chain, each of the first and second heavy chains comprising at least a hinge region, CH2 and CH3 regions, wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted, wherein the substitutions in the first and the second heavy chains are not at the same position, and wherein the amino acid positions are numbered according to EU numbering.

36. The antibody according to claim 1 or 12, wherein the antibody comprises a first heavy chain and a second heavy chain, wherein the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain, or wherein the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain.

37. The antibody according to claim 1 or 12, wherein the antibody comprises a first and a second heavy chain, and wherein the amino acid residues at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in both the first and the second heavy chains.

38. The antibody according to claim 1 or 12, wherein the antibody comprises a first and a second heavy chain, and wherein the amino acid residue at the position corresponding to position D265 in the human IgG1 heavy chain according to EU numbering is A in both the first and the second heavy chains.

39. The antibody according to claim 1, 13 or 14, wherein a) the antigen-binding region capable of binding 5T4 is humanized, and / or b) if present, the antigen-binding region capable of binding CD3 is humanized.

40. The antibody according to claim 1, 13 or 14, wherein a) the antigen-binding region capable of binding 5T4 is human, and / or b) if present, the antigen-binding region capable of binding CD3 is human.

41. The antibody according to claim 1, 13 or 14, wherein a) the antigen-binding region capable of binding 5T4 is chimeric, and / or b) if present, the antigen-binding region capable of binding CD3 is chimeric.

42. The antibody according to claim 1 or 12, wherein the antibody comprises a heavy chain, and wherein the heavy chain is modified such that the antibody induces Fc-mediated effector functions to a lesser extent than the same unmodified antibody.

43. The antibody according to claim 1 or 12, wherein the antibody comprises a kappa (κ) light chain.

44. The antibody according to claim 1 or 12, wherein the antibody comprises a lambda (λ) light chain.

45. The antibody according to claim 1 or 12, wherein the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain.

46. The antibody according to claim 13 or 14, wherein the antibody comprises a heavy chain and a lambda light chain comprising a binding region capable of binding CD3; and a heavy chain and a kappa light chain comprising a binding region capable of binding 5T4.

47. The bispecific antibody according to claim 13 or 14, which comprises an antigen-binding region capable of binding 5T4, which comprises a heavy chain variable region (VH) sequence shown in SEQ ID NO: 40 and a light chain variable region (VL) sequence shown in SEQ ID NO: 44, an antigen-binding region capable of binding CD3, which comprises a VH sequence shown in SEQ ID NO: 68 and a VL sequence shown in SEQ ID NO: 60, a first heavy chain and a second heavy chain, wherein in the first heavy chain, the amino acids at the positions corresponding to positions L234, L235, D265 and K409 of the human IgG1 heavy chain are F, E, A and R respectively, and the amino acid positions are numbered according to EU numbering, in the second heavy chain, the amino acids at the positions corresponding to positions L234, L235, D265 and F405 of the human IgG1 heavy chain are F, E, A and L respectively, and the amino acid positions are numbered according to EU numbering, an Fc region, which is of human IgG1 isotype; and A kappa light chain constant region consisting of the sequence shown in SEQ ID NO: 95, and a lambda light chain constant region consisting of the sequence shown in SEQ ID NO:

96.

48. An immunoconjugate or antibody-drug conjugate (ADC) comprising the antibody according to claim 1 or 12.

49. An immunoconjugate or antibody-drug conjugate (ADC) comprising the antibody according to claim 1 or 12, and a therapeutic moiety, wherein the therapeutic moiety is a cytotoxic agent, a cytokine, an immunosuppressant, or an antibiotic.

50. An immunoconjugate or antibody-drug conjugate (ADC) comprising the antibody according to claim 1 or 12, and a therapeutic moiety, wherein the therapeutic moiety is a chemotherapeutic drug.

51. An immunoconjugate or antibody-drug conjugate (ADC) comprising the antibody according to claim 1 or 12, and a therapeutic moiety, wherein the therapeutic moiety is a radioisotope.

52. A nucleic acid construct or nucleic acid constructs, comprising a) A nucleic acid sequence encoding a heavy chain sequence of the antibody comprising an antigen-binding region capable of binding 5T4 as defined in claim 1, and b) A nucleic acid sequence encoding a light chain sequence of the antibody comprising an antigen-binding region capable of binding 5T4 as defined in claim 1.

53. The one or more nucleic acid constructs according to claim 52, further comprising a) A nucleic acid sequence encoding a heavy chain sequence of the antibody comprising an antigen-binding region capable of binding CD3 as defined in claim 13 or 14, and b) A nucleic acid sequence encoding a light chain sequence of the antibody comprising an antigen-binding region capable of binding CD3 as defined in claim 13 or 14.

54. An expression vector or expression vectors, comprising a) A nucleic acid sequence encoding a heavy chain sequence of the antibody comprising an antigen-binding region capable of binding 5T4 as defined in claim 1, and b) A nucleic acid sequence encoding a light chain sequence of the antibody comprising an antigen-binding region capable of binding 5T4 as defined in claim 1.

55. The one or more expression vectors according to claim 54, further comprising a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined in claim 13 or 14, and b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined in claim 13 or 14.

56. A host cell comprising one or more nucleic acid constructs as defined in claim 52 or 53, or one or more expression vectors as defined in claim 54 or 55.

57. The host cell according to claim 56, wherein the host cell is of human origin or of rodent origin.

58. The host cell according to claim 56, wherein the host cell is a human embryonic kidney (HEK) cell or a Chinese hamster ovary cell.

59. The host cell according to claim 56, wherein the host cell is a HEK / Expi cell or a CHO / N50 cell.

60. A composition comprising an antibody as defined in claim 1 or 12.

61. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 or 12 - 15 and a pharmaceutically acceptable carrier.

62. Use of an antibody as defined in claim 1 or 12, a composition as defined in claim 60, or a pharmaceutical composition as defined in claim 61 in the preparation of a medicament for the treatment of cancer, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, and pancreatic cancer.

63. The use according to claim 62, wherein the cancer is cervical adenocarcinoma.

64. A method for producing an antibody as defined in claim 1 or 12, comprising the following steps: a) Culturing a host cell comprising an expression vector as defined in claim 54 or 55; and b) Purifying said antibody from the culture medium.

65. A method for producing an antibody as defined in claim 13 or 14, comprising the following steps: a) Providing an antibody capable of binding 5T4, by culturing a host cell comprising the expression vector as defined in claim 54 or 55 under conditions permitting expression of said antibody capable of binding 5T4, and purifying said antibody capable of binding 5T4 from the culture medium; b) Providing an antibody capable of binding CD3, by culturing a host cell comprising an expression vector under conditions permitting expression of said antibody capable of binding CD3, and purifying said antibody capable of binding CD3 from the culture medium, said expression vector comprising I) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding CD3 as defined in claim 13 or 14, and II) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding CD3 as defined in claim 13 or 14; c) Incubating said antibody capable of binding 5T4 with said antibody capable of binding CD3 under reducing conditions sufficient to permit cysteine in the hinge region to undergo disulfide bond isomerization, and d) Obtaining said antibody.

66. A kit-of-parts comprising an antibody as defined in claim 1 or 12; and instructions for use of the kit.

67. The kit according to claim 66, which is used as an adjunct diagnostic / for identifying, within a patient population, those patients who have a tendency to respond to treatment with an antibody as defined in claim 1 or 12 or an immunoconjugate or antibody-drug conjugate (ADC) as defined in claim 48 or 49, or for predicting the efficacy of said antibody or immunoconjugate or ADC when used to treat a patient.

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