Antibody

Abstract

Provided are antibodies that bind to 5T4, including bispecific antibodies that bind to 5T4 and CD3, pharmaceutical compositions comprising said antibodies, and uses of said antibodies for therapeutic and diagnostic procedures, particularly in the treatment of cancer. [Solution] We provide an antibody that has at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), and that can block the binding to 5T4 of an antibody that has a variable heavy chain (VH) region containing a specific sequence and a variable light chain (VL) region containing a specific sequence.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antibodies that bind to 5T4, including bispecific antibodies that bind to 5T4 and CD3. The invention further provides pharmaceutical compositions comprising said antibodies and uses of said antibodies for therapeutic and diagnostic procedures, particularly in the treatment of cancer. [Background technology]

[0002] background 5T4 (also known as trophoblast glycoprotein [TPBG] or Wnt-activated inhibitory factor 1 [WAIF1]) is a 72 kDa single-pass transmembrane protein containing eight leucine-rich repeats (LRRs) and seven potential N-glycosylation sites (Zhao et al., 2014 Structure 22, 612-620).

[0003] 5T4 expression is restricted in normal adult tissues except for the placenta (Southall et al., 1990 Br J Cancer 61, 89-95 (Non-Patent Document 2)). 5T4 is expressed in many human cancers, including kidney, cervical, ovarian, lung, prostate, and colorectal cancers (Stern and Harrop, 2017 Cancer Immunol Immunother 66, 415-426 (Non-Patent Document 3), Southall et al., 1990 Br J Cancer 61, 89-95 (Non-Patent Document 2)). 5T4 expression in tumor cells promotes tumor development by 1) promoting epithelial-mesenchymal transition (Damelin et al., 2011 Cancer Res 71, 4236-4246 (Non-Patent Document 4), Carsberg et al., 1996 Int J Cancer 68, 84-92 (Non-Patent Document 5)) 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 (Non-Patent Document 6)).

[0004] 5T4-targeting antibodies and therapies have clinical activity in several cancers known to express 5T4, including colorectal, lung, and renal cancers. For example, naptumomab estafenatox is a recombinant fusion protein consisting of a 5T4-Fab portion genetically fused to the engineered superantigen variant SEA / E-120. It is currently in clinical trials as an immunotherapy for non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and pancreatic cancer (see, e.g., Eisen, et al., 2014 Curr Oncol Rep 16, 370). TroVax® is a modified vaccinia Ankara strain (MVA-5T4) expressing a 5T4 construct, which has shown clinical benefit in colorectal cancer, prostate cancer, and renal cancer (see, for example, Stern and Harrop, 2017 Cancer Immunol Immunother 66, 415-426 (Non-Patent Document 3); Scurr et al., 2017 JAMA Oncol 12, 10 (Non-Patent Document 8)). Furthermore, anti-5T4 antibodies are described in WO2007106744 (Patent Document 1), WO03038098 (Patent Document 2), WO2011048369 (Patent Document 3), WO2013041687 (Patent Document 4), and WO2017072207 (Patent Document 5).

[0005] Although significant progress has been made in eradicating cancer, further improvements in antibody-based cancer therapies are still needed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Zhao et al.,2014 Structure 22,612-620 [Patent Document 2] Southall et al.,1990 Br J Cancer 61,89-95 [Patent Document 3] Stern and Harrop,2017 Cancer Immunol Immunother 66,415-426 [Patent Document 4] Damelin et al.,2011 Cancer Res 71,4236-4246 [Patent Document 5] Carsberg et al.,1996 Int J Cancer 68,84-92 [Patent Document 6] Kagermeier-Schenk et al.,2011 Dev Cell 21,1129-1143 [Patent Document 7] Eisen,et al.,2014 Curr Oncol Rep 16,370 [Patent Document 8] Scurr et al.,2017 JAMA Oncol 12,10 [Non-patent literature]

[0007] [Non-Patent Document 1] WO2007106744 [Non-patent document 2] WO03038098 [Non-patent document 3] WO2011048369 [Non-patent document 4] WO2013041687 [Non-patent document 5] WO2017072207 Summary of the Invention

[0008] It is an object of the present invention to provide an antibody that comprises at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), and that is capable of blocking the binding of an antibody

[0059] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:9 to 5T4.

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

[0010] In another aspect, the present invention provides a method for producing a composition comprising: a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein; The present invention relates to a nucleic acid construct comprising:

[0011] In another aspect, the present invention provides a method for producing a composition comprising: a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined herein; The present invention relates to an expression vector comprising the

[0012] In another aspect, the present invention relates to a cell comprising a nucleic acid construct or an expression vector as defined herein.

[0013] In another aspect, the present invention relates to a composition comprising the antibody of the present invention.

[0014] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody as defined herein and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present invention relates to an antibody as defined herein for use as a medicament, for example for use in the treatment of disease.

[0016] In another aspect, the present invention relates to a method of treating a disease or disorder comprising the step of administering to a subject in need thereof an antibody, composition or pharmaceutical composition as defined herein.

[0017] In another aspect, the present invention relates to a method for producing an antibody as defined herein.

[0018] In another aspect, the present invention relates to a kit-of-parts comprising an antibody as defined herein and instructions for use of said kit.

[0019] In another aspect, the present invention relates to an anti-idiotypic antibody that binds to the antigen-binding region of an antibody defined herein that is capable of binding to 5T4. [The present invention 1001] An antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), An antibody capable of blocking the binding to 5T4 of an antibody

[0059] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:9. [The present invention 1002] a) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 44

[0207] ; b) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 51

[0226] ; and c) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:9; 1001. The antibody of the present invention, which blocks the binding to 5T4 of an antibody selected from the group consisting of: [The present invention 1003] a) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 44

[0207] ; and b) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:51; The antibody of the present invention 1001 or 1002, which blocks the binding of an antibody selected from the group consisting of: [The present invention 1004] Any of the aforementioned antibodies of the invention, wherein the 5T4 is human (Homo sapiens) 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:1. [The present invention 1005] The antibody of any of claims 1001 to 1003, wherein 5T4 is the mature polypeptide sequence of cynomolgus monkey (Macaca fascicularis) 5T4, e.g., SEQ ID NO:2. [The present invention 1006] The antibody of any of claims 1001 to 1003, wherein the 5T4 is chicken (Gallus gallus) 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:3. [The present invention 1007] Any of the antibodies of the invention described above, wherein the 5T4 is human 5T4, eg, the mature polypeptide of SEQ ID NO:1, and cynomolgus 5T4, eg, the mature polypeptide of SEQ ID NO:2. [The present invention 1008] Any of the antibodies of the invention, wherein the 5T4 is human 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:1, cynomolgus monkey 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:2, and chicken 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:3. [The present invention 1009] Human 5T4, cynomolgus monkey and / or chicken 5T4 are given a K of 1E-7M or less, for example, 5E-8M or less, 1E-8M or less, 5E-9M or less, or for example, 1E-9M or less. Dand a K value in the range of, for example, 1E-7 to 5E-10 M, for example, 1E-7 to 1E-9 M, for example, 5E-8 to 5E-10 M, 5E-8 to 1E-9 M, for example, 1E-8 to 5E-10 M, 1E-8 to 1E-9 M, or for example, 1E-8 to 5E-9 M. D Any of the antibodies of the invention that are capable of binding to a target antigen with a binding affinity corresponding to a value. [The present invention 1010] 1009. An antibody of the invention, wherein the binding affinity is determined by biolayer interferometry, optionally as set forth in Example 2 herein. [The present invention 1011] The binding affinity is I) immobilizing the antibody on an anti-human IgG Fc capture biosensor at 1 μg / mL for 600 seconds; II) determining the association of 5T4ECDHis (mature protein of SEQ ID NO:99) or cynomolgus 5T4 (mature protein of SEQ ID NO:2) or recombinant cynomolgus 5T4 protein (Cusabio, Cat. No. CSB-MP024093MOV) over a period of 200 seconds and the dissociation over a period of 1000 seconds using a two-fold dilution series ranging from 100 nM to 1.56 nM; III) Relating data to a buffer control (0 nM) The antibody of any one of 1009 and 1010 of the present invention, which is determined using a biolayer interferometry method comprising: [The present invention 1012] The antibody of any of claims 1009 to 1011, wherein the binding affinity is determined using any of said antibodies of the invention that are monospecific bivalent antibodies, for example, antibodies that are full-length IgG1. [The present invention 1013] a) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO:5 and a VL region comprising the sequence shown in SEQ ID NO:9; b) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 12 and a VL region comprising the sequence shown in SEQ ID NO: 16; c) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 19 and a VL region comprising the sequence shown in SEQ ID NO: 23; d) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 26 and a VL region comprising the sequence shown in SEQ ID NO: 30

[0106] ; e) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 33 and a VL region comprising the sequence shown in SEQ ID NO: 37

[0127] ; f) an antibody

[0207] comprising a VH region comprising the sequence set forth in SEQ ID NO: 40 and a VL region comprising the sequence set forth in SEQ ID NO: 44; and g) an antibody comprising a VH region comprising the sequence set forth in SEQ ID NO: 47 and a VL region comprising the sequence set forth in SEQ ID NO: 51; an epitope or antibody binding region or binding site on 5T4 recognized by any one of the antibodies selected from the group consisting of: Any of the antibodies of the present invention, which recognizes [The present invention 1014] a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO: 86. is not an antibody binding region, binding site or epitope bound by an antibody selected from the group consisting of: a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO: 86. an antibody binding region, binding site, or epitope on 5T4 that is different from the antibody binding region, binding site, or epitope bound by an antibody selected from the group consisting of: Any of the antibodies of the present invention that bind to [The present invention 1015] Any of the antibodies of the present invention, whose binding to 5T4 is blocked by the binding to 5T4 of antibody [A3] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:86. [The present invention 1016] An antibody that binds to 5T4ECDHis (mature protein of SEQ ID NO:99) [A3], comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:86. Any of the antibodies of the present invention, wherein the antibody exhibits the following replacement: [The present invention 1017] The antibody of the invention 1014 or the invention 1015, wherein cross-blocking, or the ability of any of the antibodies of the invention to block the binding of another antibody to 5T4, is determined by a fluorescence-activated cell sorting (FACS) assay, for example in an assay performed as described in Example 5. [The present invention 1018] Cross-blocking, or the ability of any of the antibodies of the invention to block the binding of another antibody to 5T4, is determined as the ability of the unconjugated antibody to block the binding of the conjugated antibody, and optionally 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, the antibody being conjugated to fluorescein isothiocyanate (FITC), and an excess of an unconjugated antibody that targets 5T4; ii) incubating the sample at 4°C for 30 minutes, and then centrifuging the sample; iii) removing the supernatant from each sample, resuspending the cells in buffer, and determining the mean fluorescence intensity (MFI) of FITC using a flow cytometer; and iv) The percentage of binding is as follows: The difference in MFI between cells incubated with a mixture of FITC-conjugated and unconjugated antibodies and cells incubated without FITC-conjugated or unconjugated antibodies is multiplied by 100 and then divided by the difference in MFI between cells incubated with a mixture of FITC-conjugated and IgG-b12 antibodies and cells incubated without FITC-conjugated or unconjugated antibodies. The process of calculating Any of 10 14 to 10 16 antibodies of the present invention, determined by a procedure comprising: [The present invention 1019] Any of antibodies 1014 to 1017 of the present invention, wherein the ability of the antibody to block the binding of another antibody to 5T4 or the ability of the antibody to displace another antibody from binding to 5T4ECDHis (mature protein of SEQ ID NO:99) is determined using biolayer interferometry, for example in the assay described in Example 3. [The present invention 1020] The ability of an antibody to block the binding of another antibody to 5T4 or to displace another antibody from binding to 5T4 can be determined using biolayer interferometry. i) immobilizing any of the antibodies of the present invention at a concentration of 20 μg / mL in 10 mM sodium acetate buffer onto an activated amine-reactive second-generation biosensor; ii) quenching the biosensor with immobilized antibody in ethanolamine pH 8.5; iii) immersing the biosensor with immobilized antibody in a composition comprising 3.6 μg / mL (100 nM) human 5T4 ECDHis (mature protein of SEQ ID NO:99) for a period of 500 seconds, and then iv) immersing the biosensor with the immobilized antibody and 5T4ECDHis in a composition containing the other antibody targeting 5T4 at 10 μg / mL for a period of 500 seconds and determining the association response. Any of 1014 to 1018 antibodies of the present invention, determined by a procedure comprising the steps i) to iv) performed at a temperature of 30°C while shaking at 1000 rpm. [The present invention 1021] an epitope or antibody binding region on human 5T4, comprising amino acid residues R73, Y92, and R94, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; Any of the antibodies of the present invention that bind to [The present invention 1022] an epitope or antibody binding region on human 5T4, comprising amino acid residues S69, R73, Y92, and R94, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; Any of the antibodies of the present invention that bind to [The present invention 1023] an epitope or antibody binding region on human 5T4, comprising amino acid residues R73, T74, Y92, R94, and N95, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; Any one of the antibodies 1001 to 1021 of the present invention, which binds to [The present invention 1024] The antibody of any of claims 1021 to 1023, wherein the amino acid residue is directly involved in antibody binding. [The present invention 1025] Any of the antibodies of the present invention 1021 to 1024, wherein one or more of the additional amino acid residues L89, F111, L117, F138, L144, D148, and N152, in the numbering of each amino acid residue indicating their respective positions in SEQ ID NO:1, are involved in antibody binding, or indirectly involved in binding, for example, by affecting protein folding and / or by affecting the positioning of one or more amino acid residues directly involved in antibody binding. [The present invention 1026] an epitope or antibody-binding region on human 5T4, in which amino acid residues R73, Y92, and R94 are directly involved in antibody binding, and one or more of amino acid residues F111, F138, L144, and D148 are indirectly involved in said binding, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1; Any of the antibodies of the present invention that bind to [The present invention 1027] an epitope or antibody-binding region on human 5T4, in which amino acid residues S69, R73, Y92, and R94 are directly involved in antibody binding, and one or more of amino acid residues F111, F138, and D148 are indirectly involved in said binding, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1; Any one of the antibodies 1001 to 1025 of the present invention, which binds to [The present invention 1028] an epitope or antibody-binding region on human 5T4, in which amino acid residues R73, T74, Y92, R94, and N95 are directly involved in antibody binding, and amino acid residue F138 is indirectly involved in said binding, with each amino acid residue numbering indicating its respective position in SEQ ID NO:1; Any one of the antibodies 1001 to 1025 of the present invention, which binds to [The present invention 1029] Any of the antibodies of the present invention 1021 to 1028, wherein the amino acid residues contained in the epitope or antibody binding region and optionally one or more additional amino acid residues are 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. [The present invention 1030] An antibody of the invention 1029, wherein alanine scanning is performed as described in Example 16 herein, or essentially as described in Example 16 herein. [The present invention 1031] Alanine scanning i) expressing a mutant human 5T4 polypeptide, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO:1) are individually substituted with alanine, and a wild-type 5T4 polypeptide (amino acid residues 32-355 of SEQ ID NO:1), in human embryonic kidney cells, e.g., HEK293 cells, so as to obtain a sample containing 70 to 90,000 cells, e.g., 80,000 cells, for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, washing each sample twice with 150-180 μL of FACS buffer (phosphate-buffered saline + 0.1% [w / v] BSA + 0.02% [w / v] sodium azide), and resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: TIFF2026041981000002.tif9128 (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using This is carried out by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: TIFF2026041981000003.tif8128 (where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively) is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. Data are excluded from the analysis if they are predicted to be spatially distant from An antibody of any one of 1029 to 1030 of the present invention. [The present invention 1032] The non-cross-blocking 5T4-specific control antibody in step iv) an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] 1031. The antibody of the present invention, which is a bispecific antibody comprising: [The present invention 1033] Any of the antibodies of the present inventions 1001 to 1020, which bind to 5T4 such that when any one or more of amino acid residues R73, Y92 and R94, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO:1, are substituted with alanine, loss of binding occurs or binding is reduced. [The present invention 1034] Any of antibodies 1001 to 1020 and 1033 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues S69, R73, Y92 and R94, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or is reduced. [This invention 1035] Any of antibodies 1001 to 1020 and 1033 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues R73, T74, Y92, R94 and N95, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or is reduced. [The present invention 1036] Any of antibodies 1001 to 1020 and 1033 to 1035 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues L89, F111, L117, F138, L144, D148, and N152, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or binding is reduced. [This invention 1037] Any of antibodies 1001 to 1020 and 1033 to 1036 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues R73, Y92, R94, F111, F138, L144 and D148, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or binding is reduced. [The present invention 1038] Any of antibodies 1001 to 1020 and 1033 to 1036 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues S69, R73, Y92, R94, F111, F138 and D148, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or binding is reduced. [This invention 1039] Any of antibodies 1001 to 1020 and 1033 to 1036 of the present invention, which bind to 5T4 such that when any one or more of amino acid residues R73, T74, Y92, R94, N95 and F138, where the numbering of each amino acid residue indicates the respective position in SEQ ID NO: 1, are substituted with alanine, binding is lost or binding is reduced. [The present invention 1040] The antibody of any of claims 1033 to 1039, wherein the effect of the alanine substitutions is determined by alanine scanning of a polypeptide comprising amino acid residues 32 to 355 of SEQ ID NO:1. [This invention 1041] The antibody of any of 1033 to 1039, wherein the effect of alanine substitution is determined by the procedure set forth in Example 16 herein, or by the procedure essentially set forth in Example 16 herein. [The present invention 1042] Any of antibodies 1033 to 1039 of the present invention, wherein loss of binding is defined as a Z-score for binding of less than 1.5, the Z-score optionally being calculated as set forth in Example 16 of the present specification, or essentially as set forth in Example 16 of the present specification. [This invention 1043] The effect of alanine substitution is i) expressing mutant human 5T4 polypeptides, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32 to 355 of SEQ ID NO: 1) are individually substituted with alanine, and wild-type 5T4 polypeptides, in human embryonic kidney cells, e.g., HEK293 cells, so that a sample containing 70 to 90,000 cells, e.g., 80,000 cells, is obtained for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, washing each sample twice with 150-180 μL of FACS buffer (phosphate-buffered saline + 0.1% [w / v] BSA + 0.02% [w / v] sodium azide), and resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: TIFF2026041981000004.tif9128 (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using is determined by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: TIFF2026041981000005.tif8128 (where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively) is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. Data are excluded from the analysis if they are predicted to be spatially distant from The antibody of the present invention 1042. [This invention 1044] The non-cross-blocking 5T4-specific control antibody in step iii) an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] The antibody of the present invention, which is a bispecific antibody comprising: [This invention 1045] Any of the antibodies of the present invention, which, when conjugated to a cytotoxic moiety, has reduced internalization capability as demonstrated by reduced cytotoxicity compared to an antibody [H8] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:87 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:88. [The present invention 1046] Cytotoxicity can be measured using the procedure set forth in Example 7, e.g. i) providing a monovalent antibody comprising a first Fab arm of any of the antibodies of the present invention and a second Fab arm capable of binding to HIV viral protein gp120 (HIV-1 gp120), wherein the monovalent antibody is conjugated to Duostatin-3; ii) contacting breast cancer cells MDA-MB-468 (ATCC clone HTB-132) or HCC1954 (ATCC clone CRL-1338) with the monovalent antibody at 37° C. for 5 days; and iii) determining cell viability The antibody of the present invention 1045, which is determined by a procedure comprising: [This invention 1047] an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50; and h) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to the CDR1, CDR2 and CDR3 sequences set forth in any one of a) to g). Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) selected from the group consisting of: [This invention 1048] an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50; and d) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to the CDR1, CDR2 and CDR3 sequences set forth in any one of a) to c). Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) selected from the group consisting of: [This invention 1049] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH)

[0059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6, 7 and 8. [The present invention 1050] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH)

[0207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:41, 42 and 43. [This invention 1051] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH)

[0226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:48, 49 and 50. [This invention 1052] an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 24, 25 and 26, respectively; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 31, 32 and 33, respectively; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 38, 39 and 39, respectively; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 45, 46 and 47, respectively; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 52, 53 and 54, respectively; and h) a heavy chain variable region (VH) and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 sequences, each of which contains a total of 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 set forth in any one of a) to g); Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of: [This invention 1053] The six complementarity-determining regions (CDRs) of the antigen-binding region capable of binding to 5T4 are: iv) CDR sequences of SEQ ID NO: 6, 7, 8, 10, AAS and SEQ ID NO: 11; v) SEQ ID NO: 41, 42, 43, 45, DAS and CDR sequences of SEQ ID NO: 46

[0207] , or vi) CDR sequences of SEQ ID NO: 48, 49, 50, 52, DAS and SEQ ID NO: 53 Any of the aforementioned antibodies of the invention, wherein the antibody comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions when compared to [This invention 1054] The antibody of any of claims 1022 to 1029, wherein one, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acid substitutions is a conservative amino acid substitution. [This invention 1055] Any of the antibodies of the present invention, comprising one or two heavy chain variable regions comprising 6 consecutive amino acid residues of the sequence set forth in SEQ ID NO:102 (YYGMDV) in the complementarity determining region 3 (CDR3) [059, 207, 226]. [This invention 1056] The antibody of the present invention 1055, wherein the 6 consecutive amino acid residues are the most C-terminal amino acid residues in CDR3. [This invention 1057] any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises one or two heavy chain variable regions (VH) comprising 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), and a light chain variable region (VL) comprising the CDR1 sequence of SEQ ID NO:105 (QSVSSX5), the CDR2 sequence DAS, and the CDR3 sequence of SEQ ID NO:46 (QQRSNWPLT), where 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]. [This invention 1058] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) having the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6, 7 and 8, respectively, and a light chain variable region (VL) having the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:10, AAS and SEQ ID NO:11, respectively

[0059] . [This invention 1059] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:41, 42 and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, DAS and SEQ ID NO:46, respectively

[0207] . [The present invention 1060] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:52, DAS and SEQ ID NO:53, respectively

[0226] . [The present invention 1061] an antigen-binding region capable of binding to 5T4, 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; 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; 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; 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; 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; 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

[0207] ; and 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; Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) selected from the group consisting of: [The present invention 1062] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises 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

[0059] . [The present invention 1063] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises 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

[0207] . [The present invention 1064] Any of the antibodies of the present invention, wherein the antigen-binding region capable of binding to 5T4 comprises 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

[0226] . [This invention 1065] an antigen-binding region capable of binding to 5T4, 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; 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; 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; 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; 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; 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

[0207] ; 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; Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of: [The present invention 1066] an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 5 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 9; b) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 12 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 16; c) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 19 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 23; d) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 26 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 30; e) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 33 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 37; f) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44

[0207] ; and g) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 47 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 51; Any of the aforementioned antibodies of the present invention, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of: [This invention 1067] Any of the antibodies of the present invention which are full-length antibodies, such as full-length IgG1 antibodies. [The present invention 1068] Any of the antibodies of the present invention, which is a monovalent antibody. [The present invention 1069] Any of the antibodies of the present invention, which is a bivalent antibody. [The present invention 1070] Any of the antibodies of the present invention, which are monospecific antibodies. [This invention 1071] Any of the antibodies of the present invention, which are bispecific antibodies. [This invention 1072] Any of the aforementioned antibodies of the invention, which comprise an antigen-binding region of an antibody that binds to CD3, e.g., human CD3ε (epsilon), e.g., the human CD3ε (epsilon) set forth in SEQ ID NO:4. [This invention 1073] The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively [wild-type anti-CD3 (SP34 / humanized SP34, WO2015001085 (Genmab))-VH CDR sequences]; and optionally, A light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:58, GTN, and SEQ ID NO:59, respectively [wild-type anti-CD3, VL CDR sequences]. The antibody of the present invention 1072, comprising: [This invention 1074] The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising 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 to the sequence of SEQ ID NO: 57 [wild type anti-CD3 - VH full length sequence]; and optionally, a light chain variable region (VL) comprising 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 to the sequence of SEQ ID NO:60 [wild-type anti-CD3 - VL full-length sequence] The antibody of the present invention 1072 or 1073, comprising: [This invention 1075] Any of the antibodies 1072 to 1074 of the present invention, which has a lower binding affinity to human CD3ε than an antibody having an antigen-binding region comprising the VH sequence shown in SEQ ID NO:57 and the VL sequence shown in SEQ ID NO:60 [wild-type anti-CD3 (humanized SP34, WO2015001085 (Genmab)) VH and VL sequences], preferably the affinity is at least 5-fold, for example at least 10-fold, for example at least 20-fold, at least 30-fold, at least 40-fold, at least 45-fold, or for example at least 50-fold lower. [This invention 1076] The antigen-binding region has an equilibrium dissociation constant K in the range of 200 to 1000 nM, for example, in the range of 300 to 1000 nM, in the range of 400 to 1000 nM, in the range of 500 to 1000 nM, in the range of 300 to 900 nM, in the range of 400 to 900 nM, in the range of 400 to 700 nM, in the range of 500 to 900 nM, in the range of 500 to 800 nM, in the range of 500 to 700 nM, in the range of 600 to 1000 nM, in the range of 600 to 900 nM, in the range of 600 to 800 nM, or, for example, in the range of 600 to 700 nM. D The antibody of any one of 1072 to 1074 of the present invention, which binds to CD3 at [This invention 1077] The antigen-binding region has an equilibrium dissociation constant K in the range of 1 to 100 nM, for example, in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, in the range of 10 to 80 nM, in the range of 10 to 60 nM, in the range of 10 to 40 nM, or, for example, in the range of 10 to 20 nM. D The antibody of the present invention 1072 or 1075, which binds to CD3 at [This invention 1078] the antigen-binding region that binds to CD3 comprises a heavy chain variable (VH) region including a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; the heavy chain variable (VH) region, when compared to a heavy chain variable (VH) region comprising the sequence set forth in SEQ ID NO: 57, has in one of the CDR sequences an amino acid substitution at a position selected from the group consisting of T31, N57, H101, G105, S110, and Y114, numbered according to the sequence of SEQ ID NO: 57 [VH_huCD3-H1L1]; and the wild-type light chain variable (VL) region comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, GTN and SEQ ID NO: 59, respectively; Any one of the antibodies 1072 to 1077 of the present invention. [This invention 1079] An antibody of the present invention 1072, wherein the CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 contain a total of at most 1, 2, 3, 4 or 5 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 of the sequence shown in SEQ ID NO:57. [The present invention 1080] The antibody of the present invention 1072 or 1073, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 have at least 95% sequence identity, for example at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequences of CDR1, CDR2, and CDR3 of the wild-type heavy chain variable (VH) region, wherein the sequence identity is calculated based on an alignment of the amino acid sequence consisting of the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 with the amino acid sequence comprising the sequences of CDR1, CDR2, and CDR3 of the wild-type heavy chain variable (VH) region. [This invention 1081] Any of the antibodies of the present invention 1072 to 1074, wherein the antigen-binding region that binds to CD3 contains a mutation selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V. [This invention 1082] a bispecific antibody that lacks or has reduced Fc-mediated effector functions (an "inactive" antibody) and comprises the antigen-binding region of an antibody that binds to CD3; a) when purified PBMCs or T cells are used as effector cells, they are able to mediate concentration-dependent cytotoxicity of SK-OV-3 cells, e.g., when assayed as described in Example 14 herein; b) when purified T cells are used as effector cells, they are able to mediate cell concentration-dependent cytotoxicity of MDA-MB-231 when assayed, for example, as described in Example 13 herein; c) capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells, e.g., when assayed as described in Example 13 herein; d) capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; e) purified T cells, when used as effector cells, are capable of inducing cytotoxicity of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; and / or f) NOD.Cg-Prkdc inoculated with human MDA-MB-231 tumor cells scid Il2rg tm1Wjl exhibit anti-tumor activity, such as delayed tumor outgrowth, in a humanized immune hematopoietic stem cell reconstituted mouse xenograft model, such as IFN-γ / SzJ, as determined, for example, as described in Example 15; Any of the antibodies of the present invention. [This invention 1083] The ability of the antibodies to mediate concentration-dependent cytotoxicity of SK-OV-3 cells was i) isolating peripheral blood mononuclear cells (PBMCs) or T cells from healthy human donor buffy coats; ii) a first set of samples, each sample comprising PBMCs and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of PBMCs:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, 8:1 and 12:1; and a second set of samples, each sample comprising T cells and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of T cells:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, and 8:1; preparing a iii) adding the antibody to each set of samples at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; and then iv) assessing the viability of SK-OV-3 cells using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) The antibody of the present invention 1082, as determined in an in vitro cytotoxicity assay comprising: [This invention 1084] The ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising T cells and human breast adenocarcinoma MDA-MB-231 cells, wherein the ratio of T cells:MDA-MB-231 cells in the samples is 8:1; iii) adding an antibody to the sample set at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; iv) staining the T cells by incubating with fluorescently labeled antibodies against T cell activation markers, such as CD69-APC, CD25-PE-Cy7 and CD279 / PD1-BV604 antibodies for 30 minutes at 4°C; and v) analyzing the sample by flow cytometry The antibody of the present invention 1082, as determined in an assay comprising: [This invention 1085] In vitro activation of T cells in the presence of BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells, wherein the ratio of T cells:tumor cells in the samples is 4:1; iii) adding an antibody to the sample set at a concentration of 0.0128 ng / mL to 5000 ng / mL (e.g., a 5-fold dilution method) and incubating the samples at 37°C for 72 hours; iv) collecting 110 μL of T cell-containing supernatant from each sample and staining the T cells by incubating with fluorescently labeled antibodies against T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and antibodies against T cell markers, such as 69-APC, CD25-PE-Cy7, and CD279 / PD1-BV604 antibodies, at 4° C. for 30 minutes; and v) analyzing the sample by flow cytometry The antibody of the present invention 1082, which is determined by a procedure comprising: [The present invention 1086] The ability to induce cytotoxicity in BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) providing T cells isolated from a healthy human donor buffy coat; ii) providing a set of test samples and a control sample, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells adhered to the bottom of a 96-well tissue culture plate, wherein the ratio of T cells to tumor cells in the sample is 4:1; iii) adding antibody to a set of test samples at concentrations ranging from 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilutions), while control samples are left untreated or incubated with 5 μM staurosporine, and incubating all samples at 37° C. for 72 hours; iv) incubating the adherent cells in 10% (w / w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin) in RPMI-1640 medium supplemented with 10% (w / w) iron-containing donor bovine serum and penicillin / streptomycin for 4 hours at 37°C; v) Measure the absorbance of the cells and calculate the percentage of viable cells by setting the absorbance of cells incubated with staurosporine as 0% viability and untreated cells as 100% viability. Calculation process as TIFF2026041981000006.tif9150 The antibody of the present invention 1082, which is determined by a procedure comprising: [This invention 1087] an antigen-binding region capable of binding to CD3, a) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 61, 55 and 56, respectively [VH CDR1-T31P + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or b) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 63, 55 and 56, respectively [VH CDR1-T31M + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or c) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 65 and 56, respectively [VH CDR-N57E + wild-type VH CDR1, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or d) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 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, sequence GTN and sequence shown in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] e) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 69, respectively [wild-type VH CDR1,2 + VH CDR3-H101N] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 69, respectively [wild-type VL CDR1,2,3]; f) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 71, respectively [wild-type VH CDR1,2 + VH CDR3-G105P] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; g) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 73, respectively [wild-type VH CDR1,2 + VH CDR3-S110A] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or h) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 75, respectively [wild-type VH CDR1,2 + VH CDR3-S110G] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; i) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 77, respectively [wild-type VH CDR1,2 + VH CDR3-Y114V] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or j) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 79, respectively [wild-type VH CDR1,2 + VH CDR3-Y114M] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or k) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 81, respectively [wild-type VH CDR1,2 + VH CDR3-Y114R] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] The antibody of any one of the antibodies 1072 and 1078 to 1084 of the present invention, comprising: [This invention 1088] The antibody of any one of the antibodies 1072 and 1078 to 1085 of the present invention, wherein the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) [wild-type VH CDR1, 2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively, and a light chain variable region (VL) [wild-type VL CDR1, 2, 3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively. [This invention 1089] an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; The antibody 1072 of the present invention and any one of the antibodies 1078 to 1085 of the present invention. [The present invention 1090] an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 41, 42, and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45, DAS, and SEQ ID NO: 46, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; The antibody 1072 of the present invention and any one of the antibodies 1078 to 1085 of the present invention. [This invention 1091] an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 48, 49, and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 52, DAS, and SEQ ID NO: 53, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; The antibody 1047 of the present invention and any one of the antibodies 1051 to 1059 of the present invention. [This invention 1092] an antigen-binding region capable of binding to human CD3, a) the VH sequence shown in SEQ ID NO:62 [VH T31P full-length sequence] and the VL sequence shown in SEQ ID NO:60 [wild-type full-length sequence]; b) the VH sequence shown in SEQ ID NO:64 [VH T31M full length sequence] and the VL sequence shown in SEQ ID NO:60; c) the VH sequence shown in SEQ ID NO: 66 [VH N57E full length sequence] and the VL sequence shown in SEQ ID NO: 60; d) the VH sequence shown in SEQ ID NO:68 [VH H101G full length sequence] and the VL sequence shown in SEQ ID NO:60; e) the VH sequence shown in SEQ ID NO:70 [VH H101N full length sequence] and the VL sequence shown in SEQ ID NO:60; f) the VH sequence shown in SEQ ID NO: 72 [VH G105P full length sequence] and the VL sequence shown in SEQ ID NO: 60; g) the VH sequence shown in SEQ ID NO: 74 [VH S110A full length sequence] and the VL sequence shown in SEQ ID NO: 60; h) the VH sequence shown in SEQ ID NO: 76 [VH S110G full length sequence] and the VL sequence shown in SEQ ID NO: 60; i) the VH sequence shown in SEQ ID NO: 78 [VH Y114V full length sequence] and the VL sequence shown in SEQ ID NO: 60; j) the VH sequence shown in SEQ ID NO: 80 [VH Y114M full length sequence] and the VL sequence shown in SEQ ID NO: 60, and k) the VH sequence shown in SEQ ID NO:82 [VH Y114R full-length sequence] and the VL sequence shown in SEQ ID NO:60 The antibody of any one of the antibodies 1072 of the present invention and 1078 to 1091 of the present invention, which comprises a VH sequence and a VL sequence selected from the group consisting of: [This invention 1093] The antibody of any one of the antibodies 1072 and 1078 to 1092 of the present invention, wherein the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60. [This invention 1094] an antigen-binding region capable of binding to 5T4 comprising 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 [059 - VH full length sequence]; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; Any one of the antibodies 1078 to 1093 of the present invention. [This invention 1095] an antigen-binding region capable of binding to 5T4 comprising 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 [207 - VH full length sequence]; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; The antibody of the present invention 1072 and any one of the antibodies of the present invention 1078 to 1093. [This invention 1096] an antigen-binding region capable of binding to 5T4 comprising 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 [226 - VH full length sequence]; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; The antibody 1047 of the present invention and any one of the antibodies 1051 to 1064 of the present invention. [This invention 1097] an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO:9; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; The antibody 1072 of the present invention and any one of the antibodies 1078 to 1094 of the present invention. [This invention 1098] an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO: 44 [207 - VH + VL full length sequence]; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody of any one of the antibodies 1072 of the present invention, 1078 to 1093 of the present invention, and 1095 of the present invention. [This invention 1099] an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO: 51 [226 - VH + VL full length sequence]; and the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody of any one of the antibodies 1072 of the present invention, 1078 to 1093 of the present invention, and 1096 of the present invention. [The present invention 1100] Any of the antibodies of the present invention, wherein each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the variable regions comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. [The present invention 1101] An antibody of the present invention 1100, comprising two heavy chain constant regions (CH) and two light chain constant regions (CL). [The present invention 1102] comprising a first heavy chain and a second heavy chain; each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 region, and a CH3 region, wherein the first heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and the second heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, wherein the substitutions in the first heavy chain and the second heavy chain are not at the same position, and the amino acid positions are numbered according to EU numbering; The antibody of the present invention 1100 or 1101. [The present invention 1103] Any of the antibodies of the present inventions 1100 to 1102, wherein in the first heavy chain, the amino acid at a position corresponding to K409 in the human IgG1 heavy chain is R, and in the second heavy chain, the amino acid at a position corresponding to F405 in the human IgG1 heavy chain is L, or vice versa. [The present invention 1104] comprising a first heavy chain and a second heavy chain; in both the first heavy chain and the second heavy chain, the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain (EU numbering) are F and E, respectively; Any of the antibodies of the present invention. [This invention 1105] comprising a first heavy chain and a second heavy chain; the amino acid residue at the position corresponding to position D265 in a human IgG1 heavy chain (EU numbering) is A in both the first heavy chain and the second heavy chain; Any of the antibodies of the present invention. [The present invention 1106] a) the antigen-binding region capable of binding to 5T4 is humanized, and / or b) the antigen-binding region capable of binding to CD3, if present, is humanized; Any of the antibodies of the present invention. [This invention 1107] a) the antigen-binding region capable of binding to 5T4 is human, and / or b) the antigen-binding region capable of binding to CD3, if present, is human; Any of the antibodies of the present invention. [This invention 1108] a) the antigen-binding region capable of binding to 5T4 is chimeric, and / or b) the antigen-binding region capable of binding to CD3, if present, is chimeric; Any of the antibodies of the present invention. [This invention 1109] comprising a first heavy chain and optionally a second heavy chain; the first heavy chain, and, if present, the second heavy chain, are modified such that the antibody induces Fc-mediated effector function to a lesser extent than the same unmodified antibody; Any of the antibodies of the present invention. [The present invention 1110] Any of the aforementioned antibodies of the present invention, which comprises a kappa (κ) light chain. [The present invention 1111] Any of the aforementioned antibodies of the present invention, which comprise a lambda (λ) light chain. [The present invention 1112] Any of the antibodies of the invention comprising a lambda (λ) light chain and a kappa (κ) light chain, for example an antibody having a heavy chain and a lambda light chain comprising a binding region capable of binding to CD3, and a heavy chain and a kappa light chain comprising a binding region capable of binding to 5T4. [The present invention 1113] An immunoconjugate or antibody-drug conjugate (ADC) comprising any of the antibodies of the invention and a therapeutic moiety, such as a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, antibiotic, or radioisotope. [This invention 1114] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 of any one of the present inventions 1001 to 1113, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to any one of 5T4s 1072 to 1113 of the present invention. A nucleic acid construct comprising: [This invention 1115] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of the antibodies 1072 to 1113 of the present invention, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of the antibodies 1072 to 1113 of the present invention. 1114. The nucleic acid construct of the present invention, further comprising: [The present invention 1116] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 of any one of the inventions 1001 to 1112, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 of any one of the present inventions 1001 to 1112. An expression vector comprising: [This invention 1117] a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of the antibodies 1072 to 1112 of the present invention, and / or b) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of the antibodies 1072 to 1112 of the present invention. 1116. An expression vector of the present invention further comprising: [This invention 1118] For example, a cell obtained by transfecting a host cell with a nucleic acid construct of any one of the present inventions 1114 to 1115 or an expression vector of the present invention 1116 or 1117, such as a recombinant host cell, comprising the nucleic acid construct of any one of the present inventions 1114 to 1115 or the expression vector of the present invention 1116 or 1117. [This invention 1119] The cell of the invention 1118, wherein the host cell is of human origin, such as a human embryonic kidney (HEK) cell, e.g., a HEK / Expi cell, or of rodent origin, such as a Chinese hamster ovary cell, e.g., a CHO / N50 cell. [The present invention 1120] A composition comprising any one of the antibodies 1001 to 1112 of the present invention. [This invention 1121] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1112 and a pharmaceutically acceptable carrier. [This invention 1122] The antibody of any one of 1001 to 1112 of the present invention for use as a medicine. [This invention 1123] The antibody of any of claims 1001 to 1112 for use in treating a disease. [This invention 1124] The antibody for use of the present invention 1122 or the present invention 1123, wherein the disease is cancer. [Invention 1125] The antibody for use in the present invention 1124, wherein the cancer is characterized by expression of 5T4 in at least some of the tumor cells. [The present invention 1126] The antibody for use in any of claims 1122 to 1125, wherein the cancer is selected from the group consisting of kidney cancer / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine cancer / endometrial cancer / cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia. [This invention 1127] A method for treating a disease, comprising the step of administering any one of the antibodies of the present inventions 1001 to 1112, the composition of the present invention 1120, or the pharmaceutical composition of the present invention 1121 to a subject in need thereof. [This invention 1128] The method of claim 1127, which is for the treatment of cancer. [This invention 1129] 1128. The method of claim 1128, wherein the cancer is selected from the group consisting of renal / kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, and acute myeloid leukemia. [The present invention 1130] a) culturing a host cell containing an expression vector of the present invention 1114 or the present invention 1117; and b) Purifying the antibody from the culture medium A method for producing any one of the antibodies of the present inventions 1001 to 1112, comprising: [This invention 1131] a) providing an antibody capable of binding to 5T4 by culturing a host cell containing an expression vector of the present invention 1116 or the present invention 1117 under conditions allowing expression of the antibody capable of binding to 5T4, and purifying the antibody capable of binding to 5T4 from the culture medium; b) I) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of 1072 to 1123 of the present invention; and II) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 of any one of 1072 to 1112 of the present invention. providing an antibody capable of binding to CD3 by culturing a host cell containing an expression vector comprising: c) incubating an antibody capable of binding to 5T4 with an antibody capable of binding to CD3 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and d) Obtaining an antibody A method for producing any one of the antibodies of the present inventions 1001 to 1112, comprising: [This invention 1132] A kit-of-parts, such as a kit for use as a companion diagnostic / to identify patients within a patient population who have a propensity to respond to treatment with an antibody of any of inventions 1001 to 1112 or an immunoconjugate or antibody-drug conjugate (ADC) of invention 1113, or for use to predict the efficacy of said antibody or immunoconjugate or ADC when used to treat a patient, comprising an antibody of any of inventions 1001 to 1112 and instructions for use of the kit. [This invention 1133] An anti-idiotype antibody that binds to an antigen-binding region capable of binding to 5T4 of any one of 1001 to 1112 of the present invention. [Brief explanation of the drawings]

[0020] [Figure 1]Antibody displacement of IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR in combination with IgG1-5T4-A3-F405L. Antibody displacement was determined by biolayer interferometry using an Octet HTX instrument (ForteBio). IgG1-5T4-A3-F405L was immobilized on a biosensor and loaded with human 5T4 ECDHis (mature protein of SEQ ID NO:99). The loaded biosensor was then exposed to IgG1-5T4-A3-F405L, IgG1-5T4-H8-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, or IgG1-5T4-226-FEAR. This figure shows the association response (500 s) upon 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), indicating 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 (representing antibody binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex), followed by a rapid decrease in mass. This behavior 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). [Figure 2]Simultaneous binding of 5T4 antibodies to membrane-bound 5T4 was 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 5T4-expressing SK-OV-3 cells 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 binding percentage of FITC-labeled antibodies was calculated and plotted as the mean binding percentage ± standard deviation (SD). [Figure 3] Binding of 5T4 antibodies to HEK-293 cells transfected with full-length human 5T4 and full-length chicken 5T4. HEK-293 cells transiently transfected with full-length human 5T4 (SEQ ID NO:1) (A) or full-length 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 R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2, mean fluorescence intensity (MFI) was determined by flow cytometry. IgG1-b12-K409R (10 μg / mL) was included as a negative control. [Figure 4]Internalization ability of monovalent 5T4 antibody. A bispecific toxin-conjugated antibody, which recognizes 5T4 with one Fab arm and an unrelated antigen (HIV-1 gp120, not expressed on tumor cells) with the second Fab arm, was generated by controlled Fab arm exchange between unconjugated 5T4 and an HIV-1 gp120-specific IgG1-b12 antibody conjugated to Duostatin-3 at one molecule per antibody molecule. MDA-MB-468 cells (A) and HCC1954 cells (B) were incubated with a range of antibody concentrations as indicated. Cell viability was measured after 5 days. Data are expressed as the average percentage of viable cells from three replicate experiments. A monospecific bivalent IgG1-b12 conjugated to Duostatin-3 (IgG1-b12-vcDuo3) was included as a negative control. [Figure 5(I)] Binding of CD3×5T4 bispecific antibodies to full-length human 5T4 and full-length cynomolgus 5T4 transfected into HEK-293 cells. Binding of monovalent and bivalent 5T4 antibodies was analyzed using HEK-293 cells transiently transfected with full-length human 5T4 (left panel) or full-length cynomolgus 5T4 (right panel). Cells were incubated with a range of antibody concentrations as indicated. Binding was analyzed by flow cytometry after secondary labeling with FITC-conjugated goat anti-human IgG F(ab')2. A negative control antibody, IgG1-b12-K409R (3 μg / mL), was included. Data are presented as mean fluorescence intensity (MFI) values ​​± SD of two technical replicates. A. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR and IgG1-5T4-207-FEAR. B. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR and IgG1-5T4-226-FEAR. C. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR and IgG1-5T4-059-FEAR. D. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-H8-FEAR and IgG1-5T4-H8-FEAR. [Figure 5(II)-1]Binding of bispecific CD3×5T4 antibodies to cynomolgus monkey 5T4 and human 5T4 transfected into HEK-293 cells. Monovalent and bivalent binding of the 5T4 antibody was analyzed using HEK-293 cells transiently transfected with human 5T4 (left panel) or cynomolgus monkey 5T4 (right panel). Cells were incubated with a concentration series of antibodies 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-FEAL×5T4-207-FEAR and IgG1-5T4-207-FEAR. B. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR and IgG1-5T4-226-FEAR. C. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-059-FEAR and IgG1-5T4-059-FEAR. D. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-106-FEAR and IgG1-5T4-106-FEAR. E. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-076-FEAR and IgG1-5T4-076-FEAR. F. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-085-FEAR and IgG1-5T4-085-FEAR. G. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-127-FEAR and IgG1-5T4-127-FEAR. H. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR and IgG1-5T4-A1-FEAR. I. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR and IgG1-5T4-A3-FEAR. [Figure 5(II)-2] See the description of Figure 5(II)-1. [Figure 6(I)]Binding of CD3 × 5T4 bispecific antibody and 5T4 monospecific antibody to 5T4-positive human tumor cells. Binding of monovalent and bivalent 5T4 antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel) was determined by flow cytometry. Cells were incubated with a range of antibody concentrations. MFI was determined by flow cytometry after secondary labeling with FITC-conjugated goat anti-human IgG F(ab')2. A. Binding of bsIgG1-huCD3-H101G-FEAL × 5T4-207-FEAR antibody and IgG1-5T4-207-FEAR antibody to HeLa cells (left panel) or MDA-MB-231 cells (right panel). B. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-059-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-FEAL×5T4-226-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 circle). [Figure 6(II)]Binding of CD3×5T4 bispecific antibody and 5T4 monospecific antibody to HeLa cells. Monovalent and bivalent binding of 5T4 antibody to HeLa cells was determined by flow cytometry. Cells were incubated with a range of antibody concentrations. After secondary labeling with phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2, mean fluorescence intensity (MFI) was determined by flow cytometry. A. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR and IgG1-5T4-207-FEAR. B. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR and IgG1-5T4-226-FEAR. C. Binding of bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR and IgG1-5T4-059-FEAR. D. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-106-FEAR and IgG1-5T4-106-FEAR. E. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-085-FEAR and IgG1-5T4-085-FEAR. F. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-127-FEAR and IgG1-5T4-127-FEAR. G. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-A1-FEAR and IgG1-5T4-A1-FEAR. H. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-A3-FEAR and IgG1-5T4-A3-FEAR. [Figure 6(III)]Binding of CD3 × 5T4 bispecific antibody and 5T4 monospecific antibody to MDA-MB-231 cells. Monovalent and bivalent binding of 5T4 antibody to MDA-MB-231 cells was determined by flow cytometry. Cells were incubated with a range of antibody concentrations. After secondary labeling with PE-conjugated goat anti-human IgG F(ab')2, mean fluorescence intensity (MFI) was determined by flow cytometry. A. Binding of bsIgG1-huCD3-H101G-FEAL × 5T4-207-FEAR and IgG1-5T4-207-FEAR. B. Binding of bsIgG1-huCD3-H101G-FEAL × 5T4-226-FEAR and IgG1-5T4-226-FEAR. C. Binding of bsIgG1-huCD3-H101G-FEAL × 5T4-059-FEAR and IgG1-5T4-059-FEAR. D. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-106-FEAR and IgG1-5T4-106-FEAR. E. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-085-FEAR and IgG1-5T4-085-FEAR. F. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-127-FEAR and IgG1-5T4-127-FEAR. G. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-A1-FEAR and IgG1-5T4-A1-FEAR. H. Binding of bsIgG1-huCD3-H101G-FEAL x 5T4-A3-FEAR and IgG1-5T4-A3-FEAR. [Figure 7(I)]In vitro cytotoxicity induction by CD3x5T4 bispecific antibody in MDA-MB-231 cells using purified T cells as effector cells. MDA-MB-231 cells were incubated with a range of concentrations of CD3x5T4 bispecific antibody or monospecific bivalent 5T4 antibody and isolated T cells as effector cells at an effector:target cell (E:T) ratio of 8:1. Purified T cells from two different donors, Donor A (left panel) and Donor B (right panel), were used for this experiment. Cytotoxicity was determined by measuring the percentage of viable MDA-MB-231 cells after 72 hours of incubation (% viable cells = [absorbance of sample - absorbance of staurosporine-treated target cells] / [absorbance of untreated target cells - target cells of staurosporine-treated target cells] × 100). A. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, and IgG1-5T4-207-FEAR. B. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, and IgG1-5T4-226-FEAR. C. Cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, and IgG1-5T4-059-FEAR. [Figure 7(II)]IC50 values ​​of in vitro cytotoxicity induced by CD3×5T4 bispecific antibodies in MDA-MB-231 cells using purified T cells as effector cells. The IC50 values ​​of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, or bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR in MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are expressed as the mean IC50 value ±SD of two different donors. [Figure 8(I)]Induction of cytotoxicity by CD3x5T4 bispecific antibody in MDA-MB-231 cells in vitro using T cells as effector cells. MDA-MB-231 cells were incubated with a range of concentrations of 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 in this experiment. Data shown are the mean survival rate (%) ± standard error of the mean (SEM) for the three donors tested. A. T cell-mediated cytotoxicity (reduced viability) induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, and IgG1-5T4-207-FEAR. B. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, and IgG1-5T4-226-FEAR. C. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, and IgG1-5T4-059-FEAR. D. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, and IgG1-5T4-106-FEAR. E. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and IgG1-5T4-A1-FEAR. F. T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEAL×5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR, and IgG1-5T4-A3-FEAR. [Figure 8(II)]IC50 values ​​of cytotoxicity induced by CD3x5T4 bispecific antibody in MDA-MB-231 cells in vitro using T cells as effector cells. 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 presented as the mean IC50 values ​​± SD of three different donors. A. IC50 values ​​of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-FEAL×5T4-A3-FEAR. B. IC50 values ​​for T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR. [Figure 9(I)-1]In vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. MDA-MB-231 cells were incubated with a range of concentrations of CD3x5T4 bispecific antibody and monospecific bivalent 5T4 antibody, as indicated, at an E:T ratio of 8:1 with isolated T cells as effector cells. Expression of three T cell activation markers (PD1 [top panel], CD25 [middle panel], and CD69 [bottom panel]) was analyzed by flow cytometry. Two different donors, Donor A (black symbols) and Donor B (white symbols), were used in this experiment. 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-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, and IgG1-5T4-226-FEAR. C. T cell activation induced in the presence of bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, and IgG1-5T4-059-FEAR. [Figure 9(I)-2] See the description of Figure 9(I)-1. [Figure 9(II)]EC50 values ​​for in vitro T cell activation by CD3×5T4 bispecific antibodies in the presence of MDA-MB-231 cells. The EC50 values ​​for in vitro T cell activation markers (PD1, CD25, and CD69) induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, or bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR in the presence of MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are expressed as the mean ± SD of two different donors. [Figure 10(I)-1]In vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. MDA-MB-231 cells were incubated with a range of concentrations of CD3x5T4 bispecific antibody and 5T4 homodimer, and isolated T cells as effector cells, at an E:T ratio of 8:1. T cell activation was measured by the increase in the percentage of CD69+ cells within the CD4+ T cell population (left panel) and CD8+ T cell population (right panel). Three different donors were used in this experiment. Data shown are the mean CD69 upregulation percentage ± SEM for 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-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, and IgG1-5T4-226-FEAR. C. T cell activation induced in the presence of bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, and IgG1-5T4-059-FEAR. D. T cell activation induced in the presence of bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, and IgG1-5T4-106-FEAR. E. T cell activation induced in the presence of bsIgG1-huCD3-FEAL×5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and IgG1-5T4-A1-FEAR. F. T cell activation induced in the presence of bsIgG1-huCD3-FEAL×5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR, and IgG1-5T4-A3-FEAR. [Figure 10(I)-2] See the description of Figure 10(I)-1. [Figure 10(II)]EC50 values ​​for in vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. EC50 values ​​for T cell activation markers (% increase in CD69+ cells [A-B], CD25+ cells [C-D], and PD1+ cells [E-F], CD25 and CD69 cells in the CD4+ T cell population and in the CD8+ T cell population) induced in vitro by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are presented as the mean ± SD of three different donors. A. EC50 values ​​for CD69 upregulation induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-FEAL×5T4-A3-FEAR. B. EC50 values ​​for CD69 upregulation induced by bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR. C. EC50 values ​​for CD25 upregulation induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-FEAL×5T4-A3-FEAR.D. EC50 values ​​for CD25 upregulation induced by bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR. E. EC50 values ​​for PD1 upregulation induced by bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-FEAL×5T4-059-FEAR, bsIgG1-huCD3-FEAL×5T4-106-FEAR, bsIgG1-huCD3-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-FEAL×5T4-A3-FEAR. F. EC50 values ​​for PD1 upregulation induced by bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-059-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-106-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-A1-FEAR, and bsIgG1-huCD3-H101G-FEAL×5T4-A3-FEAR. [Figure 11]T cell cytokine release induced by CD3×5T4 bispecific antibodies in the presence of 5T4-positive tumor cells. MDA-MB-231 cells were incubated with 0.2 μg / mL of CD3×5T4 bispecific antibodies (bsIgG1-huCD3-FEAL×5T4-207-FEAR, bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR, bsIgG1-huCD3-FEAL×5T4-226-FEAR, bsIgG1-huCD3-H101G ... A. Tumor cell cocultures were incubated with 5T4 monospecific antibodies (IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, or IgG1-5T4-059-FEAR) at an E:T ratio of 8:1. Cytokine release was analyzed by U-PLEX assay. B. Concentrations of IL-10, IL-13, and TNF in the supernatant of T cell (from donor A)-tumor cell cocultures after 72 hours of incubation with CD3×5T4 bispecific antibody or 5T4 monospecific antibody. B. Concentrations of IL-10, IL-13 and TNF in the supernatants of T cell (from donor B)-tumor cell cocultures after 72 hours of incubation with CD3x5T4 bispecific antibody or 5T4 monospecific antibody. [Figure 12]In vitro cytotoxicity induction by CD3×5T4 bispecific antibody in SK-OV-3 cells using PBMCs as effector cells at various E:T ratios. SK-OV-3 cells were incubated with a range of concentrations of bsIgG1-huCD3-FEAL×5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR (right panel) and PBMCs 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] × 100). PBMCs from two different donors were used: A. Donor C, and B. Donor D. [Figure 13] In vitro cytotoxicity of SK-OV-3 cells by the CD3×5T4 bispecific antibody was induced using effector T cells at various E:T ratios. SK-OV-3 cells were incubated with a range of concentrations of bsIgG1-huCD3-FEAL×5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEAL×5T4-207-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. The efficiency of 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] × 100). T cells from two different donors were used: A. Donor E, and B. Donor F. [Figure 14]Anti-tumor activity of CD3×5T4 bispecific antibodies in an MDA-MB-231 xenograft model in NSG-HIS mice. A. Mean tumor size in an MDA-MB-231 xenograft model in NSG-HIS mice after treatment with PBS (vehicle control), 0.5 mg / kg bsIgG1-huCD3-FEAL×5T4-207-FEAR, or 0.5 mg / kg bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR. Tumor size was assessed by caliper measurement. Error bars indicate SEM. B. Percentage of NSG-HIS mice injected with MDA-MB-231 cells with a tumor size <500 mm3 after treatment with PBS, bsIgG1-huCD3-FEAL×5T4-207-FEAR, or bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR. [Figure 15A]Binding of directly FITC-labeled 5T4-specific antibodies to human 5T4 variants with single alanine mutations at positions 32–355 of the human 5T4 ECD was determined by flow cytometry. Binding was expressed as a Z-score (fold change) as a measure of the change in binding compared to the non-cross-blocking 5T4-specific control antibody (bsIgG1-5T4-A1-F405L×b12-FEAR-FITC) used for normalization. Numbers on the x-axis represent amino acid positions in human 5T4 (SEQ ID: 1). Residues with binding Z-scores below −1.5 (shown by the dotted line) were considered "loss of binding mutants." Residues with positive binding Z-scores are loss of binding residues relative to the non-cross-blocking 5T4-specific control antibody (bsIgG1-5T4-A1-67F-F405L×b12-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 were not evaluated because these positions contained either endogenous alanines or endogenous cysteines. Data shown are Z-scores for binding of (A) bsIgG1-b12-FEAL×5T4-059-FEAR-FITC, (B) bsIgG1-b12-FEAL×5T4-207-FEAR-FITC, (C) bsIgG1-b12-FEAL×5T4-226-FEAR-FITC, and (D) bsIgG1-5T4-A3-F405L×b12-FEAR-FITC.Buried residues with a Z-score just below -1.5, predicted to be spatially distant from most surface-exposed binding-loss residues, were excluded (L281 [Z-score: -1.57] and P326 [Z-score: -1.54] for bIgG1-b12-FEALx5T4-207-FEAR-FITC, and 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] for bsIgG1-b12-FEALx5T4-226-FEAR-FITC). [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 16(I)] In vitro cytotoxicity induction by the CD3x5T4 bispecific antibody in tumor cells of different indications using T cells as effector cells. Tumor cells were incubated with a 4:1 E:T ratio of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) at a concentration range and isolated T cells as effector cells. Cytotoxicity (reduced viability) was determined by measuring the percentage of viable tumor cells after 72 hours of incubation. Data shown are the mean viability (%) ± SEM of duplicate wells from one representative donor out of at least three donors tested. A. Cytotoxicity (reduced viability) induced in a pancreatic cancer cell line; B. Cytotoxicity (reduced viability) induced in a cervical cancer cell line. [Figure 16(II)]IC50 values ​​for 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 ​​for 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. Data are presented as the mean IC50 value ± SD of at least three different donors (see Table 10). [Figure 17(I)] In vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of tumor cells of different indications. Tumor cells were incubated with a range of 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 a 4:1 E:T ratio for 72 hours. T cell activation was measured by CD69 upregulation (% of CD69+ cells) in the CD4+ T cell population (left panel) and CD8+ T cell population (right panel). Data shown are the mean % CD69+ cells ± SD of duplicate wells from one representative donor of at least three donors tested. A. T cell activation induced by CD3x5T4 bispecific antibody in the presence of pancreatic cancer cell line BxPc-3. B. T cell activation induced by CD3x5T4 bispecific antibody in the presence of pancreatic cancer cell line PANC-1. C. T cell activation induced by CD3x5T4 bispecific antibody in the presence of cervical cancer cell line SiHa. D. T cell activation induced by CD3x5T4 bispecific antibody in the presence of cervical cancer cell line CaSki. [Figure 17(II)]EC50 values ​​for in vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of differently adapted tumor cell lines. EC50 values ​​for T cell activation (% of CD69+ cells in the CD4+ T cell population and % of CD8+ T cell population) induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in coculture with differently adapted tumor cell lines were analyzed using GraphPad Prism V7.02 software. Data are presented as the mean EC50 value ± SD for at least three different donors (see Table 10). A. EC50 values ​​for CD4+ T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of the indicated tumor cell lines. B. EC50 values ​​for CD8+ T cell activation induced by bsIgG1-huCD3-H101G-FEAL×5T4-207-FEAR in the presence of the indicated tumor cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description definition As used herein, the term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that is capable of specifically binding to an antigen under typical physiological and / or tumor-specific conditions for a meaningful period of time, such as a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3, 4, 5, 6, or 7 days or more, or other suitable functionally defined period of time (e.g., a period sufficient to induce, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period sufficient for the antibody to be internalized). The binding region that interacts with the antigen (sometimes referred to herein as a binding domain, both terms have the same meaning) comprises the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant regions of antibodies (Abs) may mediate the binding of immunoglobulins to host tissues or host factors, such as various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation.

[0022] In the context of the present invention, the term "antibody" includes monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, as well as "antibody fragments" or "fragments thereof" produced by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques, which retain the ability to specifically bind to an antigen (antigen-binding fragment) and be conjugated to a toxin. Antibodies as defined in accordance with the present invention can have any isotype, except where the disclosure herein is otherwise limited.

[0023] As indicated above, the term antibody as used herein includes antibody fragments that retain the ability to specifically interact with, e.g., bind to, an antigen, unless otherwise stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include: (i) Fab' fragments or Fab fragments, i.e., monovalent fragments consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL) and domain 1 (CH1) of the heavy chain constant region, or the monovalent antibodies described in WO2007 / 059782; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments consisting essentially of a VH domain and a CH1 domain; (iv) Fv fragments consisting essentially of a VL domain and a VH domain of a single arm of an antibody; (v) dAb fragments, see Ward et al., Nature 341 ,544-546(1989), which essentially consists of a VH domain and is also called a domain antibody, Holt et al;Trends Biotechnol.2003 Nov; 21 (11):484-90, (vi) Camel antibodies or nanobodies, Revets et al;Expert Opin Biol Ther.2005 Jan; 5 (1):111-24, and (vii) isolated complementarity-determining regions (CDRs). Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they may be joined using recombinant techniques by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)). See, e.g., Revets et al., Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24 and Bird et al., Science 242, 423-426 (1988). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or otherwise apparent from the context. While such fragments are generally encompassed within the meaning of antibody, they collectively and each independently represent unique features of the present invention and exhibit different biological properties and utilities. These and other useful antibody fragments in the context of the present invention are discussed further herein.

[0024] Antibodies can be produced in a variety of in vitro or ex vivo expression or production systems, and harvested therefrom, for example, from recombinantly modified host cells, from hybridomas, or from systems that use cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody. It should be understood that a number of different antibodies, as defined in the context of the present invention, can be provided by producing each antibody separately in the production system described above and then mixing the antibodies, or by producing several antibodies in the same production system.

[0025] As used herein, the term "immunoglobulin heavy chain" or "immunoglobulin heavy chain" refers to one of the heavy chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. As used herein, the term "immunoglobulin" refers to a group of structurally related glycoproteins consisting of two pairs of polypeptide chains: one pair of light (L) low-molecular-weight chains and one pair of heavy (H) chains, all four of which are potentially interconnected 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, NY (1989))). In the immunoglobulin structure, two heavy chains are interconnected by disulfide bonds in the so-called "hinge region." Like heavy chains, each light chain typically consists of several regions, such as a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (i.e., hypervariable regions that are hypervariable in sequence and / or can form structurally distinct loops), also called complementarity-determining regions (CDRs), interspersed with highly conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy 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)).

[0026] As used herein, the terms "half molecule," "Fab arm," and "arm" refer to one heavy-light chain pair. When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was produced by recombining half molecules from each of the first and second antibodies by any known technique, resulting in a bispecific antibody. In this context, "recombining" is not intended to be limited to any particular recombination method and thus encompasses all methods of producing bispecific antibodies described herein below, including recombination by, for example, half molecule exchange, as well as recombination at the nucleic acid level and / or by co-expression of two half molecules in the same cell.

[0027] As used herein, the term "antigen-binding region" or "binding region" refers to the region of an antibody that is capable of binding to an antigen. An antigen can be any molecule, such as a polypeptide, present on a cell, bacterium, or virion, for example. The terms "antigen" and "target" may be used interchangeably in the context of the present invention, except where the context conflicts. The terms "antigen-binding region" and "antigen-binding site" may be used interchangeably in the context of the present invention, except where the context conflicts.

[0028] The terms "blocking binding" or "blocking antibody binding" or "cross-blocking binding" or "cross-blocking binding" refer to the situation where an antibody bound to a specific antigen prevents a second antibody from binding to the same antigen, and vice versa. In the absence of the other antibody, each antibody has antigen-binding ability as determined by a significant binding response, whereas in the presence of the other antibody, one of the antibodies lacks a binding response. The ability of one antibody to block the binding of another antibody can be determined by biolayer interferometry in a classic sandwich epitope binning assay format, for example, as described in Example 3 of the present application and by Abdiche et al. (Abdiche YN, Malashock DS, Pinkerton A, Pons J. "Exploring blocking assays using Octet, ProteOn, and Biacore biosensors," Anal Biochem. 2009;386(2):172-180). Briefly, in a sandwich epitope binning assay, an antibody in solution is tested for binding to its specific antigen, which is first captured by an immobilized antibody. In the context of the present invention, if an antibody can "displace" another antibody, according to the definition of "displacement" below, the antibody does not block the binding of the other antibody. The terms "blocking binding," "blocking antibody binding," "cross-blocking binding," and "cross-blocking binding" can be used interchangeably in the context of the present invention, unless the context dictates otherwise. Preferably, the ability of one antibody to block the binding of another antibody is determined using a full-length antibody.

[0029] "Displacement" or "ability to displace" or "displacing" refers to a situation in which two antibodies disrupt each other's binding to their specific antigens by changing their binding kinetics through the formation of a transient trimolecular complex (which quickly disintegrates by retaining one antibody on the antigen and displacing the other antibody). 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). Antibody displacement can be determined by biolayer interferometry using real-time label-free biosensors in a classical sandwich assay format, as described in Abdiche et al. 2017 and herein in Example 4. Preferably, antibody displacement is determined using antibodies in IgG format.

[0030] As used herein, the term "binding" refers to the ability of a given antibody or target to bind to a predetermined antigen or target, typically within 1E s, as determined by biolayer interferometry using an antibody as the ligand and an antigen as the analyte. -6 M or less, for example, 5E -7 M or less, 1E -7 M or less, for example, 5E -8 M or less, for example, 1E -8 M or less, for example, 5E -9 M or less, or for example 1E -9 K below M Dand refers to the binding of an antibody with a binding affinity corresponding to a K for a predetermined antigen that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, such as at least 100,000-fold lower, for example, at least 100,000-fold lower, than its affinity for binding to a non-specific antigen (e.g., BSA, casein) that is neither the predetermined antigen nor a closely related antigen. D with an affinity corresponding to

[0031] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, k d k a It is obtained by dividing by

[0032] As used herein, "k d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off Also called value or off-rate.

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

[0034] As used herein, the term "5T4" refers to the protein designated 5T4, which is also known as trophoblast glycoprotein, 5T4 oncofetal antigen, 5T4 oncofetal trophoblast glycoprotein, TPBG, WAIF1, and M6P1. It is a 72-80 kDa transmembrane protein with an extensively N-linked glycosylated core. In humans (Homo sapiens), the 5T4 protein has the amino acid sequence set forth in SEQ ID NO:1 (human trophoblast glycoprotein; Uniprot accession number Q13641). In the amino acid sequence set forth 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 set forth in SEQ ID NO:2 (Uniprot accession number Q4R8Y9). In the amino acid sequence set forth 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 chicken (Gallus gallus), the 5T4 protein has the amino acid sequence set forth in SEQ ID NO:3 (Uniprot accession number R4GM46). In the sequence set forth in SEQ ID NO:3, amino acid residues 1-27 are the signal peptide, and amino acid residues 28-379 are the mature polypeptide.

[0035] As used herein, the term "CD3" refers to the human Cluster of Differentiation (CD) 3 protein, which is part of the T cell coreceptor protein complex and is composed of four distinct chains. Because CD3 is also found in other species, the term "CD3" is not limited to human CD3 unless the context contradicts it. In mammals, this complex comprises a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No. P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No. Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No. P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No. Q95LI8), and two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No. P07766; amino acid residues 1-22 are a signal peptide, and amino acid residues 23-207 are the mature CD3ε polypeptide identified herein as SEQ ID NO:4; cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No. Q95LI5; or rhesus monkey CD3ε The CD3 gene contains the CD3 ζ-chain (Human CD3ζ UniProtKB / Swiss-Prot No. G7NCB9), and the CD3 ζ-chain (Zeta) chain (Human CD3ζ UniProtKB / Swiss-Prot No. P20963, Cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No. Q09TK0). These chains associate with a molecule known as the T cell receptor (TCR) to generate an activation signal in T lymphocytes. The TCR molecule and the CD3 molecule together constitute the TCR complex.

[0036] 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 binning using biolayer interferometry, alanine scanning, or shuffle assays (using antigen constructs in which regions of the antigen have been exchanged with those of another species and determining whether the antibody still binds to the antigen). The amino acids within the antibody binding region that are involved in interactions with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and crystal structure analysis of the antibody bound to the antigen.

[0037] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface arrangements of molecules, such as amino acids, sugar side chains, or a combination thereof, and usually have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, whereas the binding to the latter is not lost in the presence of denaturing solvents. An epitope may 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 an antibody when it binds to an antigen (in other words, amino acid residues that are within or in close proximity to the footprint of a specific antibody).

[0038] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having 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 transchromosomal nonhuman animals, such as transgenic mice, whose genomes contain human heavy chain and light chain transgenes, fused to immortalized cells. Monoclonal antibodies can also be produced from recombinantly engineered host cells or systems using cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

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

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

[0041] As used herein, the term "human antibody" is intended to include antibodies having variable 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 random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other non-human species, such as mouse, have been grafted onto human framework sequences.

[0042] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which collectively form the antigenic determinant, into homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to substitute framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (backmutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions that may contain one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with favorable characteristics such as affinity and biochemical properties.

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

[0044] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 in the EU numbering system described in Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - U.S. Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991). However, the hinge region may be of any of the other subtypes described herein.

[0045] 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 in the Eu numbering system set forth in Kabat (ibid.). However, the CH1 region may be of any of the other subtypes described herein.

[0046] 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 in the EU numbering system set forth in Kabat (ibid.). However, the CH2 region may be of any of the other subtypes described herein.

[0047] 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 in the Eu numbering system set forth in Kabat (ibid.). However, the CH3 region may be of any of the other subtypes described herein.

[0048] As used herein, the term "Fc-mediated effector function" refers to a function resulting from the binding of a polypeptide or antibody to its target or antigen on a cell membrane, wherein the Fc-mediated effector function is attributable 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) antibody-mediated opsonization (binding of an antibody to a complement receptor), (xi) opsonization, and (xii) any combination of (i)-(xi).

[0049] As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is, at a minimum, unable to bind to any FcγR, unable to induce Fc-mediated cross-linking of FcγRs, or unable to induce FcγR-mediated cross-linking of target antigens by the two Fc regions of individual antibodies, or unable to bind C1q. The inactivity of an antibody Fc region can be tested using antibodies in monospecific or bispecific formats.

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

[0051] 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 using 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 the bispecific antibody to one specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

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

[0053] The term "bispecific antibody" refers to an antibody having two non-identical antigen-binding domains, such as two non-identical Fab arms or two Fab arms with non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may be on the same antigen or target or on different antigens or targets. When the epitopes are on different antigens, such antigens may be present on the same or different cells, cell types, or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, a bispecific antibody may be able to cross-link multiple antigens, for example, two different cells.

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

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

[0056] Main classification based on the structure and general chemical characterization of R TIFF2026041981000007.tif52128

[0057] Alternative physical and functional classifications of amino acid residues TIFF2026041981000008.tif88128

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

[0059] In the context of the present invention, substitutions in antibodies are indicated as follows: Original amino acid - position - replacement amino acid

[0060] The well-known amino acid nomenclature uses three-letter or one-letter codes, including the symbols "Xaa" or "X" to represent any amino acid residue. Thus, Xaa or X can refer to any 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 designation "K409R" or "Lys409Arg" means that the antibody contains a substitution of lysine with arginine at amino acid position 409.

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

[0062] In cases where the original and / or replacement amino acids may include two or more, but not all, amino acids, the two or more amino acids may be separated by "," or " / ." For example, a substitution of lysine with arginine, alanine, or phenylalanine at position 409 may be "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409→R, A or F" is.

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

[0064] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids, or with other amino acids, such as unnatural amino acids. For example, 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. This is equivalent to the designation 409X, where X designates any amino acid except the original amino acid. These substitutions may also be designated K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. This applies equally to any position referred to herein, and all such substitutions are specifically encompassed herein.

[0065] Antibodies of the invention may also include deleted amino acid residues. Such deletions may be designated as "del," and include, for example, the designation K409del. Thus, in such embodiments, lysine at position 409 has been deleted from the amino acid sequence.

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

[0067] The term "transfectoma," as used herein, includes recombinant eukaryotic host cells, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells, that express an antibody or target antigen.

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

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

[0070] As used herein, the term "internalized" or "internalization" refers to the biological process by which a molecule, such as an antibody of the present invention, is engulfed by a cell and drawn into the interior of the cell. Internalization can also be referred to as "endocytosis."

[0071] antibody In a first aspect, the present invention provides an antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), a) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO:5 and a VL region comprising the sequence shown in SEQ ID NO:9; b) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 12 and a VL region comprising the sequence shown in SEQ ID NO: 16; c) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 19 and a VL region comprising the sequence shown in SEQ ID NO: 23; d) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 26 and a VL region comprising the sequence shown in SEQ ID NO: 30

[0106] ; e) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 33 and a VL region comprising the sequence shown in SEQ ID NO: 37

[0127] ; f) an antibody

[0207] comprising a VH region comprising the sequence set forth in SEQ ID NO: 40 and a VL region comprising the sequence set forth in SEQ ID NO: 44; and g) an antibody comprising a VH region comprising the sequence set forth in SEQ ID NO: 47 and a VL region comprising the sequence set forth in SEQ ID NO: 51; The present invention provides an antibody capable of blocking the binding to 5T4 of an antibody selected from the group consisting of:

[0072] In particular, the present invention includes antibodies comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), and capable of blocking the binding of an antibody

[0059] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:9 to 5T4.

[0073] The antibody is, in particular, a) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 44

[0207] ; b) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 51

[0226] ; and An antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:9. The present invention can block the binding of an antibody selected from the group consisting of:

[0074] In a particular embodiment of the invention, the antibody comprises: a) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 44

[0207] ; and b) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:51; The present invention can block the binding of an antibody selected from the group consisting of:

[0075] The antibodies of the present invention are characterized by having specificity for or the ability to bind to human 5T4. Thus, 5T4 as referred to herein may in particular be human 5T4, e.g., the mature polypeptide of SEQ ID NO:1.

[0076] In a further embodiment, the antibodies of the invention are characterized by having specificity for or the ability to bind to cynomolgus monkey 5T4, for example having specificity for or the ability to bind to both human 5T4 and cynomolgus monkey 5T4. Cynomolgus monkey 5T4 may in particular be the mature polypeptide of SEQ ID NO:2.

[0077] In further embodiments, the antibodies of the present invention have specificity for or the ability to bind to chicken 5T4, e.g., specificity for or the ability to bind to both human 5T4 and chicken 5T4, or specificity for or the ability to bind to, e.g., human, cynomolgus monkey and chicken 5T4, wherein chicken 5T4 may, in particular, have the amino acids of the mature polypeptide of SEQ ID NO:3.

[0078] Thus, the antibodies of the present invention may have specificity or binding ability for human 5T4, eg, the mature polypeptide of SEQ ID NO:1, and cynomolgus 5T4, eg, the mature polypeptide of SEQ ID NO:2.

[0079] Furthermore, the antibodies of the present invention may have specificity or binding ability for human 5T4, e.g., the mature polypeptide of SEQ ID NO:1, cynomolgus monkey 5T4, e.g., the mature polypeptide of SEQ ID NO:2, and chicken 5T4, e.g., the mature polypeptide of SEQ ID NO:3.

[0080] The antibodies of the present invention have a K of 1E-7M or less to human 5T4, cynomolgus monkey 5T4 and / or chicken 5T4. D a K value, for example about 1E-7M or less, 5E-8M or less, about 5E-8M or less, 1E-8M or less, about 1E-8M or less, 5E-9M or less, about 5E-9M or less, for example 1E-9M or less, or for example about 1E-9M or less Dand a binding affinity corresponding to a K value, for example, in the range of 1E-7 to 5E-10M, for example, in the range of about 1E-7 to about 5E-10M, for example, 1E-7 to 1E-9M, for example, about 1E-7 to about 1E-9M, for example, 5E-8 to 5E-10M, for example, about 5E-8 to about 5E-10M, for example, 5E-8 to 1E-9M, for example, about 5E-8 to about 1E-9M, for example, 1E-8 to 5E-10M, for example, 1E-8 to 1E-9M, for example, about 1E-8 to about 5E-10M, for example, 1E-8 to 1E-9M, for example, 1E-8 to 5E-9M, or for example, in the range of about 1E-8 to about 5E-9M. D The antibody can bind with a binding affinity corresponding to the value.

[0081] While it is within the ability of one skilled in the art to determine the affinity with which an antibody binds to its target, the binding affinity of the antibodies of the invention to 5T4 may in particular be determined by biolayer interferometry, optionally as shown in Example 2 herein.

[0082] More specifically, the binding affinity of an antibody of the present invention can be determined using a procedure, such as a biolayer interferometry procedure, which comprises the following steps: I) immobilizing the antibody on an anti-human IgG Fc capture biosensor at 1 μg / mL for 600 seconds; II) determining the association of 5T4ECDHis (mature protein of SEQ ID NO:99) or cynomolgus 5T4 (mature protein of SEQ ID NO:2) or recombinant cynomolgus 5T4 protein (Cusabio, Cat. No. CSB-MP024093MOV) over a period of 200 seconds and the dissociation over a period of 1000 seconds using a two-fold dilution series ranging from 100 nM to 1.56 nM; III) Relating the data to a buffer control (0 nM).

[0083] The binding affinity of an antibody of the invention can in particular be determined using an antibody according to any one of the preceding claims which is a monospecific bivalent antibody, such as an antibody which is a full-length IgG1.

[0084] In a further embodiment of the invention, the antibody recognises or binds to an epitope or antibody binding region or binding site on 5T4, said binding site or epitope or antibody binding region comprising: a) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO:5 and a VL region comprising the sequence shown in SEQ ID NO:9; b) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 12 and a VL region comprising the sequence shown in SEQ ID NO: 16; c) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO:19 and a VL region comprising the sequence shown in SEQ ID NO:23

[0085] , d) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 26 and a VL region comprising the sequence shown in SEQ ID NO: 30

[0106] ; e) an antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 33 and a VL region comprising the sequence shown in SEQ ID NO: 37

[0127] ; f) an antibody

[0207] comprising a VH region comprising the sequence set forth in SEQ ID NO: 40 and a VL region comprising the sequence set forth in SEQ ID NO: 44; and g) an antibody comprising a VH region comprising the sequence set forth in SEQ ID NO: 47 and a VL region comprising the sequence set forth in SEQ ID NO: 51; The antibody is recognized by any one of the antibodies selected from the group consisting of:

[0085] In a further embodiment, the antibody of the invention a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO: 86. is not an antibody binding region, binding site or epitope bound by an antibody selected from the group consisting of: a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO: 86. an antibody binding region, binding site, or epitope on 5T4 that is different from the antibody binding region, binding site, or epitope bound by an antibody selected from the group consisting of: recognize or bind to

[0086] In another embodiment, the binding of an antibody of the invention to 5T4 is blocked by the binding of antibody [A3] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:86 to 5T4. The antibody comprising the VH and VL sequences set forth in SEQ ID NO:85 and SEQ ID NO:86, respectively, is antibody A3, one of three murine 5T4 antibodies disclosed in WO2007106744. In other words: antibody A3 with a single aa substitution. In the CDR sequences?

[0087] In yet another embodiment, an antibody of the invention exhibits displacement of antibody [A3] binding to 5T4 or a His-tagged extracellular domain of 5T4 (e.g., 5T4ECDHis / mature protein of SEQ ID NO:99) comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:86. This displacement behavior indicates that the antibody of the invention binds to an epitope that is distinct from, but likely adjacent to, or overlapping with, the epitope bound by antibody A3.

[0088] "Displacement" or the ability to displace bound antibody can be determined in a biolayer interferometry assay, for example, in an assay performed as described in Example 4 of the present application.

[0089] "Cross-blocking," or the ability of an antibody defined according to the present invention to block the binding of another antibody to 5T4, can be determined using a fluorescence-activated cell sorting (FACS) assay, for example in an assay performed as described in Example 5.

[0090] In particular, "cross-blocking," or the ability of an antibody of the invention to block the binding of another antibody to 5T4, is determined as the ability of an unconjugated antibody to block the binding of a conjugated antibody, and optionally, 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, the antibody being conjugated to fluorescein isothiocyanate (FITC), and an excess of an unconjugated antibody that targets 5T4; ii) incubating the sample at 4°C for 30 minutes and then centrifuging the sample; iii) removing the supernatant from each sample, resuspending the cells in buffer, and determining the mean fluorescence intensity (MFI) of FITC using a flow cytometer; and iv) The percentage of binding is as follows: The difference in MFI between cells incubated with a mixture of FITC-conjugated and unconjugated antibodies and cells incubated without FITC-conjugated or unconjugated antibodies is multiplied by 100 and then divided by the difference in MFI between cells incubated with a mixture of FITC-conjugated and IgG-b12 antibodies and cells incubated without FITC-conjugated or unconjugated antibodies. The process of calculating The procedure includes:

[0091] While one of skill in the art would be familiar with suitable techniques for determining the ability of an antibody to block the binding of another antibody to its target or to displace another antibody from binding to its target, the present application discloses suitable procedures for determining blocking and displacement of binding. Thus, in some embodiments, the ability of an antibody of the invention to block the binding of another antibody to 5T4 or to displace another antibody from binding to 5T4 can be determined using biolayer interferometry, e.g., biolayer interferometry performed as described in Example 3.

[0092] In particular, the ability of an antibody of the invention to block the binding of another antibody to 5T4 or to displace another antibody from binding to 5T4 can be determined using biolayer interferometry. i) The antibody of the present invention was applied to an activated amine-reactive second generation biosensor (activated Amine-Reactive 2 nd a step of immobilizing the sample on a biosensor (Generation biosensor); ii) quenching the biosensor with immobilized antibody in ethanolamine pH 8.5; iii) immersing the biosensor with immobilized antibody in a composition comprising 3.6 μg / mL (100 nM) human 5T4 ECDHis (mature protein of SEQ ID NO:99) for a period of 500 seconds, and then iv) immersing the biosensor with the immobilized antibody and 5T4ECDHis in a composition containing the other antibody targeting 5T4 at 10 μg / mL for a period of 500 seconds and determining the association response. The procedure can be determined by the following procedure, which includes steps i) to iv) being carried out at a temperature of 30°C while shaking at 1000 rpm.

[0093] The antibodies provided herein may bind to an epitope or antibody binding region on human 5T4 that includes amino acid residues R73, Y92, and R94, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1.

[0094] Also provided herein are antibodies that bind to an epitope or antibody binding region on human 5T4 that includes amino acid residues S69, R73, Y92, and R94, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1.

[0095] Further provided herein are antibodies that bind to an epitope or antibody binding region on human 5T4 comprising amino acid residues R73, T74, Y92, R94 and N95, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1.

[0096] Without wishing to be bound by theory, based on the results obtained in Example 16 herein, 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., by non-covalent interactions with amino acid residues within the CDR sequences of the antibody. This hypothesis is supported by the fact that these residues were identified as surface-exposed residues on the structure of 5T4 (4cnm; provided in the RCSB PDB Protein Data Bank; DOI: 10.2210 / pdb4CNM / pdb), as published in Zhao, Y., Malinauskas, T., Harlos, K., & Jones, EY (2014) "Structural insights into the inhibition of Wnt signaling by cancer antigen 5T4 / Wnt-activated inhibitory factor 1," Structure, 22(4), 612-620.

[0097] One or more of the additional amino acid residues L89, F111, L117, F138, L144, D148, and N152, with the numbering of each amino acid residue referring to their respective positions in SEQ ID NO: 1, may be involved in antibody binding, e.g., indirectly, by influencing protein folding and / or by influencing the positioning of one or more amino acid residues directly involved in antibody binding. In particular, L89, F111, L117, F138, and L144 have been identified as part of the hydrophobic core within 5T4, as described in Zhao et al., Structure, 22(4), 612-620.

[0098] Furthermore, the antibodies disclosed herein may bind to an epitope or antibody-binding region on human 5T4 in which amino acid residues R73, Y92, and R94 are directly involved in antibody binding and one or more of amino acid residues F111, F138, L144, and D148 are indirectly involved in said binding, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1.

[0099] The antibodies provided herein may bind to an epitope or antibody-binding region on human 5T4 in which amino acid residues S69, R73, Y92, and R94 are directly involved in binding of the antibody, and one or more of amino acid residues F111, F138, and D148 are indirectly involved in binding, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1.

[0100] The present disclosure also provides antibodies that bind to an epitope or antibody-binding region on human 5T4, where amino acid residues R73, T74, Y92, R94, and N95 are directly involved in antibody binding and amino acid residue F138 is indirectly involved in said binding, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1.

[0101] The amino acid residues contained in the epitope or antibody binding region, and optionally one or more additional amino acid residues indirectly involved in binding, can be identified by alanine scanning of human 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 to 355 of SEQ ID NO:1.

[0102] Alanine scanning may be performed specifically as shown in Example 16 herein, or essentially as shown in Example 16 herein.

[0103] Furthermore, alanine scanning i) expressing a mutant human 5T4 polypeptide, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO:1) are individually substituted with alanine, and a wild-type 5T4 polypeptide (amino acid residues 32-355 of SEQ ID NO:1), in human embryonic kidney cells, e.g., HEK293 cells, so as to obtain a sample containing 70 to 90,000 cells, e.g., 80,000 cells, for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, then washing each sample twice with 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 resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: TIFF2026041981000009.tif9128 (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using The method can be carried out by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: TIFF2026041981000010.tif8128 (where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively) is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. If the data are predicted to be spatially distant from

[0104] Suitable non-cross-blocking 5T4-specific control antibodies to be used in step iii) include: an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] and a bispecific antibody comprising:

[0105] The present invention provides antibodies that bind to 5T4 such that substitution of any one or more of amino acid residues R73, Y92 and R94, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1, with alanine results in loss of binding or reduced binding.

[0106] In particular, the antibody can bind to 5T4 such that loss of binding or reduced binding occurs when any one or more of amino acid residues S69, R73, Y92 and R94, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1, are substituted with alanine.

[0107] Furthermore, the antibody can bind to 5T4 such that substitution of any one or more of amino acid residues R73, T74, Y92, R94 and N95 with alanine, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1, results in loss of binding or reduced binding.

[0108] Additionally, the antibodies disclosed herein can bind to 5T4 such that substitution of any one or more of amino acid residues L89, F111, L117, F138, L144, D148, and N152 with alanine, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1, results in loss of binding or reduced binding.

[0109] Furthermore, the antibody may bind to 5T4 such that loss of binding or reduced binding occurs when any one or more of amino acid residues R73, Y92, R94, F111, F138, L144 and D148, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1, are substituted with alanine.

[0110] The antibody can bind to 5T4 such that substitution of any one or more of amino acid residues S69, R73, Y92, R94, F111, F138 and D148 with alanine, where the numbering of each amino acid residue refers to its respective position in SEQ ID NO:1, results in loss of binding or reduced binding.

[0111] In another embodiment, an antibody of the invention can bind to 5T4 such that loss of binding or reduced binding occurs when any one or more of amino acid residues R73, T74, Y92, R94, N95 and F138, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1, are substituted with alanine.

[0112] The effect of any of the above-described alanine substitutions can be determined by alanine scanning of a polypeptide comprising amino acid residues 32-355 of SEQ ID NO:1.

[0113] In particular, the effect of alanine substitution can be determined by the procedure set forth in, or essentially as set forth in, Example 16 herein.

[0114] Loss of binding is defined as a Z-score for binding less than 1.5, where the Z-score is optionally calculated as set forth in Example 16 herein or essentially as set forth in Example 16 herein.

[0115] The effect of alanine substitution was i) expressing mutant human 5T4 polypeptides, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32 to 355 of SEQ ID NO: 1) are individually substituted with alanine, and wild-type 5T4 polypeptides, in human embryonic kidney cells, e.g., HEK293 cells, so that a sample containing 70 to 90,000 cells, e.g., 80,000 cells, is obtained for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, then washing each sample twice with 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 resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: TIFF2026041981000011.tif9128 (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using can be determined by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: TIFF2026041981000012.tif8128 (where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively) is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. If the data are predicted to be spatially distant from

[0116] Suitable non-cross-blocking 5T4-specific control antibodies in step iii) of the above procedure include: an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] and a bispecific antibody comprising:

[0117] The antibodies of the present invention may be characterized by having reduced internalization potential, as indicated by reduced cytotoxicity, when conjugated to a cytotoxic moiety, compared to a similarly conjugated antibody [H8] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO: 87 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO: 88. An antibody comprising the VH and VL sequences set forth in SEQ ID NO: 87 and SEQ ID NO: 88, respectively, may be the murine 5T4 antibody mAb5T4, also referred to as the H8 antibody (Shaw et al. (2002), Biochem. J. 363:137-45, WO98 / 55607). Various chimeric or humanized versions of the antibody H8 are disclosed in WO06 / 031653.

[0118] The cytotoxicity or internalization of 5T4 antibodies that bind monovalently to 5T4 can be determined using the procedures set forth in Example 7 of the present application. In particular, cytotoxicity can be determined by: i) providing a toxin-conjugated bispecific antibody that binds monovalently to 5T4, said toxin-conjugated bispecific antibody comprising a first Fab arm of the antibody of 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; ii) incubating 5T4-positive breast cancer cells MDA-MB-468 (ATCC clone HTB-132) or HCC1954 (ATCC clone CRL-1338) with the bispecific antibody that monovalently binds to 5T4 at 37°C for 5 days; and iii) determining cell viability The concentration of the ATP can be determined in an assay comprising:

[0119] 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 variable region (VH) and light chain variable region (VL) are shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.

[0120] In certain embodiments, the antibodies of the invention comprise an antigen-binding region capable of binding to 5T4 comprising: a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50; and h) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of 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 set forth in any one of a) to g). The antibody comprises a heavy chain variable region (VH) selected from the group consisting of:

[0121] In another embodiment, the antibody of the present invention comprises an antigen-binding region capable of binding to 5T4 comprising: a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50

[0226] ; and d) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that contains a total of 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). The antibody comprises a heavy chain variable region (VH) selected from the group consisting of:

[0122] In particular, the antibody of the present invention may have an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH)

[0059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8.

[0123] Alternatively, the antibody of the present invention may be one in which the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH)

[0207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43.

[0124] Furthermore, the antibody of the present invention may have an antigen-binding region capable of binding to 5T4 that comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH)

[0226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50.

[0125] In another embodiment, the antibody of the present invention comprises an antigen-binding region capable of binding to 5T4 comprising: a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 17, 18 and 19, respectively; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 24, 25 and 26, respectively; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 31, 32 and 33, respectively; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 38, 39 and 39, respectively; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 45, DAS and SEQ ID NO: 46, respectively; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 52, 53 and 54, respectively; and h) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of 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 set forth in any one of a) to g). The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

[0126] The antibody of the present invention has an antigen-binding region capable of binding to 5T4, and the six complementarity-determining regions (CDRs) of the antigen-binding region are: i) CDR sequences of SEQ ID NO: 6, 7, 8, 10, AAS and SEQ ID NO: 11; ii) SEQ ID NO: 41, 42, 43, 45, DAS and CDR sequences of SEQ ID NO: 46

[0207] ; or iii) CDR sequences of SEQ ID NO: 48, 49, 50, 52, DAS and SEQ ID NO: 53 The antibody may be an antibody that contains a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to

[0127] Preferably one, for example two, three, four, five, six, seven, eight, nine or ten of the amino acid substitutions are conservative amino acid substitutions.

[0128] The antibody may specifically comprise one or two heavy chain variable regions comprising six consecutive amino acid residues of the sequence set forth in SEQ ID NO:102 (YYGMDV) in the complementarity determining region 3 (CDR3) [059, 207, 226]. These six consecutive amino acid residues may be the most C-terminal amino acid residues within the CDR3.

[0129] The antibody of the present invention may be an antibody [207, 226] in which the antigen-binding region capable of binding to 5T4 comprises one or two heavy chain variable regions (VH) comprising 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), and a light chain variable region (VL) comprising 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.

[0130] The antibody of the present invention may have an antigen-binding region capable of binding to 5T4 that comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:10, AAS and SEQ ID NO:11, respectively

[0059] .

[0131] Alternatively, the antibody of the present invention may have an antigen-binding region capable of binding to 5T4 that comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:41, 42, and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:45, DAS, and SEQ ID NO:46, respectively

[0207] .

[0132] In addition, the antibody of the present invention may have an antigen-binding region capable of binding to 5T4 that comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:48, 49, and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:52, DAS, and SEQ ID NO:53, respectively

[0226] .

[0133] In some embodiments, the antibody of the present invention comprises an antigen-binding region capable of binding to 5T4 comprising: 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; 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; 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; 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; 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; 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

[0207] ; and 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; The antibody comprises a heavy chain variable region (VH) selected from the group consisting of:

[0134] The antibody of the present invention may be, in particular, an antibody whose antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH)

[0059] 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.

[0135] Furthermore, the antibody of the present invention may comprise a heavy chain variable region (VH)

[0207] in which the antigen-binding region capable of binding to 5T4 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 to the sequence of SEQ ID NO:40.

[0136] In addition, the antibody of the present invention may be, in particular, an antibody comprising a heavy chain variable region (VH)

[0226] in which the antigen-binding region capable of binding to 5T4 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 to the sequence of SEQ ID NO:47.

[0137] In another embodiment, the antibody of the present invention comprises an antigen-binding region capable of binding to 5T4 comprising: 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; 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; 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; 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; 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; 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

[0207] ; 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; The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

[0138] In one embodiment, at least one binding region is 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; 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; 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; 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; 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; 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; 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; The variable heavy chain (VH) and variable light chain (VL) regions have at most 10 mutations or substitutions, at most 5 mutations or substitutions, for example at most 4 mutations or substitutions, for example at most 3 mutations or substitutions, for example at most 2 mutations or substitutions, for example at most 1 mutation or substitution, across the heavy chain variable region (VH) and light chain variable region (VL) regions selected from the group consisting of:

[0139] In some embodiments of the disclosure, said at most 10 mutations or substitutions, at most 5 mutations or substitutions, such as at most 4 mutations or substitutions, for example at most 3 mutations or substitutions, such as at most 2 mutations or substitutions, for example at most 1 mutation or substitution, are allowed across the entire variable heavy chain and the entire variable light chain. In another embodiment, said at most 10 mutations or substitutions, at most 5 mutations or substitutions, such as at most 4 mutations or substitutions, for example at most 3 mutations or substitutions, such as at most 2 mutations or substitutions, for example at most 1 mutation or substitution may not be present in any of the six CDR sequences in the variable heavy and variable light chains.

[0140] The up to 10 mutations or substitutions may be distributed throughout the entire length of the variable heavy and variable light chains of each binding region. Some or all of the mutations or substitutions may be conservative substitutions, in which an amino acid residue is substituted with an amino acid residue of the same class as defined herein above in the definition of "amino acid." For example, an acidic amino acid may be substituted for another acidic amino acid, and an aromatic residue may be substituted for another aromatic residue. Preferably, 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 substitutions.

[0141] In particular, some or all of the mutations or substitutions may be with amino acid residues that have the same physical or functional properties as the amino acid residues they replace. Amino acid residues that share physical and functional properties are described above in the definition of "amino acid," e.g., the definition of "amino acid." For example, a hydrophobic residue can be substituted for another hydrophobic amino acid residue, or a cycloalkenyl-related residue can be substituted for another cycloalkenyl-related residue.

[0142] Antibodies containing the above-disclosed substitutions or mutations may be functional variants of the VL region, VH region, or one or more CDRs as defined above by listing the SEQ ID NOs. Functional variants of the VL, VH, or CDRs used in connection with the antibodies of the invention may still enable the antibody to retain at least a substantial portion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the affinity and / or specificity / selectivity of the parent antibody, and in some cases, such 5T4 antibodies may even associate with greater affinity, selectivity, and / or specificity than the parent antibody.

[0143] In a further embodiment of the invention, the antibody comprises an antigen-binding region capable of binding to 5T4 comprising: 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; 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; 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; 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; 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; 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

[0207] ; and 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; The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

[0144] The antibodies of the invention can be full-length antibodies, such as full-length IgG1 antibodies.

[0145] Furthermore, the antibodies of the present invention can be monovalent antibodies. Alternatively, the antibodies of the present invention can be bivalent antibodies.

[0146] In another embodiment, the antibodies provided according to the present invention are monospecific antibodies.

[0147] Alternatively, the antibodies of the present disclosure can be bispecific antibodies.

[0148] It is further within the scope of the present disclosure to provide an antibody as defined above, which comprises an antigen-binding region of an antibody that binds to CD3, e.g., human CD3ε (epsilon), e.g., the human CD3ε (epsilon) set forth in SEQ ID NO:4.

[0149] In particular, the present disclosure provides a bispecific antibody comprising a first antigen-binding region of an antibody disclosed above and a second binding region that binds to CD3, e.g., human CD3, as defined above.

[0150] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody having specificity for two different epitopes, e.g., two scFvs linked in tandem by an extra peptide linker; and (iii) a dual variable domain antibody (DVD-Ig™), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond, as described in Wu et al., "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™)." TM (iv) chemically linked bispecific (Fab') fragments; (v) Tandabodies, a fusion of two single-chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibodies, a combination of scFvs and diabodies resulting in multivalent molecules; (vii) so-called "dock and lock" molecules (Dock-and-Lock®), based on the "dimerization and docking domain" in protein kinase A, which when applied to Fabs can give trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which comprise, for example, two scFvs fused to either end of a human Fab arm; and (ix) diabodies.

[0151] In one embodiment, the bispecific antibody of the invention is a diabody, a crossbody, such as a CrossMab, or a bispecific antibody obtained by controlled Fab arm exchange (such as those described in WO2011 / 131746).

[0152] 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, each containing an Fab fragment or portion of an Fab fragment of at least two different antibodies, (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or portion of an Fab fragment, (iv) Fc fusion molecules, in which a single chain Fv molecule or stabilized diabody is fused to a heavy chain constant domain, Fc region or portion thereof, (v) Fab fusion molecules, in which different Fab fragments are fused together or to a heavy chain constant domain, Fc region or portion thereof, and (vi) ScFv- and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®), in which different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein or molecule fused to a heavy chain constant domain, Fc region or portion thereof.

[0153] IgG-like molecules with complementary CH3 domain molecules include Triomab® (Trion Pharma / Fresenius Biotech, WO / 2002 / 020039), Knobs-into-Holes (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically matched (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y (Genentech), DIG body and PIG body (Pharmabcine), Strand Exchange Engineered Domain body (SEED body) (EMD Serono, WO2007110205), Biclonics (Merus), FcΔAdp (Regeneron, WO2010 / 015792), bispecific IgG1 and bispecific IgG2 (Pfizer / Rinat, WO11143545), Azymetric scaffolds (Zymeworks / Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), bivalent bispecific antibodies (Roche, WO2009 / 080254) and DuoBody® molecules (Genmab A / S, WO2011 / 131746).

[0154] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one antibody (Genentech), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybodies™ (Zyngenia), common light chain approach (Crucell / Merus, US 7,262,028), κλBodies (NovImmune), and CovX-body (CovX / Pfizer).

[0155] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig™ (Abbott, US 7,612,181), Dual domain double head 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).

[0156] 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™) (MacroGenics, WO2008157379, WO2010 / 080538), and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0157] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action Fab or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0158] Examples of scFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE®) (Micromet, Tandem Diabody (Tandab™) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), single chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies® (Ablynx), dual targeting heavy chain only domain antibodies.

[0159] In particular, the antibody of the present disclosure has an antigen-binding region that binds to CD3, a heavy chain variable region (VH) [huCD3-H1L1] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively (WO2015001085 (Genmab A / S)); and optionally, A light chain variable region (VL) [huCD3-H1L1] comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 58, GTN, and SEQ ID NO: 59, respectively. The antibody may be an antibody comprising:

[0160] In addition, the antigen-binding region that binds to CD3 is a heavy chain variable region (VH) [huCD3-H1L1] comprising 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 to the sequence of SEQ ID NO: 57; and optionally, a light chain variable region (VL) [huCD3-H1L1] comprising 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 to the sequence of SEQ ID NO:60; Also disclosed is an antibody comprising:

[0161] The present disclosure provides: an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 56, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [huCD3-H1L1]; Antibodies are further provided.

[0162] The present disclosure also provides: an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 41, 42, and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45, DAS, and SEQ ID NO: 46, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 56, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [huCD3-H1L1]; An antibody is provided.

[0163] The present disclosure also provides: an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 52, DAS and SEQ ID NO: 53, respectively [226 - VH + VL CDR1, CDR2 and CDR3 sequences]; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 56, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [huCD3-H1L1]; An antibody is provided.

[0164] The antigen-binding region that binds to CD3 has an equilibrium dissociation constant K in the range of 200 to 1000 nM, for example, in the range of 300 to 1000 nM, in the range of 400 to 1000 nM, in the range of 500 to 1000 nM, in the range of 300 to 900 nM, in the range of 400 to 900 nM, in the range of 400 to 700 nM, in the range of 500 to 900 nM, in the range of 500 to 800 nM, in the range of 500 to 700 nM, in the range of 600 to 1000 nM, in the range of 600 to 900 nM, in the range of 600 to 800 nM, or, for example, in the range of 600 to 700 nM. D can be combined with

[0165] In a further embodiment, the antibody disclosed herein has a lower affinity for binding to human CD3ε than an antibody having an antigen-binding region comprising the VH sequence as set forth in SEQ ID NO:57 and the VL sequence as set forth in SEQ ID NO:60 [huCD3-H1L1], preferably the affinity is at least 2-fold, such as at least 5-fold, such as at least 10-fold, such as at least 20-fold, at least 30-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, or at least 60-fold lower.

[0166] In particular, the antigen-binding region that binds to CD3 has an equilibrium dissociation constant K in the range of 1 to 100 nM, e.g., in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, in the range of 10 to 80 nM, in the range of 10 to 60 nM, in the range of 10 to 40 nM, or, for example, in the range of 10 to 20 nM. D can be combined with

[0167] The affinity with which antibodies of the invention bind to CD3 can be determined by biolayer interferometry using a variation of the procedure described above or the procedure set forth 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 (mature protein of SEQ ID NO:101) to the immobilized antibody is determined. Additionally, the affinity with which antibodies of the invention bind to CD3 can be determined by biolayer interferometry as provided in Example 9 herein.

[0168] It should be understood that antibodies that bind with reduced affinity to CD3, particularly human CD3, are provided in WO2017 / 009442, and any of these antibodies may serve as a basis for generating antibodies of the invention that, in addition to being able to bind 5T4, also have the ability to bind CD3 with reduced affinity. Thus, in a further aspect, the antibodies of the invention comprise: the antigen-binding region that binds to CD3 comprises a heavy chain variable (VH) region including a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; the heavy chain variable (VH) region, when compared to a heavy chain variable (VH) region comprising the sequence set forth in SEQ ID NO:57, 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, numbered according to the sequence of SEQ ID NO:57; and the wild-type light chain variable (VL) region comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, GTN and SEQ ID NO: 59, respectively; It is an antibody.

[0169] Preferably, the CDR1, CDR2 and CDR3 sequences of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 contain a total of at most 1, 2, 3, 4 or 5 amino acid substitutions when compared to the sequence shown in SEQ ID NO:57.

[0170] The amino acid sequence of CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 may have at least 95% sequence identity, for example, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the amino acid sequence of CDR1, CDR2, and CDR3 of the wild-type heavy chain variable (VH) region, where the sequence identity is calculated based on an alignment of the amino acid sequence consisting of the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 with the amino acid sequence containing the sequences of CDR1, CDR2, and CDR3 of the wild-type heavy chain variable (VH) region.

[0171] In particular, the antigen-binding region that binds to CD3 may comprise mutations selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, Y114V at positions numbered according to the reference sequence of SEQ ID NO:57.

[0172] In certain embodiments, an antibody of the invention, when it is a bispecific antibody, lacking or having reduced Fc-mediated effector function (an "inactive" antibody), and comprising the antigen-binding region of an antibody that binds CD3, a) when purified peripheral blood mononuclear cells (PBMCs) or T cells are used as effector cells, they are able to mediate concentration-dependent cytotoxicity of SK-OV-3 cells, e.g., when assayed as described in Example 14 herein; b) when purified T cells are used as effector cells, they are able to mediate cell concentration-dependent cytotoxicity of MDA-MB-231 when assayed, for example, as described in Example 13 herein; c) capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells, e.g., when assayed as described in Example 13(II) herein; d) capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; e) purified T cells, when used as effector cells, are capable of inducing cytotoxicity of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; and / or f) NOD.Cg-Prkdc inoculated with human MDA-MB-231 tumor cells scid Il2rg tm1Wjl exhibit anti-tumor activity, such as inhibition of tumor growth or delay of tumor outgrowth, in a humanized immune hematopoietic stem cell reconstituted mouse xenograft model, such as IFN-γ / SzJ, as determined, for example, as described in Example 15; It is an antibody.

[0173] Furthermore, the antibodies of the present invention do not bind to leukocyte FcγRs and do not induce CD3 antibody-dependent FcγR-mediated CD3 cross-linking upon binding to C1q in the absence of target (5T4)-specific tumor cells, as assessed by flow cytometry or ELISA.

[0174] Further disclosure of antibodies with reduced or no Fc-mediated effector functions (“inactive” antibodies) can be found below.

[0175] The ability of the antibodies to mediate concentration-dependent cytotoxicity of SK-OV-3 cells was i) isolating PBMCs or T cells from healthy human donor buffy coats; ii) a first set of samples, each sample comprising PBMCs and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of PBMCs:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, 8:1 and 12:1; and a second set of samples, each sample comprising T cells and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of T cells:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, and 8:1; preparing a iii) adding antibody to each sample set at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; and then iv) assessing the viability of SK-OV-3 cells using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) The cytotoxicity is determined in an in vitro cytotoxicity assay comprising:

[0176] The ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising T cells and human breast adenocarcinoma MDA-MB-231 cells, wherein the ratio of T cells:MDA-MB-231 cells in the samples is 8:1; iii) adding an antibody to the sample set at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; iv) staining the T cells by incubating with fluorescently labeled antibodies against T cell activation markers, such as CD69-APC, CD25-PE-Cy7 and CD279 / PD1-BV604 antibodies for 30 minutes at 4°C; and v) analyzing the T cells by flow cytometry The concentration of the ATP can be determined in an assay comprising:

[0177] APC anti-human CD69 (CD69-APC) antibody is commercially available, for example, from BioLegend (catalog numbers 310909 and 310910). CD25 monoclonal antibody, PE-Cyanine 7 (CD25-PE-Cy7), is also commercially available, for example, from ThermoFisher Scientific (catalog number 25-0259-42) and BD Biosciences (catalog number 557741). Finally, CD279 / PD1-BV604 antibody is commercially available from Genscript (catalog number A01828).

[0178] In vitro activation of T cells in the presence of BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells, wherein the ratio of T cells:tumor cells in the samples is 4:1; iii) adding an antibody to the sample set at a concentration of 0.0128 ng / mL to 5000 ng / mL (e.g., a 5-fold dilution method) and incubating the samples at 37°C for 72 hours; iv) collecting 110 μL of T cell-containing supernatant from each sample and staining the T cells by incubating with fluorescently labeled antibodies against T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and antibodies against T cell markers, such as 69-APC, CD25-PE-Cy7, and CD279 / PD1-BV604 antibodies, at 4° C. for 30 minutes; and v) analyzing the sample by flow cytometry The temperature can be determined by a procedure including:

[0179] The ability to induce cytotoxicity in BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) providing T cells isolated from a healthy human donor buffy coat; ii) providing a set of test samples and a control sample, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells adhered to the bottom of a 96-well tissue culture plate, wherein the ratio of T cells to tumor cells in the sample is 4:1; iii) adding antibody to a set of test samples at concentrations ranging from 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilutions), while control samples are left untreated or incubated with 5 μM staurosporine, and incubating all samples at 37° C. for 72 hours; iv) incubating the adherent cells in 10% (w / w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin) in RPMI-1640 medium supplemented with 10% (w / w) iron-containing donor bovine serum and penicillin / streptomycin for 4 hours at 37°C; v) Measure the absorbance of the cells and calculate the percentage of viable cells by setting the absorbance of cells incubated with staurosporine as 0% viability and untreated cells as 100% viability. Calculation process as TIFF2026041981000013.tif9128 The temperature can be determined by a procedure including:

[0180] The antibody of the present invention is particularly characterized in that the antigen-binding region capable of binding to CD3 is a) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 61, 55 and 56, respectively [VH CDR1-T31P + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or b) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 63, 55 and 56, respectively [VH CDR1-T31M + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or c) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 65 and 56, respectively [VH CDR-N57E + wild-type VH CDR1, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or d) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 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, sequence GTN and sequence shown in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] e) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 69, respectively [wild-type VH CDR1,2 + VH CDR3-H101N] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 69, respectively [wild-type VL CDR1,2,3]; f) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 71, respectively [wild-type VH CDR1,2 + VH CDR3-G105P] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; g) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 73, respectively [wild-type VH CDR1,2 + VH CDR3-S110A] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or h) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 75, respectively [wild-type VH CDR1,2 + VH CDR3-S110G] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; i) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 77, respectively [wild-type VH CDR1,2 + VH CDR3-Y114V] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or j) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 79, respectively [wild-type VH CDR1,2 + VH CDR3-Y114M] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or k) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 81, respectively [wild-type VH CDR1,2 + VH CDR3-Y114R] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] The antibody may be an antibody comprising:

[0181] In certain embodiments, the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs:54, 55, and 67, respectively, and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NO:58, sequence GTN, and sequence set forth in SEQ ID NO:59, respectively.

[0182] Furthermore, the present invention provides an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; An antibody as defined above is provided.

[0183] The present invention also provides an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 41, 42, and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45, DAS, and SEQ ID NO: 46, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; Antibodies as defined above are also provided.

[0184] Furthermore, the present invention provides an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 48, 49, and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 52, DAS, and SEQ ID NO: 53, respectively; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; An antibody as defined above is provided.

[0185] In the antibody of the present invention, the antigen-binding region capable of binding to human CD3 is a) the VH sequence [VH T31P] shown in SEQ ID NO: 62 and the VL sequence shown in SEQ ID NO: 60; b) the VH sequence [VH T31M] shown in SEQ ID NO: 64 and the VL sequence shown in SEQ ID NO: 60; c) the VH sequence shown in SEQ ID NO: 66 [VH N57E] and the VL sequence shown in SEQ ID NO: 60; d) the VH sequence shown in SEQ ID NO:68 [VH H101G] and the VL sequence shown in SEQ ID NO:60; e) the VH sequence shown in SEQ ID NO: 70 [VH H101N] and the VL sequence shown in SEQ ID NO: 60; f) the VH sequence shown in SEQ ID NO: 72 [VH G105P] and the VL sequence shown in SEQ ID NO: 60; g) the VH sequence shown in SEQ ID NO: 74 [VH S110A] and the VL sequence shown in SEQ ID NO: 60; h) the VH sequence shown in SEQ ID NO: 76 [VH S110G] and the VL sequence shown in SEQ ID NO: 60; i) the VH sequence shown in SEQ ID NO: 78 [VH Y114V] and the VL sequence shown in SEQ ID NO: 60; j) the VH sequence shown in SEQ ID NO: 80 [VH Y114M] and the VL sequence shown in SEQ ID NO: 60, and k) the VH sequence shown in SEQ ID NO:82 [VH Y114R] and the VL sequence shown in SEQ ID NO:60 The VH and VL sequences may be selected from the group consisting of:

[0186] In particular, the antibody of the present invention may be an antibody whose antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO:68 [VH H101G] and the VL sequence shown in SEQ ID NO:60.

[0187] In some embodiments, the antibody of the invention comprises: an antigen-binding region capable of binding to 5T4 comprising 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 an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G] and the VL sequence shown in SEQ ID NO: 60; It is something.

[0188] In another embodiment, the antibody of the invention comprises: an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising a 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 an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G] and the VL sequence shown in SEQ ID NO: 60; It is something.

[0189] In yet another embodiment, the antibody of the invention comprises: an antigen-binding region capable of binding to 5T4 comprising 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 an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G] and the VL sequence shown in SEQ ID NO: 60; It is something.

[0190] As is well known to those skilled in the art, each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. This structure may also be found in the antibodies of the present invention. Furthermore, the antibodies of the present invention may comprise two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0191] In certain embodiments, the antibody of the present invention comprises a first heavy chain and a second heavy chain, e.g., a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, a CH2 region, and a CH3 region. Stable heterodimeric antibodies can be obtained in high yields by so-called Fab arm exchange, as described, for example, in WO2008 / 119353 and WO2011 / 131746, or based on two homodimeric starting proteins containing only slight asymmetric mutations in the CH3 region. Thus, in some embodiments of the invention, the antibody comprises a first heavy chain having at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, and a second heavy chain having at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, wherein the substitutions in the first and second heavy chains are not in the same position, and the amino acid positions are numbered according to EU numbering.

[0192] In particular embodiments, the invention provides antibodies in which in the first heavy chain the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R and in the second heavy chain the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L, or vice versa.

[0193] In some embodiments, the antibodies of the present invention comprise, in addition to the antigen-binding region, an Fc region consisting of the Fc sequences of two heavy chains. The first and second Fc sequences can each be of any isotype, for example, 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, or IgG4 isotype, or a mixed isotype, for example, a human IgG1 isotype.

[0194] The antibodies of the present invention may contain modifications in the Fc region to render the antibody inactive or non-activating. Thus, in the antibodies disclosed herein, one or both heavy chains may be modified so that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody comprising unmodified first and second heavy chains. Fc-mediated effector function may be measured by determining Fc-mediated CD69 expression on T cells (i.e., CD69 expression as a result of CD3 antibody-mediated Fcγ receptor-dependent CD3 cross-linking) via binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of FcγRs. In particular, the heavy chain constant sequence may be modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type (unmodified) antibody, where Fc-mediated CD69 expression is determined in a PBMC-based functional assay, for example, as described in Example 3 of WO2015001085. Modification of the heavy and light chain constant sequences may also result in reduced C1q binding to the antibody. The reduction may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the unmodified antibody. C1q binding may also be determined by ELISA. Additionally, the Fc region may be modified such that antibody-mediated Fc-mediated T cell proliferation 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 the unmodified antibody, where T cell proliferation is measured in a PBMC-based functional assay.

[0195] Examples of amino acid positions that can be modified include positions L234 and L235 in an IgG1 isotype antibody. Thus, the antibodies of the present invention may comprise a first heavy chain and a second heavy chain in which the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain (EU numbering) are F and E, respectively.

[0196] 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). Therefore, the antibodies of the present invention may comprise a first heavy chain and a second heavy chain in which the amino acid residue at the position corresponding to position D265 in the human IgG1 heavy chain according to EU numbering is A in both heavy chains. A further embodiment of the present invention provides antibodies in which 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 at least one, for example both, of the first and second heavy chains. In the present application, antibodies having a combination of the three amino acid substitutions L234F, L235E and D265A, and additionally having the K409R or F405L mutations disclosed herein above, will be named with the suffix "FEAR" or "FEAL", respectively.

[0197] The amino acid sequence of a wild-type IgG1 heavy chain constant region is identified herein as SEQ ID NO: 89. Consistent with the embodiments disclosed above, an antibody of the invention may comprise an IgG1 heavy chain constant region carrying an F405L substitution and having the amino acid sequence set forth in SEQ ID NO: 90 and / or an IgG1 heavy chain constant region carrying a K409R substitution and having the amino acid sequence set forth in SEQ ID NO: 94.

[0198] The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E, and D265A substitutions is identified herein 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 identified herein 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 identified herein as SEQ ID NO: 93.

[0199] The present invention provides a) the antigen-binding region capable of binding to 5T4 is humanized, and / or b) the antigen-binding region capable of binding to CD3, if present, is humanized; Antibodies are further provided.

[0200] The present invention also provides a) the antigen-binding region capable of binding to 5T4 is human, and / or b) the antigen-binding region capable of binding to CD3, if present, is human; An antibody is provided.

[0201] Furthermore, the present invention provides a) the antigen-binding region capable of binding to 5T4 is chimeric, and / or b) the antigen-binding region capable of binding to CD3, if present, is chimeric; An antibody is provided.

[0202] In some embodiments of the invention, the antibody comprises a kappa (κ) light chain. In particular embodiments of the invention involving bispecific antibodies, the kappa light chain comprises the CDR1, CDR2 and CDR3 sequences of the 5T4 antibody light chain disclosed above.

[0203] In a further embodiment of the invention, the antibody of any one of the preceding claims comprises a lambda (λ) light chain. In a particular embodiment of the invention involving a bispecific antibody, the lambda light chain comprises the CDR1, CDR2 and CDR3 sequences of the CD3 antibody light chain disclosed above, in particular the CDR1, CDR2 and CDR3 sequences of the CD3 antibody with reduced affinity for CD3 disclosed above. The amino acid sequence of the kappa light chain constant region is included herein as SEQ ID NO:95, and the amino acid sequence of the lambda light chain constant region is included herein as SEQ ID NO:96.

[0204] In certain embodiments, the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain, e.g., an antibody having a heavy chain and a lambda light chain that includes a binding region capable of binding to CD3, and a heavy chain and a kappa light chain that includes a binding region capable of binding to 5T4.

[0205] Immunoconjugates In another aspect, the present invention provides an immunoconjugate or antibody-drug conjugate (ADC) comprising an antibody as defined above and a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, an antibiotic, or a radioisotope. Generally, those skilled in the art have numerous cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, antibiotics, and radioisotopes at their disposal, and the optimal choice of therapeutic moiety will depend on the desired application of the immunoconjugate. For certain applications, a preferred cytotoxic agent may be a microtubule-disrupting agent, e.g., a duostatin, such as duostatin-3.

[0206] nucleic acid construct A further aspect of the present invention is a method for producing a pharmaceutical composition comprising the steps of: a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above; The present invention relates to a nucleic acid construct comprising:

[0207] The nucleic acid construct is a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above; It may further include:

[0208] Expression vector Another aspect of the invention provides expression vectors comprising nucleic acids encoding the heavy and / or light chain sequences of an antibody of the invention. In particular, the expression vectors comprise: a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 as defined above; may include:

[0209] The expression vector is a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined above; It may further include:

[0210] In a further embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody, eg, a human IgG1,κ monoclonal antibody.

[0211] In the context of the present invention, an expression vector can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate 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 anti-5T4 antibody-encoding nucleic acid is expressed in a naked DNA or RNA vector, e.g., comprising linear expression elements (see, e.g., Sykes and Johnston, Nat Biotech, 2004). 17, 355-59 (1997)), compacted nucleic acid vectors (e.g., those described in US Pat. No. 6,077,835 and / or WO 00 / 70087), plasmid vectors such as pBR322, pUC19 / 18, or pUC118 / 119, "midge" minimal size nucleic acid vectors (e.g., Schakowski et al., Mol Ther 3, 793-800 (2001)) or contained in a precipitation nucleic acid vector construct, such as the CaP04 precipitation construct (see, e.g., 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 methods for their use are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0212] In one embodiment, the vector is suitable for expressing an anti-5T4 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), and pET vector (Novagen, Madison, Wisconsin).

[0213] Additionally or alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0214] The nucleic acid construct and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of directing a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretory leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization / anchoring sequences (e.g., membrane permeabilization sequences, GPI anchoring sequences), etc.

[0215] In the expression vectors of the present invention, the anti-5T4 antibody-encoding nucleic acid can contain or be accompanied by any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an effective poly(A) termination sequence, an origin of replication for plasmid production in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid can also contain an inducible promoter rather than a constitutive promoter, such as CMV IE (though those skilled in the art will recognize that such a term is actually a descriptor of the degree of gene expression under certain conditions).

[0216] In one embodiment, an expression vector encoding an anti-5T4 antibody is placed into and / or delivered to the host cell or animal by a viral vector.

[0217] Cells and host cells In a further aspect, the present invention provides a cell comprising a nucleic acid construct as defined herein above or an expression vector as defined herein above. It should be understood that the cell may be obtained by transfecting a host cell with said nucleic acid construct or expression vector, e.g. a recombinant host cell.

[0218] The host cells can be of human origin, such as human embryonic kidney (HEK) cells, e.g., HEK / Expi cells. Alternatively, the host cells can be of rodent origin, such as Chinese hamster ovary cells, e.g., CHO / N50 cells. Additionally, the host cells can be of bacterial origin.

[0219] The cell may contain a nucleic acid sequence encoding an antibody of the present invention, or a portion thereof, stably integrated into the genome of the cell. Alternatively, the cell may contain a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence encoding the expression of an anti-5T4 antibody of the present invention, or a portion thereof. In particular, the host cell may contain a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence encoding the expression of an anti-5T4 antibody, or a portion thereof.

[0220] composition A further aspect of the present invention provides a composition comprising an antibody, such as a bispecific antibody or immunoconjugate as defined above. The composition may be a pharmaceutical composition comprising the antibody, bispecific antibody or immunoconjugate and a pharmaceutically acceptable carrier.

[0221] Pharmaceutical compositions can be formulated using carriers, excipients, and / or diluents, as well as any other components suitable for pharmaceutical compositions, such as 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. Pharmaceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, should be suitable for the antibody or antibody conjugate of the present invention and the selected mode of administration. The suitability of carriers and other components of pharmaceutical compositions is determined based on the lack of a significant negative effect on the desired biological properties of the selected compound of the present invention or pharmaceutical composition (e.g., a substantial inability to affect antigen binding [e.g., a relative inhibition of 10% or less, a relative inhibition of 5% or less, etc.]).

[0222] Pharmaceutical compositions of the present invention may include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0223] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including factors well known in the medical arts, such as the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, and the age, sex, weight, condition, general health, and medical history of the patient being treated.

[0224] 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.

[0225] 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 (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0226] Pharmaceutically acceptable carriers include sterile aqueous solution or sterile aqueous dispersion, and sterile powder for extemporaneously preparing sterile injectable solution or sterile injectable dispersion.The use of such media and agents for pharmaceutically active substances is known in the art.Any conventional media or agents can be considered for use in the pharmaceutical compositions of the present invention, unless they are incompatible with active compounds.

[0227] 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, alpha-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0228] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0229] The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, preservatives, and buffers, which can enhance the shelf life or efficacy of the pharmaceutical composition. The compounds of the present invention can be prepared using 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, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes, 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*, JR Robinson, ed., *Marcel Dekker, Inc., New York, 1978.

[0230] In one embodiment, the compound of the present invention can be formulated to ensure proper distribution in vivo.Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions, and sterile powders for extemporaneously preparing sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Any conventional media or agent is contemplated for use in the pharmaceutical compositions of the present invention, unless it is incompatible with the active compound.Other active compounds or therapeutic compounds may also be incorporated into the composition.

[0231] Injectable pharmaceutical compositions are typically sterile and stable under manufacturing 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 containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent, for example, with one or a combination of the ingredients listed above, as needed, followed by microfiltration sterilization. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients, for example, those selected from those listed above. In the case of sterile powders for preparing sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization), which yield powders from a previously sterile-filtered solution of the active ingredient and any other desired ingredients.

[0232] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above as needed, followed by microfiltration sterilization.Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients selected from those listed above.In the case of sterile powders for preparing sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization), which produce powders from a solution of the active ingredient and any other desired ingredients that has been previously sterile-filtered.

[0233] Pharmaceutical compositions of the invention may contain one antibody, bispecific antibody or antibody-drug conjugate (ADC) of the invention, or may contain a combination of an antibody, bispecific antibody or ADC of the invention with another therapeutic compound, or may contain a combination of compounds of the invention.

[0234] The pharmaceutical composition can be administered by any suitable route and mode. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0235] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally, i.e., by a mode of administration other than enteral administration and topical administration, usually by injection, including epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion. In particular, the pharmaceutical compositions of the present invention may be administered by intravenous or subcutaneous injection or infusion.

[0236] Uses and Therapeutic Applications The present invention further provides antibodies, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADCs) as defined herein for use as pharmaceuticals. The anti-5T4 antibodies or immunoconjugates of the present invention can be used in the treatment or prevention of diseases or disorders involving cells expressing 5T4. In particular, bispecific antibodies of the present invention, i.e., antibodies comprising antigen-binding regions capable of binding to 5T4 and CD3, may be useful in therapeutic situations where specific targeting and T cell-mediated killing of cells expressing 5T4 is desired, and may be more efficient than conventional anti-5T4 antibodies in certain such indications and situations.

[0237] In one embodiment, disclosed herein are antibodies, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADCs) of the present invention for use in the treatment of cancer. The antibodies, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADCs) can be used, in particular, to treat cancers characterized by expression of 5T4 in at least a portion of tumor cells.

[0238] The cancer may be 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, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia, among others.

[0239] Additionally, the present invention relates to the use of an antibody of the present invention for the manufacture of a medicament for treating cancer, such as a cancer selected from the group consisting of renal / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, and acute myeloid leukemia.

[0240] In a further aspect, the present invention provides a method of treating a disease comprising administering to a subject in need thereof an antibody, immunoconjugate, composition, e.g., a pharmaceutical composition or antibody-drug conjugate (ADC), of the invention.

[0241] In a particular embodiment of the invention, the method is a method for treating cancer. The method of the invention particularly comprises: a) selecting a subject suffering from a cancer comprising tumor cells that express 5T4 and / or a cancer known to express 5T4; and b) administering to said subject an antibody, such as a bispecific antibody, or a pharmaceutical composition or an antibody-drug conjugate (ADC) of the invention. may include:

[0242] The cancer may be 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, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia, among others.

[0243] The dosage regimen in the above-mentioned treatment and use methods is adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be increased or decreased accordingly depending on the requirements of the therapeutic situation. Parenteral compositions can be formulated into dosage unit forms for ease of administration and uniformity of dosage.

[0244] Effective dosages and dosing regimens for antibodies depend on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the invention is about 0.001-10 mg / kg, such as about 0.001-5 mg / kg, for example, about 0.001-2 mg / kg, for example, 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 for a therapeutically effective amount of an antibody of the invention is about 0.1-100 mg / kg, for example, 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.

[0245] A physician of ordinary skill in the art can readily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian could start the dosage 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 dosage until the desired effect is achieved. Generally, a suitable daily dose of an antibody of the invention will be that amount of compound that is the lowest dose effective to produce a therapeutic effect. Administration can be, for example, parenteral, e.g., intravenous, intramuscular, or subcutaneous. In one embodiment, the antibody can be administered by infusion at a weekly dosage calculated in mg / m2. Such dosage can be based on the mg / kg dosage given above, e.g., according to the following formula: Dose (mg / kg) × 70:1.8. Such administration can be repeated, e.g., 1 to 8 times, e.g., 3 to 5 times. Administration can be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. In one embodiment, the antibody may be administered by slow continuous infusion over an extended period of time, for example, greater than 24 hours, to reduce toxic side effects.

[0246] In one embodiment, the antibody may be administered in a weekly dosage calculated as a fixed dose, up to eight times, e.g., four to six times if administered once a week. Such a regimen may be repeated one or more times as needed, e.g., after six or twelve months. Such dosages may be based on the mg / kg dosages provided above, assuming a body weight of 70 kg. Dosages may be determined or adjusted by measuring the amount of the antibody of the invention in the blood after administration, e.g., by collecting a biological sample and using an anti-idiotypic antibody targeting the 5T4 antigen-binding region of the antibody of the invention.

[0247] In one embodiment, the antibody can be administered as a maintenance treatment, for example, once a week for six months or more.

[0248] Antibodies may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer is in remission.

[0249] The antibodies of the invention can also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is for use in combination with one or more additional therapeutic agents, such as cytotoxic, chemotherapeutic, or antiangiogenic agents.

[0250] antibody production Also provided herein are methods for producing antibodies, such as bispecific antibodies, of the present invention. a) culturing a host cell containing an expression vector as defined herein, and b) Purifying the antibody from the culture medium

[0023] A method for producing the antibody of the present invention is provided, comprising:

[0251] In an embodiment of the invention in which an antibody comprises a binding region capable of binding to 5T4 and a binding region capable of binding to CD3, the antibody comprises a) providing an antibody capable of binding to 5T4 by culturing a host cell comprising an expression vector as defined herein under conditions that allow expression of the antibody capable of binding to 5T4, and purifying the antibody capable of binding to 5T4 from the culture medium; b) I) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined herein above, and II) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 as defined herein above; providing an antibody capable of binding to CD3 by culturing a host cell containing an expression vector comprising: c) incubating an antibody capable of binding to 5T4 with an antibody capable of binding to CD3 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and d) Obtaining an antibody It can be produced using a method comprising:

[0252] kit The present invention further provides a kit of parts, for example a kit for use as a companion diagnostic / for use in identifying patients within a patient population who have a propensity to respond to treatment with an antibody as defined herein above or an immunoconjugate or antibody-drug conjugate (ADC) as defined herein above, or for use in predicting the efficacy or anti-tumor activity of said antibody or immunoconjugate or ADC when used to treat a patient, comprising an antibody as defined herein above and instructions for use of the kit.

[0253] Anti-idiotype antibodies In a further aspect, the present invention relates to an anti-idiotypic antibody that binds to an antibody comprising at least one antigen-binding region capable of binding to 5T4, i.e., an antibody of the invention described herein. In a particular embodiment, the anti-idiotypic antibody binds to an antigen-binding region capable of binding to 5T4.

[0254] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genotype as the source of the anti-5T4 monoclonal antibody with the monoclonal antibody from which the anti-Id is to be prepared. The immunized animal is typically able to recognize and react to the idiotypic determinants of the immunizing antibody by producing antibodies against these idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in U.S. Pat. No. 4,699,880. Such antibodies are a further feature of the present invention.

[0255] Anti-Id antibodies can also be used as "immunogens" to induce immune responses in yet other animals that produce so-called anti-anti-Id antibodies. Anti-anti-Id antibodies can be epitopically identical to the original monoclonal antibody that induced them. Thus, by using antibodies against the idiotypic determinants of a monoclonal antibody, it is possible to identify other cells expressing antibodies of identical specificity. Anti-Id antibodies can be modified (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to the 5T4-specific antibodies of the present invention. For example, monoclonal anti-Id antibodies can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will typically contain anti-anti-Id antibodies with binding characteristics similar, if not identical, to those of the original / parent anti-5T4 antibody.

[0256] array TIFF2026041981000014.tif58158TIFF2026041981000015.tif224158TIFF2026 041981000016.tif226158TIFF2026041981000017.tif232158TIFF20260419810 00018.tif231158TIFF2026041981000019.tif192158TIFF2026041981000020.t if215158TIFF2026041981000021.tif234158TIFF2026041981000022.tif44158

[0257] The present invention is further illustrated by the following examples which should not be construed as further limiting. [Example]

[0258] Example 1: Generation of 5T4 antibody and screening materials Expression constructs for 5T4 The following codon-optimized constructs were generated to express various full-length 5T4 variants: human 5T4 (Uniprot accession number Q13641), cynomolgus monkey 5T4 (Uniprot accession number Q4R8Y9), and chicken 5T4 (Uniprot accession number R4GM46). In addition, the following codon-optimized constructs were generated for various 5T4 extracellular domain (ECD) variants: human 5T4 ECD (aa 1-355 of Uniprot accession number Q13641) with a C-terminal His tag (5T4ECDHis) (SEQ ID NO:99) and human 5T4 ECD (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, and 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.

[0259] The constructs contained appropriate restriction sites for cloning and the optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999;234(2):187-208). Full-length human 5T4 and cynomolgus 5T4 codon-optimized constructs were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen). Full-length chicken 5T4 codon-optimized constructs were cloned into the mammalian expression vector pSB, which contains Sleeping Beauty inverted terminal repeats flanking an expression cassette consisting of the CMV promoter and HSV-TK polyA signal.

[0260] Generation of HEK-293F cell lines transiently expressing full-length human, cynomolgus monkey, or chicken 5T4 Freestyle™ 293-F (a HEK-293 subclone [HEK-293F] adapted to growth in suspension and chemically defined Freestyle medium) cells were obtained from Invitrogen (catalog no. R790-07) and transfected with the codon-optimized constructs described above using 293fectin (Invitrogen, catalog no. 12347-019) according to the manufacturer's instructions.

[0261] Purification of His-tagged 5T4 5T4ECDHis (mature protein of SEQ ID NO:99) was expressed in HEK-293F cells as described above. 5T4ECD91-FcRbHis was expressed using the Expi293F expression platform (Thermo Fisher Scientific, Waltham, MA, USA, catalog no. A14527) essentially as described by the manufacturer.

[0262] The His tag allows purification by immobilized metal affinity chromatography, in which Co binds to a chelator immobilized on a chromatography resin. 2+The supernatant containing the His-tagged protein was incubated with this resin in batch mode (i.e., in solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant either do not bind or bind weakly compared to the His-tagged protein. After incubation, the beads were recovered from the supernatant and loaded onto a column. The column was washed to remove weakly bound proteins. The strongly bound His-tagged protein was then purified by Co 2+ The protein was eluted with a buffer containing imidazole, which competes with the binding of His to the ATP. The eluate was removed by buffer exchange on a desalting column.

[0263] immunization 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) with 20 μg of 5T4ECDHis protein in Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA, catalog no. S6322) alternately at 14-day intervals. A total of eight immunizations were administered: four IP and four SC immunizations.

[0264] To generate the antibodies IgG1-5T4-076 and IgG1-5T4-059, HCo12-BalbC(IgG1-5T4-076) and HCo20-BalbC(IgG1-5T4-059) transgenic mice (Bristol-Myers Squibb) were immunized with 20 μg of 5T4ECDHis protein in Sigma adjuvant system, IP and SC, alternating at 14-day intervals. A total of eight immunizations were performed: four IP and four SC immunizations.

[0265] To generate antibody IgG1-5T4-085, HCo17-BalbC transgenic mice were immunized IP and SC with 20 μg of 5T4ECDHis protein and 20 μg of 5T4ECD91-FcRbHis mature protein in Sigma adjuvant system, 14 days apart. A total of eight immunizations were performed: four IP and four SC immunizations.

[0266] To generate the antibodies IgG1-5T4-106 and IgG1-5T4-127, HCo12-BalbC (IgG1-5T4-106) and HCo17-BalbC (IgG1-5T4-127) transgenic mice were immunized IP and SC with 20 μg of 5T4ECD91-FcRbHis mature protein in Sigma adjuvant system at 14-day intervals. A total of eight immunizations were performed: four IP and four SC immunizations.

[0267] Mice with 5T4-specific antibody titers determined by the antigen-specific screening Fluorometric Microvolume Assay Technology (FMAT) (described below) at least twice in a row were boosted with 10 μg of 5T4ECDHis or 10 μg of 5T4ECD91-FcRbHis (intravenously in PBS), and splenocytes and lymph node cells from these mice were fused 3–4 days later.

[0268] Homogeneous antigen-specific screening assay The presence of 5T4 antibodies in the serum of immunized mice or in HuMAb (human monoclonal antibody) hybridoma or transfectoma cultures was determined by a homogeneous antigen-specific screening assay using FMAT (Applied Biosystems, Foster City, CA, USA), for which a combination of four cell-based assays was used.

[0269] Serum from immunized mice or supernatant samples of hybridoma or transfectoma cultures were analyzed for binding of human antibodies 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 no. SVP-60-5) coated with 5T4ECD91-FcRBHis, and HEK-293 wild-type cells (negative control).

[0270] Samples were added to the cells and allowed to bind to 5T4. HuMAb binding was then detected using a fluorescent conjugate (AffiniPure goat anti-human IgG Fc gamma-Alexa Fluor® 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 considered positive if the counts were greater than 50 and the counts x fluorescence was at least three times that of the negative control.

[0271] HuMAb hybridoma generation HuMAb mice (described above) that had developed sufficient antigen-specific titers were sacrificed, and the spleens and lymph nodes adjacent to the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells with a mouse myeloma cell line (SP2.0 cells) was performed using a CytoPulse CEEF 50 Electrofusion System (Cellectis, Paris, France) essentially according to the manufacturer's instructions. Antigen-positive primary wells were then subcloned using the ClonePix system (Genetix, Hampshire, UK). To do so, specific primary well hybridomas were seeded in a semi-solid medium consisting of 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA). Subclones were retested for 5T4 binding according to the antigen-specific binding assay described above and scanned using the IsoCyte system (Molecular Devices). To select the best-producing clones per primary well for further expansion, IgG levels were measured using the Octet system (Fortebio, Menlo Park, USA). Further expansion and culture of the resulting HuMAb hybridomas was performed according to standard protocols (e.g., those described in Coligan JE, Bierer, BE, Margulies, DH, Shevach, EM, and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).

[0272] Sequence analysis of the 5T4 antibody variable domain and cloning into an expression vector 2~5×10 6Total RNA was prepared from 100 ng of 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 directly cloned in frame into the p33G1f and p33kappa expression vectors (pcDNA3.3-based vectors containing codon-optimized human IgG1m(f) constant domain and kappa constant domain, respectively) by ligation-independent cloning (Aslanidis, C. and PJ de Jong, Nucleic Acids Res 1990;18(20):6069-74). The variable domains from these expression vectors were sequenced, and the CDRs were annotated according to the IMGT definition (Lefranc MP et al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucleic Acids Res. 36, W503-508 (2008)). Clones with the correct open reading frame (ORF) were expressed and tested for antigen binding. The lead panel was ordered as codon-optimized sequences (GeneArt, Thermo Fisher Scientific) and produced in the Expi293 expression system according to the manufacturer's instructions (Thermo Fisher Scientific). Antibodies from these supernatants were purified and used for functional characterization. The sequences of the resulting lead clones are shown in the table above.

[0273] 5T4 control antibody In some examples, comparative antibodies against 5T4 (IgG1-5T4-H8, IgG1-5T4-A3, and IgG1-5T4-A1) previously described in WO2007 / 106744 were used. Codon-optimized antibody coding sequences were synthesized and cloned into the pCDNA3.3 expression vector (Thermo Fisher Scientific).

[0274] IgG1-b12 antibody In some examples, antibody b12 (Barbas, C. F. J. Mol. Biol. 1993 Apr 5;230(3):812-23), an HIV-1 gp120-specific antibody, was used as a negative control. A codon-optimized antibody coding sequence for this control antibody was synthesized and cloned into the pCDNA3.3 expression vector (Thermo Fisher Scientific). The sequences of the variable heavy (VH) and variable light (VL) chains are included herein as SEQ ID NO:97 and SEQ ID NO:98, respectively.

[0275] Example 2: Determining the binding affinity of 5T4-specific antibodies using biolayer interferometry The affinity of the 5T4 antibody for recombinant 5T4 protein was determined using label-free biolayer interferometry on an Octet HTX instrument (ForteBio, Portsmouth, UK). 5T4 antibody (1 μg / mL) was immobilized on an anti-human IgG Fc capture biosensor (ForteBio) for 600 seconds. After a baseline measurement (100 seconds), the association (200 seconds) and dissociation (1000 seconds) of human 5T4 ECDHis (mature protein of SEQ ID NO:99) or recombinant cynomolgus 5T4 protein (Cusabio, catalog number CSB-MP024093MOV) in sample diluent (ForteBio) were determined using a two-fold dilution series (ranging from 100 nM to 1.56 nM) starting from 3.58 μg / mL (100 nM) human 5T4 ECDHis or 3.99 μg / mL (100 nM) cynomolgus 5T4, with shaking at 30°C and 1000 rpm. Data were analyzed using Data Analysis software v9.0.0.12 (ForteBio). Values ​​from reference wells containing only sample diluent during the association and dissociation steps were subtracted from the values ​​from wells containing antigen, separately for each antibody. The Y-axis was aligned to the last 10 seconds of baseline, and interstep correction alignment to dissociation and Savitzky-Golay filtering were applied. Responses of <0.05 nm were excluded from the analysis. Data were fitted using a 1:1 model and a global full fit with an association time of 200 seconds and a dissociation time of 1000 seconds or 50 seconds as the window of interest. A fit with the full dissociation time (1000 seconds) as the window of interest was used as the default. R of the fit 2 Based on the values ​​and visual inspection, a dissociation time of 50 seconds was used as the window of interest for IgG1-5T4-127-FEAR.

[0276] Table 1 shows the association rate constant, k, of the 5T4 antibody to human 5T4ECDHis determined by biolayer interferometry. a (1 / Ms), dissociation rate constant k d(1 / s) and the equilibrium dissociation constant K D (M) is shown. The affinity range of the antibodies for human 5T4 is 1.3 x 10 -9 ~2.7×10 -8 The response of IgG1-5T4-085-FEAR was measured to be less than 0.05 nm, which prevented proper fitting of the data (low R for these fits). 2 Furthermore, the response of IgG1-5T4-076-FEAR could not be fitted properly. These data are shown in italics.

[0277] Table 2 shows the association rate constant k for cynomolgus monkey 5T4 determined by biolayer interferometry. a (1 / Ms), dissociation rate constant k d (1 / s) and the equilibrium dissociation constant K D (M) is shown. The affinity range of the antibody for cynomolgus 5T4 is 1.1 x 10 -9 ~4.1×10 -8 The responses of IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR, and IgG1-5T4-H8-FEAR were smaller than 0.05 nm, which prevented proper fitting of the data (low R 2 Furthermore, the response of IgG1-5T4-076-FEAR could not be fitted properly. These data are shown in italics.

[0278] Table 1. Binding affinities of monospecific bivalent 5T4 antibodies to the human 5T4 extracellular domain as determined by label-free biolayer interferometry. TIFF2026041981000023.tif92128

[0279] Table 2. Binding affinity of monospecific bivalent 5T4 antibodies to the cynomolgus monkey 5T4 extracellular domain as determined by label-free biolayer interferometry. TIFF2026041981000024.tif92128

[0280] Example 3: Cross-blocking of 5T4 antibody determined by biolayer interferometry Antibody cross-blocking analysis (epitope binning) was performed using biolayer interferometry on an Octet HTX instrument (ForteBio). 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) according to the manufacturer's instructions. After a baseline measurement (100 seconds) in sample diluent (ForteBio), the biosensor containing the immobilized antibody was loaded with 100 nM (3.6 μg / mL) human 5T4 ECDHis (mature protein of SEQ ID NO:99) for 500 seconds. The association response of a second 5T4 antibody (10 μg / mL) was then determined for 500 seconds. The biosensor was regenerated by three 5-second exposures to 10 mM glycine pH 2.5 followed by sample diluent. Starting from the baseline step, measurements were repeated with a new set of second 5T4 antibodies. Each biosensor was used 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 to the association step, and Savitzky-Golay filtering was applied. To correct for dissociation of 5T4 ECDHis from the immobilized antibody, the response of the sample diluent during the association step was subtracted from the association response of the second antibody. The corrected association responses were plotted in matrix format. In general, responses >0.1 nM were considered for non-blocking antibody pairs (white), and responses between -0.1 and 0.1 nM were considered for blocking antibody pairs (dark gray). In 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 displaces 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). Visual inspection of the data curves by an expert was sometimes required to assign blocking, non-blocking, or displacing properties to antibody pairs.

[0281] Cross-blocking experiments were performed with 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.

[0282] None of the antibodies (except for 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. Antibodies IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, and IgG1-5T4-127-FEAR (as well as IgG1-5T4-A3-F405L itself) also blocked binding of IgG1-5T4-A3-F405L to 5T4ECDHis, whereas antibodies IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR did not block binding of IgG1-5T4-A3-F405L to 5T4ECDHis. Antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR, in combination with IgG1-5T4-A3-F405L, showed antibody displacement, as described in more detail in Example 4.

[0283] Table 3. Antibody cross-blocking determined by biolayer interferometry Column 1 shows immobilized antibodies, and row 1 shows antibodies in solution. Corrected association responses of antibodies in solution are shown. Cross-blocking antibodies are shown in dark grey, and displacing antibody combinations are shown in light grey with an asterisk. Non-blocking antibody combinations are unlabeled (clear background). TIFF2026041981000025.tif129128

[0284] Example 4: Antibody displacement of IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR combined with IgG1-5T4-A3-F405L Antibody displacement was demonstrated using biolayer interferometry 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 a baseline measurement (100 seconds) in sample diluent (ForteBio), the biosensor containing the immobilized IgG1-5T4-A3-F405L antibody was loaded with 100 nM (3.6 μg / mL) human 5T4ECDHis (mature protein of SEQ ID NO:99) for 500 seconds. The association response of the 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 then determined for 500 seconds. Experiments were performed at 30°C using a shaker speed of 1000 rpm. Data were analyzed using Data Analysis software v9.0.0.12 (ForteBio). To correct for dissociation of human 5T4 ECDHis from immobilized IgG1-5T4-A3-F405L, the buffer control response was subtracted from the second antibody response, the Y-axis was aligned to the association step, and Savitzky-Golay filtering was applied.

[0285] As shown in Figure 1, IgG1-5T4-A3-F405L showed no binding, representing cross-blocking (self-blocking) with IgG1-5T4-A3-F405L. IgG1-5T4-H8-FEAR showed binding to 5T4ECDHis and therefore no cross-blocking with IgG1-5T4-A3-F405L. IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR initially showed positive responses (representing binding to the IgG1-5T4-A3-F405L-5T4ECDHis complex rather than cross-blocking with IgG1-5T4-A3-F405L), which then decreased to below the self-blocking response of IgG1-5T4-A3-F405L. This demonstrates the loss of mass from the IgG1-5T4-A3-F405L-5T4ECDHis complex and indicates that IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR induce dissociation of human 5T4ECDHis from IgG1-5T4-A3-F405L after binding to the complex. This phenomenon, described as antibody displacement, indicates that epitopes are close together 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.

[0286] Example 5: Simultaneous binding of 5T4 antibodies to membrane-bound 5T4 measured by flow cytometry 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 assessed by flow cytometry. IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T4-226-FEAR were conjugated to fluorescein isothiocyanate (FITC, Thermo Fisher Scientific) according to the manufacturer's instructions. SK-OV-3 cells (50,000 cells per condition), expressing approximately 20,000 5T4 molecules per cell, were incubated with a mixture of 10 μg / mL of 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 of FITC-conjugated 5T4 antibodies (IgG1-5T4-H8-FEAR-FITC, IgG1-5T4-207-FEAR-FITC, and IgG1-5T4-226-FEAR-FITC). The combinations tested are listed in Table 4. After 30 minutes of incubation at 4°C, 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: ([MFI of cells with Ab-FITC and unconjugated Ab − MFI of cells without Ab-FITC or unconjugated Ab] × 100) / (MFI of cells with Ab-FITC and isotype control − MFI of cells without Ab-FITC or unconjugated Ab).

[0287] Figure 2 shows that binding of IgG1-5T4-H8-FEAR-FITC, IgG1-5T4-207-FEAR-FITC, and IgG1-5T4-226-FEAR-FITC was blocked in the presence of 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 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. This demonstrates that the antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind to different epitopes on 5T4 compared to IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L.

[0288] Table 4. List of antibody combinations used in flow cytometry experiments TIFF2026041981000026.tif94152

[0289] Example 6: Binding of 5T4 antibody to human or chicken 5T4 transfected HEK-293 cells Binding of the 5T4 antibody to HEK-293 cells (generated as described in Example 1) transiently transfected with full-length human or chicken 5T4 was analyzed by flow cytometry. Cells (5x10 4Cells (1000 cells / well) were incubated with serial dilutions of 5T4 antibody (ranging from 0.01 to 10 μg / mL in 3-fold dilution steps) in 50 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer) in a 96-well polystyrene round-bottom plate (Greiner Bio-One, Cat. No. 650180) for 30 min at 4°C. After washing twice in staining buffer, cells were incubated in 50 μL of R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (1:500 in staining buffer, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, Cat. No. 109-116-098) for 30 min at 4°C. Cells were washed twice in staining buffer, resuspended in 20 μL of staining buffer, and analyzed using an iQue screener (Intellicyt Corporation, USA). Binding curves were analyzed by nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0290] Figure 3A 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 3B shows that 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, but IgG1-5T4-A3-F405L showed negligible binding to HEK-293 cells transfected with full-length chicken 5T4. The negative control antibody IgG1-b12-K409R showed no binding to full-length human or chicken 5T4 transfected HEK-293 cells at a concentration of 10 μg / mL.

[0291] Example 7: Internalization ability of 5T4 antibody in tumor cells Experiments were performed to characterize the internalization ability of monovalent 5T4 antibodies. Intracellular payload delivery and resulting cytotoxicity were used as a readout for 5T4 antibody internalization after target binding. A bispecific toxin-conjugated antibody, which recognizes 5T4 with one Fab arm and an unrelated antigen (HIV-1 gp120, not expressed on tumor cells) with the second Fab arm, was generated by controlled Fab arm exchange between unconjugated 5T4 antibody and an IgG1-b12 antibody (specific for HIV-1 gp120) conjugated to the microtubule-disrupting agent Duostatin-3. The resulting bispecific Duostatin-3 conjugated antibody carries one toxin molecule per antibody (drug-to-antibody ratio of 1). Serial dilutions (0.00152–10 μg / mL, 3x) of a duostatin-3-conjugated bispecific antibody that binds monovalently to 5T4 were added to MDA-MB-468 cells (breast cancer cell line, ATCC, clone HTB-132) or HCC1954 cells (breast cancer cell line, ATCC, clone CRL-2338) seeded in flat-bottom 96-well tissue culture plates (5,000 cells / well, Greiner-bio-one, The Netherlands, catalog no. 655180). After incubating the cells at 37°C for 5 days, cell viability was assessed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA, catalog no. G7570) according to the manufacturer's instructions. Cytotoxicity curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 software (GraphPad Software, San Diego, CA, USA).

[0292] Figure 4 shows the cytotoxicity of Duostatin-3-conjugated bispecific antibodies monovalently binding to 5T4 in MDA-MB-468 cells (A) or HCC1954 cells (B). bsIgG1-5T4-H8-FEAR×b12-vcDuo3 exhibited a high ability to induce cytotoxicity, indicating the antibody's effective internalization. In contrast, bsIgG1-5T4-076-FEAR×b12-vcDuo3, bsIgG1-5T4-085-FEAR×b12-vcDuo3, and bsIgG1-5T4-127-FEAR×b12-vcDuo3 did not induce any cytotoxicity, and the dose-response curves were similar to those of the nonbinding IgG1-b12-vcDuo3 control antibody. This indicates poor internalization of these antibodies after binding to membrane-bound 5T4. bsIgG1-5T4-059-FEAR×b12-vcDuo3, bsIgG1-5T4-106-FEAR×b12-vcDuo3, bsIgG1-5T4-207-FEAR×b12-vcDuo3, and bsIgG1-5T4-226-FEAR×b12-vcDuo3 induced intermediate cytotoxicity in both cell lines tested, indicating that these monovalent 5T4 antibodies induced internalization, but not to the same extent as bsIgG1-5T4-H8-FEAR×b12-vcDuo3.

[0293] Example 8: Humanized CD3 antibodies to generate CD3x5T4 bispecific antibodies 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 with the mutations L234F, L235E, D265A, and F405L, which allow for the generation of bispecific antibodies by controlled Fab arm exchange, as described above, as well as amino acid substitutions in the Fc domain that disrupt interaction with the IgG Fc receptor (Fc gamma receptor [FcγR]) and complement. These mutations have previously been demonstrated to have no effect on target binding of the antibody into which they are introduced (see, e.g., US2015 / 0337049).

[0294] The production of humanized antibody IgG1-huCD3-H1L1-H101G is described in Example 2 of WO2017 / 009442. IgG1-huCD3-H1L1-H101G will be referred to as "IgG1-huCD3-H101G." Antibody IgG1-huCD3-H101G-FEAL is a variant thereof with amino acid substitutions L234F, L235E, D265A, and F405L, as described herein above.

[0295] Example 9: Determination of CD3 binding affinity using biolayer interferometry The binding affinities of selected CD3 antibodies, including IgG1-huCD3 and IgG1-huCD3-H101G, were determined as described in Example 7 of WO2017 / 009442.

[0296] Briefly, the binding affinity of selected CD3 antibodies in the IgG1-huCD3-FEAL format to recombinant soluble CD3ε (CD3E27-GSKa) (mature protein of SEQ ID NO:101) was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio). An anti-human Fc capture biosensor (ForteBio, catalog no. 18-5060) was loaded with hIgG (1 mg / mL) for 600 seconds. After a baseline measurement (200 seconds), the association (1000 seconds) and dissociation (2000 seconds) of CD3E27-GSKa were determined using a CD3E27-GSKa concentration range of 27.11 μg / mL to 0.04 μg / mL (1000 nM to 1.4 nM) in 3-fold dilution steps (sample diluent, ForteBio, catalog no. 18-5028). The theoretical molecular mass of CD3E27-GSKa, based on the amino acid sequence, i.e., 27.11 kDa, was used for calculations. Experiments were performed at 30°C with shaking at 1000 rpm. Each antibody was tested in at least two independent experiments. Data were analyzed using ForteBio Data Analysis Software v8.1 using a 1:1 model and a global full fit with an association time of 1000 seconds and a dissociation time of 100 seconds. Data traces were corrected by subtracting a reference curve (measurement with antibody on biosensor, sample diluent only), aligning the Y-axis to the last 10 seconds of baseline, and applying step-by-step correction and Savitzky-Golay filtering. Data traces with a response <0.05 nM were excluded from the analysis.

[0297] Table 5 shows the association rate constant k for recombinant CD3ε determined by biolayer interferometry. a (1 / Ms), dissociation rate constant k d (1 / s) and the equilibrium dissociation constant K D (M) shows the IgG1-huCD3-FEAL against recombinant CD3ε. D : 638nM) compared to D The binding affinity was 15 nM.

[0298] Table 5. Binding affinities of monospecific bivalent CD3 antibodies to recombinant CD3ε determined by label-free biolayer interferometry. TIFF2026041981000027.tif37128

[0299] Example 10: Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific antibodies were generated in vitro using DuoBody® platform technology, i.e., 2-MEA-induced Fab arm exchange as described in WO2011147986, WO2011131746, and WO2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5:962-973). To enable the production of bispecific antibodies by this method, IgG1 molecules were generated that carried a single mutation in the CH3 domain: the F405L mutation in one parental IgG1 antibody (i.e., CD3 antibody) and the K409R mutation in the other parental IgG1 antibody (i.e., 5T4 antibody or control HIV-1 gp120-specific antibody). In addition to these mutations, the parent IgG1 antibody contained substitutions, namely L234F, L235E, D265A (FEA), that result in an Fc domain that is unable to interact with the IgG Fc receptor (Fc gamma receptor) and complement.

[0300] To generate bispecific antibodies, the two parent antibodies were dissolved in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4 2- , 1.8mM H2PO4 - , 163.9mM Na + , 140.3 mM Cl -Equal masses of the antibodies were mixed in 1000 mM NaCl, pH 7.4. 2-Mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM, and the reaction mixture was incubated at 31°C for 5 hours. To allow reoxidation of interchain disulfide bonds and formation of intact bispecific antibodies, 2-MEA was removed by dialysis into PBS buffer using a Slide-A-Lyzer carriage with a 10 kDa molecular weight cutoff (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0301] The following antibodies were used in the examples:

[0302] CD3 antibody IgG1-huCD3-FEAL (having VH and VL sequences shown in SEQ ID NO:57 and SEQ ID NO:60) IgG1-huCD3-H101G-FEAL (having the VH and VL sequences shown in SEQ ID NO:68 and SEQ ID NO:60)

[0303] 5T4 antibody IgG1-5T4-207-FEAR (having VH and VL sequences shown in SEQ ID NO:40 and SEQ ID NO:44) IgG1-5T4-226-FEAR (having VH and VL sequences shown in SEQ ID NO:47 and SEQ ID NO:51) IgG1-5T4-059-FEAR (having VH and VL sequences shown in SEQ ID NO:5 and SEQ ID NO:9) IgG1-5T4-076-FEAR (having VH and VL sequences shown in SEQ ID NO:12 and SEQ ID NO:16) IgG1-5T4-085-FEAR (having VH and VL sequences shown in SEQ ID NO:19 and SEQ ID NO:23) IgG1-5T4-106-FEAR (having VH and VL sequences shown in SEQ ID NO:26 and SEQ ID NO:30) IgG1-5T4-127-FEAR (having VH and VL sequences shown in SEQ ID NO:33 and SEQ ID NO:37) IgG1-5T4-H8-FEAR (based on the 5T4 antibody H8 from Wyeth (WO2007 / 106744 and US2010 / 0173382); with VH and VL sequences shown in SEQ ID NO:87 and SEQ ID NO:88) IgG1-5T4-A1-F405L (based on 5T4 antibody A1 from Wyeth (WO2007 / 106744 and US8044178); with VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO:84) IgG1-5T4-A1-FEAR (based on 5T4 antibody A1 from Wyeth (WO2007 / 106744 and US8044178); with VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO:84) IgG1-5T4-A3-F405L (based on the 5T4 antibody A3 from Wyeth (WO2007 / 106744 and US8759495); with VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO:86) IgG1-5T4-A3-FEAR (based on the 5T4 antibody A3 from Wyeth (WO2007 / 106744 and US8759495); with VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO:86)

[0304] bispecific antibody TIFF2026041981000028.tif132128

[0305] Fluorescein isothiocyanate (FITC)-labeled bispecific antibodies TIFF2026041981000029.tif32128

[0306] Duostatin-3 conjugated bispecific antibody TIFF2026041981000030.tif54128

[0307] Non-binding control antibody IgG-b12 is an HIV-1 gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23) that is used in some examples as a negative, non-binding control second arm of the bispecific antibody. IgG1-b12-F405L is the variant with the substitution F405L. IgG1-b12-FEAL is a variant thereof with mutations that allow the generation of bispecific antibodies by controlled Fab arm exchange, namely L234F, L235E, D265A and F405L, as well as substitutions that result in an Fc domain that is unable to interact with the IgG Fc receptor (Fc gamma receptor) and complement. IgG1-b12-K409R is the variant with the substitution K409R. IgG1-b12-FEAR is a variant thereof with mutations that allow the generation of bispecific antibodies by controlled Fab arm exchange, namely L234F, L235E, D265A and K409R, as well as substitutions that result in an Fc domain that is unable to interact with the IgG Fc receptor (Fc gamma receptor) and complement.

[0308] Example 11: Binding of CD3x5T4 bispecific antibody to cynomolgus monkey 5T4 and human 5T4 expressed in HEK-293 cells The binding of bispecific monovalent CD3x5T4 and monospecific bivalent 5T4 antibodies to the plasma membrane of HEK-293 cells transiently transfected with human 5T4 or cynomolgus 5T4 (generated as described in Example 1) was analyzed by flow cytometry.

[0309] Cells (3 × 10 4Cells (10...

Claims

1. An antibody comprising at least one antigen-binding region capable of binding to 5T4 (trophoblast glycoprotein), An antibody capable of blocking the binding to 5T4 of antibody [059] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:

9.

2. a) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:44 [207]; b) an antibody [226] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:51; and c) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:5 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:9 [059] The antibody of claim 1, which blocks the binding of an antibody selected from the group consisting of:

3. a) an antibody [207] comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:40 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:44; and b) an antibody comprising a variable heavy chain (VH) region comprising the sequence set forth in SEQ ID NO:47 and a variable light chain (VL) region comprising the sequence set forth in SEQ ID NO:51 [226] 3. The antibody of claim 1 or claim 2, which blocks the binding of an antibody selected from the group consisting of:

4. 10. The antibody of any one of the preceding claims, wherein the 5T4 is human (Homo sapiens) 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:

1.

5. 4. The antibody of any one of claims 1 to 3, wherein the 5T4 is the mature polypeptide sequence of cynomolgus monkey (Macaca fascicularis) 5T4, e.g., SEQ ID NO:

2.

6. 4. The antibody of any one of claims 1 to 3, wherein the 5T4 is chicken (Gallus gallus) 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:

3.

7. 10. The antibody of any one of the preceding claims, wherein the 5T4 is human 5T4, e.g., the mature polypeptide of SEQ ID NO:1, and cynomolgus 5T4, e.g., the mature polypeptide of SEQ ID NO:

2.

8. The antibody of any one of the preceding claims, wherein the 5T4 is human 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:1, cynomolgus monkey 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:2, and chicken 5T4, e.g., the mature polypeptide sequence of SEQ ID NO:

3.

9. Human 5T4, cynomolgus monkey and / or chicken 5T4 are given a K of 1E-7M or less, for example, 5E-8M or less, 1E-8M or less, 5E-9M or less, or for example, 1E-9M or less. D and can bind with a binding affinity corresponding to a K value, for example in the range of 1E-7 to 5E-10 M, for example in the range of 1E-7 to 1E-9 M, for example in the range of 5E-8 to 5E-10 M, 5E-8 to 1E-9 M, for example in the range of 1E-8 to 5E-10 M, 1E-8 to 1E-9 M, or for example in the range of 1E-8 to 5E-9 M. D 4. The antibody of claim 1, wherein the antibody is capable of binding with a binding affinity corresponding to a value.

10. 10. The antibody of claim 9, wherein the binding affinity is determined by biolayer interferometry, optionally as described in Example 2 herein.

11. The binding affinity is I) immobilizing the antibody on an anti-human IgG Fc capture biosensor at 1 μg / mL for 600 seconds; II) determining the association of 5T4ECDHis (mature protein of SEQ ID NO:99) or cynomolgus 5T4 (mature protein of SEQ ID NO:2) or recombinant cynomolgus 5T4 protein (Cusabio, Cat. No. CSB-MP024093MOV) over a 200 second period and the dissociation over a 1000 second period using a two-fold dilution series ranging from 100 nM to 1.56 nM; III) Relating data to a buffer control (0 nM) The antibody of any one of claims 9 and 10, wherein the antibody is determined using biolayer interferometry comprising:

12. 12. The antibody of any one of claims 9 to 11, wherein the binding affinity is determined using an antibody of any one of the preceding claims that is a monospecific bivalent antibody, such as an antibody that is a full-length IgG1.

13. a) an antibody [059] comprising a VH region comprising the sequence set forth in SEQ ID NO:5 and a VL region comprising the sequence set forth in SEQ ID NO:9; b) an antibody [076] comprising a VH region comprising the sequence set forth in SEQ ID NO:12 and a VL region comprising the sequence set forth in SEQ ID NO:16; c) an antibody [085] comprising a VH region comprising the sequence set forth in SEQ ID NO:19 and a VL region comprising the sequence set forth in SEQ ID NO:23; d) an antibody comprising a VH region comprising the sequence set forth in SEQ ID NO:26 and a VL region comprising the sequence set forth in SEQ ID NO:30 [106]; e) an antibody [127] comprising a VH region comprising the sequence set forth in SEQ ID NO:33 and a VL region comprising the sequence set forth in SEQ ID NO:37; f) an antibody [207] comprising a VH region comprising the sequence set forth in SEQ ID NO: 40 and a VL region comprising the sequence set forth in SEQ ID NO: 44; and g) an antibody comprising a VH region comprising the sequence set forth in SEQ ID NO: 47 and a VL region comprising the sequence set forth in SEQ ID NO: 51 [226] an epitope or antibody binding region or binding site on 5T4 recognized by any one of the antibodies selected from the group consisting of: The antibody of any one of the preceding claims, which recognizes

14. a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO:

86. is not an antibody binding region, binding site or epitope bound by an antibody selected from the group consisting of: a) an antibody [H8] comprising a VH region comprising the sequence set forth in SEQ ID NO: 87 and a VL region comprising the sequence set forth in SEQ ID NO: 88; b) an antibody [A1] comprising a VH region comprising the sequence set forth in SEQ ID NO: 83 and a VL region comprising the sequence set forth in SEQ ID NO: 84; and c) An antibody [A3] comprising a VH region comprising the sequence shown in SEQ ID NO: 85 and a VL region comprising the sequence shown in SEQ ID NO:

86. an antibody binding region, binding site, or epitope on 5T4 that is different from the antibody binding region, binding site, or epitope bound by an antibody selected from the group consisting of: The antibody of any one of the preceding claims, which binds to

15. An antibody described in any one of the above claims, wherein binding to 5T4 is blocked by binding to 5T4 of antibody [A3] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:

86.

16. An antibody that binds to 5T4ECDHis (the mature protein of SEQ ID NO:99) [A3], comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:85 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:

86.

10. The antibody of claim 1, wherein the antibody exhibits the following replacement:

17. An antibody described in claim 14 or claim 15, wherein cross-blocking or the ability of an antibody described in any one of the preceding claims to block the binding of another antibody to 5T4 is determined by a fluorescence-activated cell sorting (FACS) assay, for example in an assay performed as described in Example 5.

18. Cross-blocking, or the ability of an antibody of any one of the preceding claims to block the binding of another antibody to 5T4, is determined as the ability of the unconjugated antibody to block the binding of the conjugated antibody, and optionally 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, said antibody being conjugated to fluorescein isothiocyanate (FITC), and an excess of an unconjugated antibody that targets 5T4; ii) incubating the sample at 4°C for 30 minutes and then centrifuging the sample; iii) removing the supernatant from each sample, resuspending the cells in buffer, and determining the mean fluorescence intensity (MFI) of FITC using a flow cytometer; and iv) The percentage of binding is as follows: The difference in MFI between cells incubated with a mixture of FITC-conjugated and unconjugated antibodies and cells incubated without FITC-conjugated or unconjugated antibodies is multiplied by 100 and then divided by the difference in MFI between cells incubated with a mixture of FITC-conjugated and IgG-b12 antibodies and cells incubated without FITC-conjugated or unconjugated antibodies. The process of calculating The antibody of any one of claims 14 to 16, wherein the antibody is determined by a procedure comprising:

19. An antibody according to any one of claims 14 to 17, wherein the ability of the antibody to block the binding of another antibody to 5T4 or to displace another antibody from binding to 5T4ECDHis (mature protein of SEQ ID NO:99) is determined using biolayer interferometry, for example in the assay described in Example 3.

20. The ability of an antibody to block the binding of another antibody to 5T4 or to displace another antibody from binding to 5T4 can be determined using biolayer interferometry. i) immobilizing the antibody of any one of the preceding claims on an activated amine-reactive second generation biosensor in an amount of 20 μg / mL in 10 mM sodium acetate buffer; ii) quenching the biosensor with immobilized antibody in ethanolamine pH 8.5; iii) immersing the biosensor with immobilized antibody in a composition comprising 3.6 μg / mL (100 nM) human 5T4 ECDHis (mature protein of SEQ ID NO:99) for a period of 500 seconds, and then iv) immersing the biosensor with the immobilized antibody and 5T4ECDHis in a composition containing the other antibody targeting 5T4 at 10 μg / mL for a period of 500 seconds and determining the association response. The antibody of any one of claims 14 to 18, wherein steps i) to iv) are carried out at a temperature of 30°C with shaking at 1000 rpm.

21. an epitope or antibody binding region on human 5T4, comprising amino acid residues R73, Y92, and R94, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; The antibody of any one of the preceding claims, which binds to

22. an epitope or antibody binding region on human 5T4, comprising amino acid residues S69, R73, Y92, and R94, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; The antibody of any one of the preceding claims, which binds to

23. an epitope or antibody binding region on human 5T4, comprising amino acid residues R73, T74, Y92, R94, and N95, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO:1; The antibody of any one of claims 1 to 21, which binds to

24. The antibody of any one of claims 21 to 23, wherein the amino acid residues are directly involved in antibody binding.

25. 25. The antibody of any one of claims 21 to 24, wherein one or more of the additional amino acid residues L89, F111, L117, F138, L144, D148, N152, with the numbering of each amino acid residue indicating their respective positions in SEQ ID NO: 1, are involved in antibody binding, or indirectly involved in binding, such as by affecting protein folding and / or by affecting the positioning of one or more amino acid residues directly involved in antibody binding.

26. an epitope or antibody-binding region on human 5T4, in which amino acid residues R73, Y92, and R94 are directly involved in antibody binding, and one or more of amino acid residues F111, F138, L144, and D148 are indirectly involved in said binding, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1; The antibody of any one of the preceding claims, which binds to

27. an epitope or antibody-binding region on human 5T4, in which amino acid residues S69, R73, Y92, and R94 are directly involved in antibody binding, and one or more of amino acid residues F111, F138, and D148 are indirectly involved in said binding, with each amino acid residue numbering referring to its respective position in SEQ ID NO:1; The antibody of any one of claims 1 to 25, which binds to

28. an epitope or antibody-binding region on human 5T4, in which amino acid residues R73, T74, Y92, R94, and N95 are directly involved in antibody binding, and amino acid residue F138 is indirectly involved in said binding, with each amino acid residue numbering indicating its respective position in SEQ ID NO:1; The antibody of any one of claims 1 to 25, which binds to

29. The antibody of any one of claims 21 to 28, wherein the amino acid residues contained in the epitope or antibody binding region and optionally one or more additional amino acid residues are 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.

30. 30. The antibody of claim 29, wherein the alanine scanning is performed as described in Example 16 herein, or essentially as described in Example 16 herein.

31. Alanine scanning i) expressing mutant human 5T4 polypeptides, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO:1) are individually substituted with alanine, and wild-type 5T4 polypeptides (amino acid residues 32-355 of SEQ ID NO:1), in human embryonic kidney cells, e.g., HEK293 cells, so as to obtain a sample containing 70 to 90,000 cells, e.g., 80,000 cells, for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of antibody conjugated to fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, followed by washing each sample twice with 150-180 μL of FACS buffer (phosphate-buffered saline + 0.1% [w / v] BSA + 0.02% [w / v] sodium azide) and resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using This is carried out by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively. is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. Data are excluded from the analysis if they are predicted to be spatially distant from An antibody according to any one of claims 29 to 30.

32. The non-cross-blocking 5T4-specific control antibody in step iv) an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and -An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] 32. The antibody of claim 31, which is a bispecific antibody comprising:

33. 21. The antibody of any one of claims 1 to 20, which binds to 5T4 such that when any one or more of amino acid residues R73, Y92 and R94, with each amino acid residue numbering referring to its respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

34. 34. The antibody of any one of claims 1 to 20 and claim 33, which binds to 5T4 such that when any one or more of amino acid residues S69, R73, Y92 and R94, with each amino acid residue numbering referring to its respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

35. 34. The antibody of any one of claims 1 to 20 and claim 33, which binds to 5T4 such that when any one or more of amino acid residues R73, T74, Y92, R94 and N95, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

36. 36. The antibody of any one of claims 1 to 20 and claims 33 to 35, which binds to 5T4 such that when any one or more of amino acid residues L89, F111, L117, F138, L144, D148, N152, with the numbering of each amino acid residue indicating their respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

37. 37. The antibody of any one of claims 1 to 20 and claims 33 to 36, which binds to 5T4 such that when any one or more of amino acid residues R73, Y92, R94, F111, F138, L144 and D148, with the numbering of each amino acid residue referring to their respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

38. 37. The antibody of any one of claims 1 to 20 and claims 33 to 36, which binds to 5T4 such that when any one or more of amino acid residues S69, R73, Y92, R94, F111, F138 and D148, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

39. 37. The antibody of any one of claims 1 to 20 and claims 33 to 36, which binds to 5T4 such that when any one or more of amino acid residues R73, T74, Y92, R94, N95 and F138, with the numbering of each amino acid residue referring to its respective position in SEQ ID NO: 1, are substituted with alanine, there is loss of binding or reduced binding.

40. The antibody of any one of claims 33 to 39, wherein the effect of the alanine substitution is determined by alanine scanning of a polypeptide comprising amino acid residues 32 to 355 of SEQ ID NO:

1.

41. 40. The antibody of any one of claims 33 to 39, wherein the effect of the alanine substitution is determined by the procedure set forth in Example 16 herein, or by the procedure essentially set forth in Example 16 herein.

42. 40. The antibody of any one of claims 33 to 39, wherein loss of binding is defined as a Z-score for binding of less than 1.5, optionally calculated as set forth in Example 16 herein, or essentially as set forth in Example 16 herein.

43. The effect of alanine substitution is i) expressing mutant human 5T4 polypeptides, in which all amino acid residues except cysteine ​​and alanine in the extracellular domain of human 5T4 (corresponding to amino acid residues 32 to 355 of SEQ ID NO: 1) are individually substituted with alanine, and wild-type 5T4 polypeptides, in human embryonic kidney cells, e.g., HEK293 cells, so as to obtain a sample containing 70 to 90,000 cells, e.g., 80,000 cells, for each mutant 5T4 or wild-type 5T4; ii) incubating the cells in each sample with 20 μL of antibody conjugated to fluorescein isothiocyanate (FITC)-conjugated antibody (3 μg / mL in FACS buffer) for 40 minutes at room temperature, followed by washing each sample twice with 150-180 μL of FACS buffer (phosphate-buffered saline + 0.1% [w / v] BSA + 0.02% [w / v] sodium azide) and resuspending the cells in each sample in 30 μL of FACS buffer; iii) For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) for the viable single cell population in that sample, and the data for each test antibody was calculated using the following formula: (where "aa position" refers to the position mutated to alanine) normalizing the binding intensity to that of a non-cross-blocking 5T4-specific control antibody using is determined by a procedure including To express the loss or gain of antibody binding, a Z-score is calculated: where μ and σ are the mean and standard deviation of the normalized gMFI calculated from all variants, respectively. is calculated according to The gMFI of the control antibody for a particular 5T4 variant is the average gMFI (from all variants). 対照Ab Average gMFI of 対照Ab If lower than -2.5 × SD, data were excluded from the analysis and, optionally, If a residue binds with a Z-score just below -1.5 (e.g., between -1.5 and -1.8, e.g., between -1.5 and -1.7, or e.g., between -1.5 and -1.6), the residue is predicted to be buried, and the residue is The majority of residues predicted to be surface exposed, for which loss of binding or reduced binding is determined. Data are excluded from the analysis if they are predicted to be spatially distant from The antibody of claim 42.

44. The non-cross-blocking 5T4-specific control antibody in step iii) an antigen-binding region [A1] comprising the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 84, and -An antigen-binding region comprising the VH sequence shown in SEQ ID NO: 97 and the VL sequence shown in SEQ ID NO: 98 [B12] 44. The antibody of claim 43, which is a bispecific antibody comprising:

45. The antibody of any one of the preceding claims, which, when conjugated to a cytotoxic moiety, has reduced internalization capability, as indicated by reduced cytotoxicity, compared to antibody [H8] comprising a variable heavy chain (VH) region comprising the sequence shown in SEQ ID NO:87 and a variable light chain (VL) region comprising the sequence shown in SEQ ID NO:

88.

46. Cytotoxicity can be measured using the procedure set forth in Example 7, e.g. i) providing a monovalent antibody comprising a first Fab arm of the antibody of any one of the preceding claims and a second Fab arm capable of binding to the HIV viral protein gp120 (HIV-1 gp120), said antibody being conjugated to Duostatin-3; ii) contacting breast cancer cells MDA-MB-468 (ATCC clone HTB-132) or HCC1954 (ATCC clone CRL-1338) with the monovalent antibody at 37° C. for 5 days; and iii) determining cell viability The antibody of claim 45, wherein the antibody is determined by a procedure comprising:

47. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) [059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15 [076]; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22 [085]; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29 [106]; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36 [127]; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43 [207]; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50 [226]; and h) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to the CDR1, CDR2 and CDR3 sequences set forth in any one of a) to g). The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) selected from the group consisting of:

48. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) [059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43 [207]; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50 [226]; and d) a heavy chain variable region (VH) comprising a CDR1, CDR2 and CDR3 sequence that comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to the CDR1, CDR2 and CDR3 sequences set forth in any one of a) to c). The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) selected from the group consisting of:

49. An antibody described in any one of the above claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) [059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6, 7 and 8.

50. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH) [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 41, 42 and 43.

51. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of heavy chain variable regions (VH) [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 48, 49 and 50.

52. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively [059]; b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 13, 14 and 15, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 17, DAS and SEQ ID NO: 18, respectively [076]; c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 21 and 22, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 24, DAS and SEQ ID NO: 25, respectively [085]; d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, 28 and 29, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 31, 32 and 33, respectively [106]; e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 34, 35 and 36, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 38, DAS and SEQ ID NO: 39, respectively [127]; f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 45, DAS and SEQ ID NO: 46, respectively [207]; g) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 52, DAS and SEQ ID NO: 53, respectively [226]; and h) a heavy chain variable region (VH) and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 sequences, each of which comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions compared to the CDR1, CDR2 and CDR3 sequences set forth in any one of a) to g); The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

53. The six complementarity-determining regions (CDRs) of the antigen-binding region capable of binding to 5T4 are: iv) CDR sequences of SEQ ID NO:6, 7, 8, 10, AAS and SEQ ID NO:11 [059]; v) SEQ ID NO: 41, 42, 43, 45, DAS and CDR sequences of SEQ ID NO: 46 [207], or vi) SEQ ID NO: 48, 49, 50, 52, DAS and CDR sequences of SEQ ID NO: 53 [226] 6. The antibody of claim 1, wherein the antibody comprises a total of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or at most 10 amino acid substitutions when compared to

54. 30. The antibody of any one of claims 22 to 29, wherein one, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acid substitutions are conservative amino acid substitutions.

55. The antibody of any one of the preceding claims, comprising one or two heavy chain variable regions [059, 207, 226] comprising 6 consecutive amino acid residues of the sequence shown in SEQ ID NO: 102 (YYGMDV) in the complementarity determining region 3 (CDR3).

56. The antibody of claim 55, wherein the six consecutive amino acid residues are the most C-terminal amino acid residues within CDR3.

57. The antigen-binding region capable of binding to 5T4 is selected from the CDR1 sequence of SEQ ID NO:41 (GGSFSGYY), the CDR2 sequence of SEQ ID NO:103 (IDHSX 1 ST) and the CDR2 sequence of SEQ ID NO:104 (AX 2 WFGELX 3 X 4 YYYGMDV) and one or two heavy chain variable regions (VH) containing the CDR3 sequence of SEQ ID NO: 105 (QSVSSX 5 and a light chain variable region (VL) comprising the CDR1 sequence of SEQ ID NO: 46 (QQRSNWPLT), the CDR2 sequence DAS, and the CDR3 sequence of SEQ ID NO: 46 (QQRSNWPLT), wherein: X 1 is G or E, and X 2 is A or G, and X 3 is W or Y, and X 4 is D or H, and X 5 is Y or F [207, 226].

58. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:10, AAS and SEQ ID NO:11, respectively [059].

59. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:41, 42 and 43, respectively, and a 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].

60. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:48, 49 and 50, respectively, and a 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].

61. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) [059] 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; 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 [076]; 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 [085]; 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 [106]; 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 [127]; 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 [207]; and 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 [226] The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) selected from the group consisting of:

62. 59. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises 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.

63. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises 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 [207].

64. The antibody of any one of the preceding claims, wherein the antigen-binding region capable of binding to 5T4 comprises 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 [226].

65. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or comprising 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 comprising 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 [059]; 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 [076]; 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 [085]; 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 [106]; 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 [127]; 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]; 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] The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

66. an antigen-binding region capable of binding to 5T4, a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:9 [059]; b) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 12 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 16 [076]; c) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 19 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 23 [085]; d) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:26 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:30 [106]; e) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 33 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 37 [127]; f) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 [207]; and g) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 and a light chain variable region (VL) comprising the sequence of SEQ ID NO:51 [226] The antibody of any one of the preceding claims, comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

67. The antibody of any one of the preceding claims, which is a full-length antibody, such as a full-length IgG1 antibody.

68. The antibody of any one of the preceding claims, which is a monovalent antibody.

69. The antibody of any one of the preceding claims, which is a bivalent antibody.

70. The antibody of any one of the preceding claims, which is a monospecific antibody.

71. The antibody of any one of the preceding claims, which is a bispecific antibody.

72. 10. The antibody of any one of the preceding claims, comprising an antigen-binding region of an antibody that binds to CD3, such as human CD3ε (epsilon), such as the human CD3ε (epsilon) set forth in SEQ ID NO:

4.

73. The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively [wild-type anti-CD3 (SP34 / humanized SP34, WO2015001085 (Genmab))-VH CDR sequences]; and optionally, a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:58, GTN, and SEQ ID NO:59, respectively [wild-type anti-CD3, VL CDR sequences]; 73. The antibody of claim 72, comprising:

74. The antigen-binding region that binds to CD3 is a heavy chain variable region (VH) comprising 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 to the sequence of SEQ ID NO: 57 [wild type anti-CD3 - VH full length sequence]; and optionally, a light chain variable region (VL) comprising 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 to the sequence of SEQ ID NO:60 [wild-type anti-CD3 - VL full-length sequence] 74. The antibody of claim 72 or claim 73, comprising:

75. 75. The antibody of any one of claims 72 to 74, which has a lower affinity for binding to human CD3ε than an antibody having an antigen-binding region comprising the VH sequence as set forth in SEQ ID NO:57 and the VL sequence as set forth in SEQ ID NO:60 (wild type anti-CD3 (humanized SP34, WO2015001085 (Genmab)) VH and VL sequences), preferably the affinity is at least 5 fold lower, such as at least 10 fold lower, such as at least 20 fold lower, at least 30 fold lower, at least 40 fold lower, at least 45 fold lower or such as at least 50 fold lower.

76. The antigen-binding region has an equilibrium dissociation constant K in the range of 200 to 1000 nM, e.g., in the range of 300 to 1000 nM, in the range of 400 to 1000 nM, in the range of 500 to 1000 nM, in the range of 300 to 900 nM, in the range of 400 to 900 nM, in the range of 400 to 700 nM, in the range of 500 to 900 nM, in the range of 500 to 800 nM, in the range of 500 to 700 nM, in the range of 600 to 1000 nM, in the range of 600 to 900 nM, in the range of 600 to 800 nM, or for example in the range of 600 to 700 nM. D 75. The antibody of any one of claims 72 to 74, which binds to CD3 at

77. The antigen-binding region has an equilibrium dissociation constant K in the range of 1 to 100 nM, e.g., in the range of 5 to 100 nM, in the range of 10 to 100 nM, in the range of 1 to 80 nM, in the range of 1 to 60 nM, in the range of 1 to 40 nM, in the range of 1 to 20 nM, in the range of 5 to 80 nM, in the range of 5 to 60 nM, in the range of 5 to 40 nM, in the range of 5 to 20 nM, in the range of 10 to 80 nM, in the range of 10 to 60 nM, in the range of 10 to 40 nM, or for example in the range of 10 to 20 nM. D 76. The antibody of claim 72 or claim 75, which binds to CD3 at

78. the antigen-binding region that binds to CD3 comprises a heavy chain variable (VH) region including a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; the heavy chain variable (VH) region has, when compared to a heavy chain variable (VH) region comprising the sequence set forth in SEQ ID NO:57, 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, numbered according to the sequence of SEQ ID NO:57 [VH_huCD3-H1L1]; and the wild-type light chain variable (VL) region comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, GTN and SEQ ID NO: 59, respectively; 78. The antibody of any one of claims 72 to 77.

79. The antibody of claim 72, wherein the CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 contain a total of at most 1, 2, 3, 4 or 5 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 of the sequence shown in SEQ ID NO:

57.

80. An antibody according to claim 72 or claim 73, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD3 have at least 95% sequence identity, for example at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity, to the amino acid sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain variable (VH) region, wherein the sequence identity is calculated based on an alignment of the amino acid sequence consisting of the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable (VH) region of the antigen-binding region that binds to CD with an amino acid sequence comprising the sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain variable (VH) region.

81. 75. The antibody of any one of claims 72 to 74, wherein the antigen-binding region that binds to CD3 comprises a mutation selected from the group consisting of T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V.

82. a bispecific antibody that lacks or has reduced Fc-mediated effector functions (an "inactive" antibody) and comprises the antigen-binding region of an antibody that binds to CD3; a) when purified PBMCs or T cells are used as effector cells, they are able to mediate concentration-dependent cytotoxicity of SK-OV-3 cells, e.g., when assayed as described in Example 14 herein; b) when purified T cells are used as effector cells, they are able to mediate cell concentration-dependent cytotoxicity of MDA-MB-231 when assayed, for example, as described in Example 13 herein; c) capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells, e.g., when assayed as described in Example 13 herein; d) capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, CaSki and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; e) purified T cells, when used as effector cells, are capable of inducing cytotoxicity of BxPC-3, PANC-1, Ca Ski and / or SiHa tumor cells, e.g., when assayed as described in Example 17 herein; and / or f) NOD.Cg-Prkdc cells inoculated with human MDA-MB-231 tumor cells scid Il2rg tm1Wjl exhibit anti-tumor activity, such as delayed tumor outgrowth, in a humanized immune hematopoietic stem cell reconstituted mouse xenograft model, such as IFN-γ / SzJ, as determined, for example, as described in Example 15; An antibody according to any one of the preceding claims.

83. The ability of the antibodies to mediate concentration-dependent cytotoxicity of SK-OV-3 cells was i) isolating peripheral blood mononuclear cells (PBMCs) or T cells from healthy human donor buffy coats; ii) a first set of samples, each sample comprising PBMCs and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of PBMCs:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, 8:1, and 12:1; and a second set of samples, each sample comprising T cells and human ovarian adenocarcinoma SK-OV-3 cells, the ratios of T cells:SK-OV-3 cells in the samples being 1:2, 1:1, 2:1, 4:1, and 8:1; preparing a iii) adding the antibody to each set of samples at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; and then iv) assessing the viability of SK-OV-3 cells using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) 83. The antibody of claim 82, wherein the cytotoxicity is determined in an in vitro cytotoxicity assay comprising:

84. The ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising T cells and human breast adenocarcinoma MDA-MB-231 cells, wherein the ratio of T cells:MDA-MB-231 cells in the samples is 8:1; iii) adding antibody to the sample set at a concentration ranging from 0.0128 ng / mL to 1000 ng / mL and incubating the samples at 37°C for 72 hours; iv) staining the T cells by incubating with fluorescently labeled antibodies against T cell activation markers, such as CD69-APC, CD25-PE-Cy7 and CD279 / PD1-BV604 antibodies, for 30 minutes at 4°C; and v) analyzing the sample by flow cytometry The antibody of claim 82, wherein the antibody is determined in an assay comprising:

85. In vitro activation of T cells in the presence of BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) isolating T cells from healthy human donor buffy coats; ii) providing a set of samples, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells, wherein the ratio of T cells:tumor cells in the samples is 4:1; iii) adding antibody to the sample set at a concentration of 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilutions) and incubating the samples at 37°C for 72 hours; iv) collecting 110 μL of T cell-containing supernatant from each sample and staining the T cells by incubating with fluorescently labeled antibodies against T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and antibodies against T cell markers, such as 69-APC, CD25-PE-Cy7, and CD279 / PD1-BV604 antibodies, at 4° C. for 30 minutes; and v) analyzing the sample by flow cytometry The antibody of claim 82, wherein the antibody is determined by a procedure comprising:

86. The ability to induce cytotoxicity in BxPC-3, PANC-1, CaSki and / or SiHa tumor cells i) providing T cells isolated from a healthy human donor buffy coat; ii) providing a set of test samples and a control sample, each sample comprising the T cells and BxPC-3, PANC-1, Ca Ski, or SiHa tumor cells adhered to the bottom of a 96-well tissue culture plate, wherein the ratio of T cells to tumor cells in the sample is 4:1; iii) adding antibody to a set of test samples at concentrations ranging from 0.0128 ng / mL to 5000 ng / mL (e.g., 5-fold dilutions), while control samples are left untreated or incubated with 5 μM staurosporine, and incubating all samples at 37° C. for 72 hours; iv) incubating the adherent cells in 10% (w / w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin) in RPMI-1640 medium supplemented with 10% (w / w) iron-containing donor bovine serum and penicillin / streptomycin for 4 hours at 37°C; v) Measure the absorbance of the cells and calculate the percentage of viable cells by setting the absorbance of cells incubated with staurosporine as 0% viability and untreated cells as 100% viability. The process is calculated as The antibody of claim 82, wherein the antibody is determined by a procedure comprising:

87. an antigen-binding region capable of binding to CD3, a) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 61, 55 and 56, respectively [VH CDR1-T31P + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or b) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 63, 55 and 56, respectively [VH CDR1-T31M + wild-type VH CDR2, 3] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1, 2, 3]; or c) a heavy chain variable region (VH) [VH CDR-N57E + wild-type VH CDR1, 3] comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 65 and 56, respectively, and a light chain variable region (VL) [wild-type VL CDR1, 2, 3] comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 58, 69, and 59, respectively; or d) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 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 set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] e) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 69, respectively [wild-type VH CDR1,2 + VH CDR3-H101N] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 69, respectively [wild-type VL CDR1,2,3]; f) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 71, respectively [wild-type VH CDR1,2 + VH CDR3-G105P] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; g) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 73, respectively [wild-type VH CDR1,2 + VH CDR3-S110A] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or h) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 75, respectively [wild-type VH CDR1,2 + VH CDR3-S110G] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 58, 59, and 59, respectively [wild-type VL CDR1,2,3]; i) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 77, respectively [wild-type VH CDR1,2 + VH CDR3-Y114V] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or j) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 79, respectively [wild-type VH CDR1,2 + VH CDR3-Y114M] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3]; or k) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 54, 55 and 81, respectively [wild-type VH CDR1,2 + VH CDR3-Y114R] and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 58, sequence GTN and sequence set forth in SEQ ID NO: 59, respectively [wild-type VL CDR1,2,3] 85. The antibody of any one of claims 72 and 78 to 84, comprising:

88. 86. The antibody of any one of claims 72 and 78 to 85, wherein the antigen-binding region capable of binding to CD3 comprises a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively, and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 59, respectively.

89. an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, AAS and SEQ ID NO: 11, respectively [059]; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; An antibody according to any one of claims 72 and 78 to 85.

90. an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 45, DAS and SEQ ID NO: 46, respectively [207]; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; An antibody according to any one of claims 72 and 78 to 85.

91. an antigen-binding region capable of binding to 5T4 comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 52, DAS and SEQ ID NO: 53, respectively [226]; and an antigen-binding region capable of binding to CD3 comprising a heavy chain variable region (VH) [wild-type VH CDR1,2 + VH CDR3-H101G] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 54, 55 and 67, respectively; and a light chain variable region (VL) [wild-type VL CDR1,2,3] comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 58, 59, and 60, respectively; An antibody according to any one of claims 47 and 51 to 59.

92. an antigen-binding region capable of binding to human CD3, a) the VH sequence shown in SEQ ID NO:62 [VH T31P full-length sequence] and the VL sequence shown in SEQ ID NO:60 [wild-type full-length sequence]; b) the VH sequence shown in SEQ ID NO:64 [VH T31M full length sequence] and the VL sequence shown in SEQ ID NO:60; c) the VH sequence shown in SEQ ID NO: 66 [VH N57E full length sequence] and the VL sequence shown in SEQ ID NO: 60; d) the VH sequence shown in SEQ ID NO: 68 [VH H101G full length sequence] and the VL sequence shown in SEQ ID NO: 60; e) the VH sequence shown in SEQ ID NO:70 [VH H101N full length sequence] and the VL sequence shown in SEQ ID NO:60; f) the VH sequence shown in SEQ ID NO: 72 [VH G105P full length sequence] and the VL sequence shown in SEQ ID NO: 60; g) the VH sequence shown in SEQ ID NO: 74 [VH S110A full length sequence] and the VL sequence shown in SEQ ID NO: 60; h) the VH sequence shown in SEQ ID NO: 76 [VH S110G full length sequence] and the VL sequence shown in SEQ ID NO: 60; i) the VH sequence shown in SEQ ID NO: 78 [VH Y114V full length sequence] and the VL sequence shown in SEQ ID NO: 60; j) the VH sequence shown in SEQ ID NO: 80 [VH Y114M full-length sequence] and the VL sequence shown in SEQ ID NO: 60, and k) the VH sequence shown in SEQ ID NO:82 [VH Y114R full-length sequence] and the VL sequence shown in SEQ ID NO:60 92. The antibody of any one of claims 72 and 78 to 91, comprising a VH sequence and a VL sequence selected from the group consisting of:

93. 93. The antibody of any one of claims 72 and 78 to 92, wherein the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown in SEQ ID NO: 68 [VH H101G full length sequence] and the VL sequence shown in SEQ ID NO:

60.

94. an antigen-binding region capable of binding to 5T4 comprising 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 [059 - VH full-length sequence]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; 94. The antibody of any one of claims 78 to 93.

95. an antigen-binding region capable of binding to 5T4, comprising 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 [207 - VH full length sequence]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody according to any one of claims 72 and 78 to 93.

96. an antigen-binding region capable of binding to 5T4 comprising 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 [226 - VH full length sequence]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody according to any one of claims 47 and 51 to 64.

97. an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO:9 [059]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody according to any one of claims 72 and 78 to 94.

98. an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO: 44 [207 - VH + VL full length sequence]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody described in any one of claims 72, 78 to 93 and 95.

99. an antigen-binding region capable of binding to 5T4 comprising 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 heavy chain light 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 SEQ ID NO: 51 [226 - VH + VL full length sequence]; and an antigen-binding region capable of binding to human CD3 comprising the VH sequence shown in SEQ ID NO: 68 [VH H101G full-length sequence] and the VL sequence shown in SEQ ID NO: 60; An antibody described in any one of claims 72, 78 to 93 and 96.

100. An antibody according to any one of the preceding claims, wherein each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.

101. 101. The antibody of claim 100, comprising two heavy chain constant regions (CH) and two light chain constant regions (CL).

102. comprising a first heavy chain and a second heavy chain; each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 region, and a CH3 region, wherein the first heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and the second heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, wherein the substitutions in the first heavy chain and the second heavy chain are not at the same position, and the amino acid positions are numbered according to EU numbering; An antibody according to claim 100 or claim 101.

103. 103. The antibody of any one of claims 100 to 102, wherein in said first heavy chain the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R and in said second heavy chain the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L, or vice versa.

104. comprising a first heavy chain and a second heavy chain; in both the first heavy chain and the second heavy chain, the amino acid residues at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain (EU numbering) are F and E, respectively; An antibody according to any one of the preceding claims.

105. comprising a first heavy chain and a second heavy chain; the amino acid residue at the position corresponding to position D265 in a human IgG1 heavy chain (EU numbering) is A in both the first heavy chain and the second heavy chain; An antibody according to any one of the preceding claims.

106. a) the antigen-binding region capable of binding to 5T4 is humanized; and / or b) the antigen-binding region capable of binding to CD3, if present, is humanized; An antibody according to any one of the preceding claims.

107. a) the antigen-binding region capable of binding to 5T4 is human; and / or b) the antigen-binding region capable of binding to CD3, if present, is human; An antibody according to any one of the preceding claims.

108. a) the antigen-binding region capable of binding to 5T4 is chimeric; and / or b) the antigen-binding region capable of binding to CD3, if present, is chimeric; An antibody according to any one of the preceding claims.

109. comprising a first heavy chain and optionally a second heavy chain; the first heavy chain, and, if present, the second heavy chain, are modified such that the antibody induces Fc-mediated effector function to a lesser extent than the same unmodified antibody; An antibody according to any one of the preceding claims.

110. The antibody of any one of the preceding claims, comprising a kappa (κ) light chain.

111. The antibody of any one of the preceding claims, comprising a lambda (λ) light chain.

112. The antibody of any one of the preceding claims, comprising a lambda (λ) light chain and a kappa (κ) light chain, such as an antibody having a heavy chain and a lambda light chain comprising a binding region capable of binding to CD3 and a heavy chain and a kappa light chain comprising a binding region capable of binding to 5T4.

113. An immunoconjugate or antibody-drug conjugate (ADC) comprising the antibody of any one of the preceding claims and a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, an antibiotic, or a radioisotope.

114. a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 according to any one of claims 1 to 113, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 according to any one of claims 72 to 113; A nucleic acid construct comprising:

115. a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 113, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 113 115. The nucleic acid construct of claim 114, further comprising:

116. a) a nucleic acid sequence encoding the heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 according to any one of claims 1 to 112, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to 5T4 according to any one of claims 1 to 112. An expression vector comprising:

117. a) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 112, and / or b) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 112 117. The expression vector of claim 116, further comprising:

118. A cell comprising a nucleic acid construct of any one of claims 114 to 115 or an expression vector of claim 116 or claim 117, such as a cell, e.g. a recombinant host cell, obtained by transfecting a host cell with a nucleic acid construct of any one of claims 114 to 115 or an expression vector of claim 116 or claim 117.

119. 119. The cell of claim 118, wherein the host cell is of human origin, such as a human embryonic kidney (HEK) cell, such as a HEK / Expi cell, or of rodent origin, such as a Chinese hamster ovary cell, such as a CHO / N50 cell.

120. A composition comprising an antibody according to any one of claims 1 to 112.

121. 113. A pharmaceutical composition comprising the antibody of any one of claims 1 to 112 and a pharmaceutically acceptable carrier.

122. 113. The antibody of any one of claims 1 to 112 for use as a medicament.

123. 113. The antibody of any one of claims 1 to 112 for use in the treatment of a disease.

124. 124. The antibody for use according to claim 122 or claim 123, wherein the disease is cancer.

125. 125. The antibody for use of claim 124, wherein the cancer is characterized by expression of 5T4 in at least some of the tumor cells.

126. 126. The antibody for use of any one of claims 122 to 125, wherein the cancer is selected from the group consisting of renal / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia.

127. 122. A method of treating a disease, comprising administering to a subject in need thereof the antibody of any one of claims 1 to 112, the composition of claim 120, or the pharmaceutical composition of claim 121.

128. 128. The method of claim 127, for the treatment of cancer.

129. 129. The method of claim 128, wherein the cancer is selected from the group consisting of renal / renal cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine / endometrial / cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, and acute myeloid leukemia.

130. a) culturing a host cell comprising the expression vector of claim 114 or claim 117; and b) Purifying the antibody from the culture medium 113. A method for producing an antibody according to any one of claims 1 to 112, comprising:

131. a) providing an antibody capable of binding to 5T4 by culturing a host cell comprising the expression vector of claim 116 or claim 117 under conditions that allow expression of the antibody capable of binding to 5T4, and purifying the antibody capable of binding to 5T4 from the culture medium; b) I) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 1123, and II) A nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to CD3 according to any one of claims 72 to 112. providing an antibody capable of binding to CD3 by culturing a host cell containing an expression vector comprising: c) incubating an antibody capable of binding to 5T4 with an antibody capable of binding to CD3 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and d) Obtaining antibodies 113. A method for producing an antibody according to any one of claims 1 to 112, comprising:

132. 114. A kit-of-parts, such as a kit for use as a companion diagnostic / to identify patients within a patient population who have a property that will respond to treatment with the antibody of any one of claims 1 to 112 or the immunoconjugate or antibody-drug conjugate (ADC) of claim 113, or for use in predicting the efficacy of said antibody or immunoconjugate or ADC when used to treat a patient, comprising an antibody of any one of claims 1 to 112 and instructions for use of said kit.

133. An anti-idiotype antibody that binds to an antigen-binding region capable of binding to 5T4 according to any one of claims 1 to 112.

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