5T4 binders and uses thereof

Abstract

The present disclosure provides 5T4 binding agents (e.g., antibodies, including multispecific antibodies such as bispecific antibodies and antibody-drug conjugates) and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 341,944, filed on May 13, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a computer-readable sequence listing submitted with this application in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted with this application is named "14529-107-228_SEQ_LISTING.xml", created on May 8, 2023, and is 102,425 bytes in size.

[0005] field

[0006] The present disclosure generally relates to binding agents, such as antibodies or fragments thereof, that bind 5T4, including human 5T4, and methods of use thereof.

[0007] background

[0008] 5T4 is an N-glycosylated transmembrane 72kDa glycoprotein containing 8 leucine-rich repeats. 5T4, also known as Wnt-activated inhibitory factor 1 or WAIF1, is commonly referred to as carcinoembryonic antigen or trophoblast glycoprotein (TPBG) because it is expressed in the fetal trophoblast (where it was first discovered). 5T4 is present in tumors including colorectal tumors, ovarian tumors and gastric tumors. Its expression is used as a prognostic aid. The expression of 5T4 in normal tissues is very limited, but it is widely present throughout the development of malignant tumors. Although its limited expression seems to make 5T4 a target in cancer treatment, the therapeutic success of binding agents and vaccines targeting 5T4 has not yet been achieved.

[0009] Thus, there remains a need in the art for agents that can target 5T4 to treat, prevent, or ameliorate 5T4-mediated diseases, disorders, or conditions, including those involving tumor cells expressing 5T4.

[0010] Overview

[0011] The present disclosure provides 5T4 binding agents, including human 5T4 binding agents. Such agents include antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies and antibody-drug conjugates (ADCs) that bind to 5T4. In some embodiments, such binding agents bind to the same epitope of human 5T4 as antibodies comprising CDRs described herein (e.g., Tables 1-3). In some embodiments, such binding agents bind to the same epitope of human 5T4 as antibodies comprising heavy chain variable regions and light chain variable regions described herein (e.g., Tables 1-3).

[0012] The present disclosure also provides compositions comprising 5T4 binding agents. In some embodiments, such compositions include antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies and ADCs that bind to 5T4. In some embodiments, such compositions include antibodies and ADCs that bind to substantially the same epitopes of human 5T4 as antibodies comprising CDRs described herein (e.g., Tables 1-3). In some embodiments, such compositions include antibodies and ADCs that bind to substantially the same epitopes of human 5T4 as antibodies comprising heavy chain variable regions and light chain variable regions described herein (e.g., Tables 1-3).

[0013] The present disclosure also provides methods for treating, preventing or alleviating 5T4-mediated diseases, disorders or conditions (including one or more symptoms of 5T4-mediated diseases, disorders or conditions) with 5T4-binding agents or compositions comprising the agents. Such compositions include antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies and ADCs that bind to 5T4. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A and Figure 1B Sequence alignment of the heavy chain variable region and light chain variable region (including VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of mAbA4, mAbA15 and mAbA17 is shown. The boundaries of the CDRs are indicated by Kabat, AbM, Chothia, Contact, IMGT and AHon numbering.

[0016] Detailed Description

[0017] The present disclosure provides 5T4 binding agents. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) and ADCs that bind to 5T4, including antibodies and ADCs that bind to human 5T4. Such binding agents can be used to treat, prevent or alleviate 5T4-mediated diseases, disorders or conditions (including one or more symptoms of diseases, disorders or conditions) compositions and methods. 5T4-mediated diseases, disorders and conditions include a variety of cancers, including but not limited to any cancer in which tumor cells express or overexpress 5T4. As used herein, the term "overexpression" means more genetic products that are transcribed and translated than normal (such as in normal cells), and its process is usually a feature of cancer cells. In addition, the 5T4 binding agents described herein, such as 5T4 binding antibodies (e.g., monospecific or multispecific antibodies, including bispecific antibodies) and 5T4 binding ADCs can be used to kill and / or remove tumor cells. The 5T4 binding agents described herein, such as 5T4 binding antibodies (e.g., monospecific or multispecific antibodies, including bispecific antibodies) and 5T4 binding ADCs can be used for compositions and methods for treating cancer.

[0018] Unless otherwise indicated, the term "5T4" refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native 5T4 from any vertebrate source, including mammals, such as primates (e.g., humans, cynomolgus monkeys (cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). 5T4, also known as "5T4 oncotrophoblast glycoprotein" or "Wnt activation inhibitory factor 1" or "5T4 oncotrophoblast glycoprotein" or "5T4 carcinoembryonic antigen" or "WAIF1" or "M6P1" or "5T4-AG" or "5T4AG" or "TPBG", is a glycoprotein encoded by the TPBG gene. The term 5T4 encompasses "full-length" 5T4, as well as any form of 5T4 or any fragment thereof produced by processing in a cell. In some embodiments, 5T4 comprises a signal sequence. In some embodiments, 5T4 does not include a signal sequence. In some embodiments, the term 5T4 refers to a fragment of full-length 5T4 that comprises the 5T4 extracellular domain. The term 5T4 also encompasses naturally occurring variants of 5T4, such as SNP variants, splice variants, and allelic variants.

[0019] The full-length amino acid sequence of human 5T4 is provided below (exemplary signal sequence = italic text; exemplary extracellular domain = underlined text):

[0020]

[0021] The full-length amino acid sequence of cynomolgus monkey (cyno) 5T4 is provided below (exemplary signal sequence = italicized text; exemplary extracellular domain = underlined text):

[0022]

[0023] The full-length amino acid sequence of mouse 5T4 is provided below (exemplary signal sequence = italic text; exemplary extracellular domain = underlined text):

[0024]

[0025] As used herein, the term "binding agent" or its grammatical equivalent refers to a molecule (e.g., an antibody) having one or more antigen binding sites that bind an antigen. In some embodiments, a 5T4 binding agent as described herein is an antibody, antibody fragment, or other peptide-based molecule that binds 5T4 (such as human 5T4), as well as a conjugate (e.g., ADC) of an antibody, antibody fragment, or peptide-based molecule.

[0026] The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and in the broadest sense, and specifically encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with multiple or single epitope specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human antibody forms with full-length heavy and / or light chains. As used herein, VHH refers to domain antibodies derived from the variable region of only heavy chain antibodies. Exemplary single domain antibodies include, but are not limited to, antibodies naturally lacking light chains, such as those from camelid species (e.g., llamas), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, and cattle. VHH can also be derived from other species besides Camelidae, which can produce heavy chain antibodies that naturally lack light chains. Antibodies also include antibody fragments (and / or polypeptides comprising antibody fragments) that retain 5T4 binding characteristics. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of antibodies, for example, Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual variable domain antibodies, single variable domains, linear antibodies, V regions, multispecific antibodies formed by antibody fragments, F(ab)2, Fd, Fc, double antibodies, double double antibodies, disulfide-linked Fv (dsFv), single domain antibodies (e.g., nanobodies) or other fragments (e.g., fragments consisting of variable regions of heavy and light chains that are non-covalently coupled). In general, the variable (V) region domain can be any suitable arrangement of immunoglobulin heavy chain (VH) and / or light chain (VL) variable domains. For example, antibody also includes a tetrameric antibody comprising two heavy chains and two light chain molecules, an antibody light chain monomer and an antibody heavy chain monomer. Therefore, for example, the V region domain can be a dimer and contain a VHH-VHH, VH-VH, VH-VL or VL-VL dimer in conjunction with 5T4. If desired, VH and VL can be covalently coupled directly or through a joint to form a single-chain Fv (scFv). For ease of reference, scFv protein is referred to herein as being included in the category "antibody fragment". Another form of antibody fragment is a peptide comprising one or more complementary determining regions (CDR) of an antibody. CDR (also referred to as "minimum recognition unit" or "hypervariable region") can be obtained by constructing a polynucleotide encoding one or more CDRs of interest.Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," In Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," In Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be incorporated into, for example, single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of novel antigen receptors (v-NARs), and dual single chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, antibodies comprising VH and / or VL contain light chain and / or heavy chain constant regions, for example, one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include epitope binding fragments of any of the above. The antibodies described herein may be any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0027] The term "monospecific" when referring to a binding agent (eg, an antibody or ADC) as used herein refers to a binding agent that has one or more binding sites, each of which binds to the same epitope of the same antigen.

[0028] When used with respect to a binding agent (e.g., an antibody or ADC), the term "bispecific" refers to a binding agent that is capable of specifically binding to at least two different antigenic determinants, such as two binding sites, each of which is formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or each binding site is formed by a pair of VHH domains that bind to different antigens or different epitopes on the same antigen. Such bispecific binding agents (e.g., antibodies or ADCs) may have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binding agent (e.g., antibody or ADC) formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., an antibody or ADC) comprises two antigen binding sites, each can bind to a different antigenic determinant. Such bispecific binding agents (eg, antibodies or ADCs) can bind to two different epitopes on the same antigen (eg, epitopes on 5T4).

[0029] In the context of two or more nucleic acids or polypeptides, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (if necessary, introducing gaps) to obtain maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, when compared and aligned using a sequence comparison algorithm or by visual inspection to measure maximum correspondence, two nucleic acids or polypeptides are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, and in some embodiments at least 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residue identity. In some embodiments, the identity is present in the region of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues or any integer value therebetween of the amino acid sequence. In some embodiments, the identity is present in a region longer than 60-80 residues, for example, at least about 80-100 residues, and in some embodiments, the sequence is substantially the same in the total length of the sequence being compared, such as the coding region of a target protein or an antibody. In some embodiments, the identity is present in the region of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases or any integer value therebetween of the nucleotide sequence. In some embodiments, the identity is present in a region longer than 60-80 bases, for example, at least about 80-1000 bases or more, and in some embodiments, the sequence is substantially the same in the total length of the sequence being compared (e.g., the nucleotide sequence encoding the target protein).

[0030] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain of similar chemical properties. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). For example, a substitution of phenylalanine for tyrosine is a conservative substitution. Typically, conservative substitutions in the sequence of a polypeptide, soluble protein, and / or antibody of the present disclosure will not eliminate binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence to a target binding site. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0031] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may include non-amino acids (e.g., interrupted by non-amino acids). The term also encompasses amino acid polymers that are natural or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as connection or conjugation to a portion such as a labeling component or a drug (e.g., a toxin) (directly or indirectly). This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides may exist as single chains or single-chain dimers.

[0032] As used herein, an "antigen" is a portion or molecule containing an epitope to which a binding agent (e.g., an antibody or ADC) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen to which a binding agent (e.g., an antibody or ADC) described herein binds is 5T4 (e.g., human 5T4) or a fragment thereof, including a fragment comprising one or more domains of 5T4.

[0033] As used herein, "epitope" is a term in the art, and refers to a local region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear or discontinuous epitope. For example, in the case of a polypeptide antigen, an epitope can be a continuous amino acid of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more discontinuous regions of a polypeptide (a "conformation", a "non-linear" or "discontinuous" epitope), such as human 5T4. It will be appreciated by those skilled in the art that, in general, a linear epitope may or may not be dependent on a secondary, tertiary or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids, whether or not they are folded in a native three-dimensional protein structure. In other embodiments, an antibody requires that the amino acid residues constituting the epitope exhibit a specific conformation (e.g., bend, twist, curn or fold) in order to recognize and bind the epitope.

[0034] An antibody binds to an "epitope" or "substantially the same epitope" or "the same epitope" as a reference antibody when the two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space. The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competition assay, which can be configured in many different formats, for example, using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate, or expressed on the surface of cells, and a radioactive, fluorescent, or enzymatic label is used to measure the ability of an unlabeled antibody to block the binding of a labeled antibody.

[0035] "Epitope binning" is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition properties of different antibodies, using competitive assays combined with computational processes to cluster antibodies based on their epitope recognition properties and identify antibodies with different binding specificities. As shown in Example 5, additional details about epitope binning and methods for determining epitope binding of antibodies are described herein.

[0036] As used herein, the terms "specific binding," "specific recognition," "immunospecific binding," "selective binding," "immunospecific recognition," and "immunospecific" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope) as such binding is understood by those of skill in the art. In some embodiments, "specific binding" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, with a longer duration, with a greater affinity, or some combination of the above, than with alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, Biacore TM , KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), OctetQK384 system (ForteBio, Menlo Park, CA) or other determinations known in the art. In some embodiments, when an antibody or antigen-binding domain binds an antigen with an affinity higher than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), the antibody or antigen-binding domain binds or specifically binds to the antigen. Typically, the specific or selective reaction will be at least twice the background signal or noise, and can be more than 10 times the background. See, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed. 1989) for discussion on antibody specificity. In some embodiments, the degree of binding of an antibody or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of an antibody or antigen-binding domain to its specific target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen has a Ka of at least 2 log, 2.5 log, 3 log, 4 log or greater than the Ka of the molecule that binds to another antigen. In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins. In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other non-5T4 proteins. In some embodiments, "specific binding" means, for example, that a polypeptide or molecule binds to an antigen with a K of about 0.1 mM or less, but more typically less than about 1 μM. D In some embodiments, "specific binding" means that a polypeptide or molecule binds to a protein or target with a K of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. DBinding to target. Due to the sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets in more than one species. Similarly, due to the homology in certain regions of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding, such as binding to a single target. Therefore, in some embodiments, a polypeptide or molecule can specifically bind to more than one target. In some embodiments, multiple targets can be bound by the same antigen binding site on a polypeptide or molecule. For example, in some cases, an antibody can include two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and include at least two antigen binding sites with different specificities. Usually, but not necessarily, reference to "binding" means "specific binding".

[0037] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding agent, such as an antibody or ADC) and its binding partner (e.g., an antigen, such as 5T4). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y can generally be expressed in terms of the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies generally bind antigen more slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen more quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. In one embodiment, "K D ” or “K D The K value can be measured by biolayer interferometry (BLI) using, for example, the Octet QK384 system (ForteBio, Menlo Park, CA). Alternatively, the K value can also be measured in a radiolabeled antigen binding assay (RIA). D For example, using the Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293: 865-881), or using Biacore TM Surface plasmon resonance (SPR) measurements using, for example, Biacore TM -2000 or Biacore TM-3000(Biacore TM , Inc., Piscataway, NJ). "On-rate" or "rate of binding" or "association rate" or "k on and “off-rate” or “rate of dissociation” or “dissociation rate” or “k off " can also be used, for example, OctetQK384 system (ForteBio, Menlo Park, CA) or Biacore TM -2000 or Biacore TM -3000(Biacore TM , Inc., Piscataway, NJ) were measured using the same SPR or BLI technology as described above.

[0038] When used in the context of a 5T4 binder (e.g., an antibody or ADC), the term "competition" means a binder that competes for the same epitope or binding site on a target, including competition between such binders as determined by an assay in which the binder under study prevents or inhibits specific binding of a reference molecule (e.g., a reference ligand or a reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., 5T4). Many types of competitive binding assays can be used to determine whether a test binder competes with a reference molecule for binding to 5T4 (e.g., human 5T4). Examples of assays that can be used include solid phase direct or indirect radioimmunoassays (RIA); solid phase direct or indirect enzyme immunoassays (EIA); sandwich competition assays (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung et al., (1990) Virology 176:546-552); solid phase direct label assays; solid phase direct label sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1996). Harbor Press); solid phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Molec. Immunol. 25: 7-15) and direct labeling RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32: 77-82). Typically, such assays involve the use of purified antigen (e.g., 5T4, such as human 5T4) bound to a solid surface or cell that carries an unlabeled test antigen binding protein (e.g., a test 5T4 antibody or ADC) or a labeled reference antigen binding protein (e.g., a reference 5T4 antibody or ADC). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to an adjacent epitope (e.g., a similar epitope or an overlapping epitope) that is sufficiently close to the epitope bound by the reference antibody to allow steric hindrance of the antibodies to occur. Typically, when the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 20%, such as at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0039] As used herein, the term "constant region" or "constant domain" is an antibody term well known in the art and refers to an antibody portion, such as the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding of the antibody to an antigen but may exhibit various effector functions, such as interactions with Fc receptors. The term includes portions of immunoglobulin molecules having amino acid sequences that are generally more conserved relative to immunoglobulin variable domains.

[0040] In some embodiments, antibody "effector function" refers to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody and that vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0041] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to its carboxyl terminus. For example, during the production or purification of an antibody, or by recombinant engineering of a nucleic acid encoding the heavy chain of an antibody, the C-terminal lysine in the Fc region (according to residue 447 of the EU numbering system) can be removed. Exemplary Fc region sequences are provided below (CH2 domain = bold text; CH3 domain = underlined text):

[0042]

[0043] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain), and can be assessed using various assays as disclosed.

[0044] A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of an Fc region found in nature and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, including or combined with other sequences, such as variable region sequences). Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A allotypes and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0045] "Variant Fc region" comprises an amino acid sequence that is different from a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution, such as about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, and preferably about one to about five amino acid substitutions, compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein may have at least about 80% homology with the native sequence Fc region and / or with the Fc region of the parent polypeptide, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may have a loss of effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are provided below (CH2 domain = bold text, amino acid changes are underlined; CH3 domain = underlined text):

[0046]

[0047] As used herein, the term "heavy chain", when used to refer to an antibody, refers to a polypeptide chain of about 50 kDa-70 kDa, wherein the amino terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion includes one or more constant regions. "Heavy chain" can refer to any of the different types based on the amino acid sequence of the constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0048] As used herein, the term "light chain", when used to refer to an antibody, refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. There are two different types based on the amino acid sequence of the constant domain, such as kappa (K) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0049] The terms "antigen binding fragment", "antigen binding domain", "antigen binding region" and similar terms refer to the portion of an antibody that contains amino acid residues (e.g., CDRs) that interact with an antigen and confer specificity and affinity to the binding fragment, domain or region for the antigen. As used herein, "antigen binding fragment" includes "antibody fragments" that contain a portion of an antibody that includes one or more CDRs, such as an antigen binding region or variable region of an antibody.

[0050] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, internal antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0051] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen binding sites that bind to a 5T4 antigen.

[0052] The antibody can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibody described herein is an IgG antibody (e.g., human IgG) or its class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass.

[0053] In some embodiments, the antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequence of the H chain is identical and the amino acid sequence of the L chain is identical. In some embodiments, the antibody is a 2-chain antibody unit comprising a VHH-VHH pair, wherein the amino acid sequence of the VHH is identical. In some embodiments, the H chain and / or the L chain comprises a constant region, such as a human constant region. In some embodiments, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such antibodies comprises a gamma heavy chain constant region, such as a human gamma heavy chain constant region. In some embodiments, such antibodies comprise an IgG constant region, such as a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0054] The antibody or fragment thereof can preferentially bind to 5T4, such as human 5T4, which means that the antibody or fragment thereof binds to 5T4 with a greater affinity than it binds to an unrelated control protein and / or binds to human 5T4 with a greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof can specifically recognize and bind to 5T4 or a portion thereof. "Specific binding" means that the affinity of the antibody or fragment thereof to bind to 5T4 is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000 or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof can essentially exclusively bind to 5T4 (e.g., being able to distinguish 5T4 from other known polypeptides, for example, by a measurable difference in binding affinity). In some embodiments, a 5T4 binding agent (e.g., an antibody or ADC) can react with a 5T4 sequence other than a human 5T4 sequence (e.g., a cynomolgus monkey 5T4 sequence).

[0055] The term "variable region", "variable domain" refers to a part of the light chain or heavy chain of an antibody, which is usually located at the amino terminal of the light chain or heavy chain, and has a length of about 120 to 130 amino acids in the heavy chain and a length of about 100 to 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that the sequences of certain segments of the variable region are widely different between antibodies. The V region mediates antigen binding and defines the specificity of a specific antibody to its specific antigen. However, variability is not evenly distributed within the 110 amino acid range of the variable region. Instead, the V region consists of a less variable (e.g., relatively unchanged) segment called a framework region (FR) of about 15-30 amino acids, separated by a shorter region with greater variability (e.g., extreme variability) called a "hypervariable region" or alternatively a "complementarity determining region". The variable regions of the heavy and light chains each contain four frameworks (FR1, FR2, FR3, and FR4), mainly adopting a β-folded configuration connected by three hypervariable regions, which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable regions in each chain are closely held together by the framework, and together with the hypervariable regions from the other chain, contribute to the formation of the antigen binding site of the antibody (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody-dependent cellular toxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the antibody. The sequence of the variable region is widely different between different antibodies. The variability of the sequence is concentrated in the CDR, and the less variable part in the variable region is called the framework region (FR). The CDR of the light and heavy chains is mainly responsible for the interaction of the antibody with the antigen. In specific embodiments, the variable region is a human variable region.

[0056] As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region" or "CDR" refer to regions of an antibody variable region whose sequence is hypervariable and / or forms structurally defined loops. Typically, an antibody comprises six hypervariable regions: three in VH (H1 or VH CDR1, H2 or VH CDR2, H3 or VH CDR3), and three in VL (L1 or VL CDR1, L2 or VL CDR2, L3 or VL CDR3). Many descriptions of hypervariable regions are in use and are encompassed herein. The Kabat CDR is based on sequence variability and is the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Whereas Chothia refers to the position of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDR and Chothia structural loops and are used in the AbM antibody modeling software of Oxford Molecular (see, e.g., Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on analysis of available complex crystal structures. The residues from each of these hypervariable regions or CDRs are mentioned below.

[0057] A universal numbering system, ImMunoGeneTics, has been developed and widely adopted Information system (Lefranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)). IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of humans and other vertebrates. In this article, CDR is referred to in terms of amino acid sequence and position in the light chain or heavy chain. Since the "position" of CDR within the structure of the immunoglobulin variable domain is conserved between species and is present in structures called loops, CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequence according to structural features. This information can be used for grafting and replacing CDR residues from immunoglobulins from one species into an acceptor framework, usually from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plückthun, J. Mol. Biol. 309: 657-670 (2001). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also shown below. The various systems known in the art or described herein represent different ways of delineating CDRs, and they are generally considered equivalent when they are used to define the same antibody. The exemplary system shown herein combines Kabat and Chothia.

[0058] Exemplary IMGT Kabat AbM Chothia Contact <![CDATA[V H CDR1]]> 26-35 27-38 31-35 26-35 26-32 30-35 <![CDATA[V H CDR2]]> 50-65 56-65 50-65 50-58 53-55 47-58 <![CDATA[V H CDR3]]> 95-102 105-117 95-102 95-102 96-101 93-101 <![CDATA[V L CDR1]]> 24-34 27-38 24-34 24-34 26-32 30-36 <![CDATA[V L CDR2]]> 50-56 56-65 50-56 50-56 50-52 46-55 <![CDATA[V L CDR3]]> 89-97 105-117 89-97 89-97 91-96 89-96

[0059] The hypervariable region may comprise the following "extended hypervariable region": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region" or "CDR" are used interchangeably.

[0060] The term "vector" refers to a material for carrying or containing a nucleic acid sequence, including, for example, introducing a nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or a marker operable for stable integration into the host cell chromosome. In addition, the vector may include one or more selective marker genes and appropriate expression control sequences. For example, the selective marker gene that may be included provides resistance to antibiotics or toxins, supplements nutritional deficiency defects, or provides key nutrients that are not available in the culture medium. The expression control sequence may include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more nucleic acid molecules are to be co-expressed (for example, antibody heavy chain and light chain or antibody VH and VL), two nucleic acid molecules may, for example, be inserted into a single expression vector or a separate expression vector. For single vector expression, the encoding nucleic acid may be operably connected to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods known in the art may be used to confirm that nucleic acid molecules are introduced into a host cell. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in sufficient amounts to produce the desired product (e.g., a 5T4 binder as described herein), and it will also be understood that methods well known in the art can be used to optimize expression levels to obtain sufficient expression.

[0061] "5T4-mediated disease," "5T4-mediated disorder," and "5T4-mediated condition" are used interchangeably and refer to any disease, disorder, or condition associated with or characterized by cells expressing 5T4, such as tumor cells expressing 5T4. 5T4-mediated diseases include cancer, including but not limited to cancers that express or overexpress 5T4.

[0062] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or potential causes, prevent the occurrence of symptoms and / or their potential causes, and / or improve or remedy damage caused by or associated with a disease, disorder, or condition. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0063] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., an antibody or ADC described herein or any other agent described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder or condition and / or symptoms associated therewith. A therapeutically effective amount of an agent (including a therapeutic agent) can be an amount necessary to (i) reduce or improve the development or progression of a given disease, disorder or condition, (ii) reduce or improve the recurrence, development or onset of a given disease, disorder or condition, and / or (iii) improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein). A "therapeutically effective amount" of a substance / molecule / agent of the present disclosure (e.g., a 5T4 antibody or ADC) can vary according to factors such as the disease state, age, sex, and weight of an individual and the ability of the substance / molecule / agent to elicit a desired response in an individual. A therapeutically effective amount encompasses an amount in which any toxic or deleterious effects of a substance / molecule / agent are exceeded by a therapeutically beneficial effect. In certain embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (eg, ADC or drug) effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0064] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of a disease, disorder or condition, or reducing the likelihood of the onset (or recurrence) of a disease, disorder or condition or associated symptoms.

[0065] The full therapeutic or preventive effect does not necessarily occur by administering one dose, and may occur only after administering a series of doses. Thus, a therapeutically or preventively effective amount may be administered in one or more administrations.

[0066] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0067] As used herein, "carrier" includes carriers, excipients, stabilizers, or preservatives that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. The carrier is typically an aqueous pH buffered solution. Examples of carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG) and PLURONICS TM . The term "carrier" can also refer to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete)), an excipient, or a vehicle administered with a therapeutic agent. Such a carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary carrier. Saline solutions, as well as aqueous dextrose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition can also include a small amount of a wetting agent or emulsifier, or a pH buffer. The composition can take the form of a solution, a suspension, an emulsion, a tablet, a pill, a capsule, a powder, a sustained release formulation, etc. Oral compositions (including formulations) may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions including pharmaceutical compounds may contain a preventive or therapeutically effective amount of a 5T4 binding agent (e.g., an antibody or ADC), such as in an isolated or purified form, together with a suitable amount of a carrier, to provide a form for appropriate administration to a subject (e.g., a patient). The formulation should be suitable for the mode of administration.

[0068] In some embodiments, the disclosure provides 5T4 binding agents that can be used as therapeutic agents herein. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) and ADCs that bind to 5T4. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heterologous conjugate antibodies, as well as variants thereof with increased or decreased affinity or other properties.

[0069] In some embodiments, described herein are 5T4 binders (e.g., antibodies or ADCs) that bind to 5T4 (including 5T4 polypeptides, 5T4 polypeptide fragments, 5T4 peptides, or 5T4 epitopes). In some embodiments, the 5T4 binder is a human or humanized antibody (e.g., comprising a human constant region) that binds to 5T4 (including 5T4 polypeptides, 5T4 polypeptide fragments, 5T4 peptides, or 5T4 epitopes). In some embodiments, the 5T4 binder (e.g., antibody or ADC), such as a human 5T4 binder, can bind to 5T4 expressed on the surface of a mammalian (e.g., human) cell (including a tumor cell expressing 5T4). In some embodiments, the 5T4 binder (e.g., antibody or ADC) binds to an extracellular epitope (e.g., 5T4 epitope) of 5T4 exposed on a cell (e.g., a tumor cell). In some embodiments, described herein are 5T4 binders (e.g., antibodies or ADCs) that bind to 5T4 (such as human 5T4 or a portion thereof). In some embodiments, 5T4 is human 5T4. In some embodiments, the 5T4-binding agent is a human 5T4-binding agent (eg, an antibody or ADC that binds human 5T4).Exemplary amino acid sequences of human 5T4 are described herein.

[0070] In some embodiments, a 5T4-binding agent (e.g., an antibody or ADC) described herein competes for binding to 5T4 (e.g., human 5T4) with a 5T4-binding agent (e.g., an antibody or ADC) comprising a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any antibody described herein, e.g., the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Tables 1-3. Thus, in some embodiments, the 5T4-binding agents (e.g., antibodies or ADCs) described herein compete for binding to 5T4 (such as human 5T4) with 5T4-binding agents (e.g., antibodies or ADCs) comprising one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from: (a) an antibody designated as mAbA4; (b) an antibody designated as mAbA15; and (c) an antibody designated as mAbA17, as shown in Tables 1-3. In some embodiments, the 5T4-binding agents (e.g., antibodies or ADCs) described herein compete for binding to 5T4 (such as human 5T4) with 5T4-binding agents (e.g., antibodies or ADCs) comprising one, two, and / or three VH CDRs and one, two, and / or three VL CDRs from: (a) an antibody designated as mAbA4; (b) an antibody designated as mAbA15; and (c) an antibody designated as mAbA17; as shown in Tables 1-3. In some embodiments, the 5T4-binding agents (e.g., antibodies or ADCs) described herein compete for binding to 5T4 (such as human 5T4) with 5T4-binding agents (e.g., antibodies or ADCs) comprising the VH region and VL region from (a) the antibody designated mAbA4; (b) the antibody designated mAbA15; and (c) the antibody designated mAbA17, as shown in Tables 1-3. In some embodiments, the 5T4-binding agents (e.g., antibodies or ADCs) described herein compete for binding to 5T4 (such as human 5T4) with 5T4-binding agents (e.g., antibodies or ADCs) comprising (a) a VH region comprising the amino acid sequence of SEQ ID NO: 25 and a VL region comprising the amino acid sequence of SEQ ID NO: 26; (b) a VH region comprising the amino acid sequence of SEQ ID NO: 44 and a VL region comprising the amino acid sequence of SEQ ID NO: 45; and (c) a VH region comprising the amino acid sequence of SEQ ID NO: 62 and a VL region comprising the amino acid sequence of SEQ ID NO: 63.

[0071] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein comprise the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of any antibody described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 depicted in Tables 1-3. Thus, in some embodiments, the 5T4 binding agents (e.g., antibodies or ADCs) described herein comprise one, two and / or three heavy chain CDRs and / or one, two and / or three light chain CDRs from: (a) an antibody designated as mAbA4; (b) an antibody designated as mAbA15; and (c) an antibody designated as mAbA17, as shown in Tables 1-3. In some embodiments, the 5T4-binding agents (e.g., antibodies or ADCs) described herein comprise one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from: (a) the antibody designated mAbA4; (b) the antibody designated mAbA15; and (c) the antibody designated mAbA17, as shown in Tables 1-3.

[0072] In some embodiments, the 5T4 binder (e.g., antibody or ADC) comprises a VH region of any one of the binders described herein, comprising VH CDR1, VH CDR2 and / or VH CDR3, and / or a VL region comprising VL CDR1, VLCDR2 and / or VL CDR3 (see, e.g., any one of Tables 1-3). Thus, in some embodiments, the 5T4 binder (e.g., antibody or ADC) described herein comprises one, two and / or three heavy chain CDRs and / or one, two and / or three light chain CDRs from Table 1. In some embodiments, the 5T4 binder (e.g., antibody or ADC) described herein comprises one, two and / or three heavy chain CDRs and / or one, two and / or three light chain CDRs from Table 2. In some embodiments, the 5T4 binder (e.g., antibody or ADC) described herein comprises one, two and / or three heavy chain CDRs and / or one, two and / or three light chain CDRs from Table 3.

[0073] The antibody designated mAbA4 comprises the VH sequence of SEQ ID NO:25 and the VL sequence of SEQ ID NO:26.

[0074] The antibody designated mAbA15 comprises the VH sequence of SEQ ID NO:44 and the VL sequence of SEQ ID NO:45.

[0075] The antibody designated mAbA17 comprises the VH sequence of SEQ ID NO:62 and the VL sequence of SEQ ID NO:63.

[0076]

[0077]

[0078]

[0079] In some embodiments, the 5T4 binders described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4 binders, comprise a VH region or a VH domain. Additionally or alternatively, in some embodiments, the 5T4 binders described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4 binders, comprise a VL region or a VL domain. In some embodiments, the 5T4 binders described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4 binders, have a combination of: (i) a VH domain or a VH region; and / or (ii) a VL domain or a VL region.

[0080] In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise a heavy chain having the following combination: (i) a VH as described in any one of Tables 1-3; and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences:

[0081] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 69).

[0082] Another exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences:

[0083] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 70).

[0084] In some embodiments, the 5T4 binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4 binding agents, comprise a light chain having the following combination: (i) a VL domain as described in any one of Tables 1-3; and (ii) a light chain constant domain (CL). An exemplary light chain (e.g., for pairing with an IgG heavy chain) comprises any VL sequence described herein and the following CL amino acid sequence:

[0085] RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 71).

[0086] In some embodiments, the 5T4 binders described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4 binders, comprise (a) a heavy chain having (i) a VH as described in any one of Tables 1-3 and (ii) a combination of one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3); and (b) a light chain having (i) a VL as described in any one of Tables 1-3 and (ii) a combination of a light chain constant domain (CL or CL1) in IgG form. Exemplary 5T4 binders (e.g., antibodies or ADCs) comprise an IgG heavy chain and a light chain, the IgG heavy chain comprising any VH sequence as described herein and an amino acid sequence of SEQ ID NO: 69 or 70, and the light chain comprising any VL sequence as described herein and an amino acid sequence of SEQ ID NO: 71.

[0087] In some embodiments, the 5T4-binding agents (e.g., antibodies, such as bispecific antibodies or ADCs) described herein, including human 5T4-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 1. In some embodiments, the 5T4-binding agents (e.g., antibodies, such as bispecific antibodies or ADCs) described herein, including human 5T4-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 2. In some embodiments, the 5T4-binding agents (e.g., antibodies, such as bispecific antibodies or ADCs) described herein, including human 5T4-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 3. In some embodiments, the 5T4-binding agents (e.g., antibodies, such as bispecific antibodies or ADCs) described herein, including human 5T4-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Tables 1, 2, and / or 3.

[0088] In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VHCDR1, VH CDR2, VH CDR3, listed in Table 1. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VLCDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3, listed in Table 1. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs listed in Table 1, e.g., VH CDR1, VH CDR2, VH CDR3, and one or more CDRs, including three VL CDRs listed in Table 1, e.g., VL CDR1, VL CDR2 and / or VL CDR3.

[0089] In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VHCDR1, VH CDR2, VH CDR3, listed in Table 2. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VLCDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3, listed in Table 2. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs listed in Table 2, e.g., VH CDR1, VH CDR2, VH CDR3, and one or more CDRs, including three VL CDRs listed in Table 2, e.g., VL CDR1, VL CDR2 and / or VL CDR3.

[0090] In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, listed in Table 3. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VLCDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3, listed in Table 3. In other embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs), including human 5T4-binding agents, comprise one or more CDRs, including three VH CDRs listed in Table 3, e.g., VH CDR1, VH CDR2, VH CDR3, and one or more CDRs, including three VL CDRs listed in Table 3, e.g., VL CDR1, VL CDR2 and / or VL CDR3.

[0091] In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies) comprise one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61. In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies, or ADCs) comprise two or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61. In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies, or ADCs) comprise three or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61. In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs) comprise four or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61. In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs) comprise five or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61. In some embodiments, the 5T4-binding agents described herein (e.g., antibodies, such as bispecific antibodies or ADCs) comprise six or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-43, or 46-61.

[0092] In some embodiments, the 5T4-binding agent (e.g., antibody, such as a bispecific antibody or ADC) described herein comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3. In other embodiments, the 5T4-binding agent (e.g., antibody, such as a bispecific antibody or ADC) described herein comprises one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3. In other embodiments, the 5T4-binding agent (e.g., antibody, such as a bispecific antibody or ADC) described herein comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3 and one or more VL CDRs listed in Tables 1-3. Thus, in some embodiments, the 5T4-binding agent (e.g., antibody, such as a bispecific antibody or ADC) described herein comprises a VH CDR1 having an amino acid sequence of any one of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 31, 34, 35, 39, 46, 50, 53, and 57. In some embodiments, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VH CDR2 having an amino acid sequence of any one of SEQ ID NOs: 2, 8, 14, 19, 24, 28, 32, 36, 40, 43, 47, 51, 54, 58, and 61. In some embodiments, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VH CDR3 having an amino acid sequence of any one of SEQ ID NOs: 3, 9, 15, 20, 29, 33, 37, 41, 48, 52, 55, and 59. In some embodiments, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from a VH CDR1, a VH CDR2, a VH CDR3 depicted in any one of the amino acid sequences depicted in Tables 1-3. In some embodiments, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VL CDR1 having an amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, and 21. In another embodiment, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VL CDR2 having an amino acid sequence of any one of SEQ ID NOs: 5, 11, and 22. In some embodiments, a 5T4-binding agent (e.g., an antibody, such as a bispecific antibody, or ADC) described herein comprises a VL CDR3 having an amino acid sequence of any one of SEQ ID NOs: 6, 17, 23, 30, 38, 42, 49, 56, and 60.In some embodiments, a 5T4-binding agent described herein (e.g., an antibody, such as a bispecific antibody or ADC) comprises a VL CDR1 and / or VL CDR2 and / or VL CDR3 independently selected from the VL CDR1, VL CDR2, VL CDR3 depicted in any one of the amino acid sequences depicted in Tables 1-3.

[0093] In some embodiments, a 5T4-binding agent described herein (e.g., an antibody, such as a bispecific antibody or ADC) comprises a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 35, and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58, and (v) SEQ ID NO: NO: 24, 43 or 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55, and (iv) SEQ ID NO: 20, 41 or 59; and / or a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16, and (iv) SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1 NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56, and (iii) SEQ ID NO: 23, 42 or 60.

[0094] In some embodiments, a 5T4-binding agent described herein (e.g., an antibody, such as a bispecific antibody or ADC) comprises a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 35, and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58, and (v) SEQ ID NO: NO: 24, 43 or 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55, and (iv) SEQ ID NO: 20, 41 or 59.

[0095] In some embodiments, a 5T4-binding agent described herein (e.g., an antibody, such as a bispecific antibody or ADC) comprises a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16, and (iv) SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 6, 30, or 49, (ii) SEQ ID NO: 17, 38, or 56, and (iii) SEQ ID NO: 23, 42, or 60.

[0096] In some embodiments, described herein are antibodies or fragments thereof that bind to 5T4, wherein the antibodies or fragments thereof comprise: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 35, and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58, and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH having an amino acid sequence selected from the group consisting of: CDR3: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55, and (iv) SEQ ID NO: 20, 41 or 59; and / or a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16, and (iv) SEQ ID NO: 21; (2) a VLCDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VLCDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56, and (iii) SEQ ID NO: NO: 23, 42 or 60.

[0097] In some embodiments, described herein are antibodies or fragments thereof that bind to 5T4, wherein the antibodies or fragments thereof comprise a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 35, and (v) SEQ ID NO: 18, 39 or 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28 or 47, (ii) SEQ ID NO: 8, 32 or 51, (iii) SEQ ID NO: 14, 36 or 54, (iv) SEQ ID NO: 19, 40 or 58, and (v) SEQ ID NO: 24, 43 or 61; and (3) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27 or 46, (ii) SEQ ID NO: 7, 31 or 50, (iii) SEQ ID NO: 12, 34 or 53, (iv) SEQ ID NO: 13 or 35, and (v) SEQ ID NO: 24, 43 or 61. CDR3: (i) SEQ ID NO: 3, 29 or 48, (ii) SEQ ID NO: 9, 33 or 52, (iii) SEQ ID NO: 15, 37 or 55, and (iv) SEQ ID NO: 20, 41 or 59.

[0098] In some embodiments, the present invention describes an antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises a light chain variable (VL) region comprising: (1) a VLCDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16, and (iv) SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 6, 30 or 49, (ii) SEQ ID NO: 17, 38 or 56, and (iii) SEQ ID NO: 23, 42 or 60.

[0099] In some embodiments, antibodies or fragments thereof that bind to 5T4 are described herein and include all three heavy chain complementary determining regions (CDRs) and / or all three light chain CDRs from the following: an antibody designated as mAbA4, which includes a VH sequence of SEQ ID NO: 25 and a VL sequence of SEQ ID NO: 26; an antibody designated as mAbA15, which includes a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 45; or an antibody designated as mAbA17, which includes a VH sequence of SEQ ID NO: 62 and a VL sequence of SEQ ID NO: 63. In some embodiments, the antibody or fragment thereof includes all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated as mAbA4. In some embodiments, the antibody or fragment thereof includes all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated as mAbA15. In some embodiments, the antibody or fragment thereof includes all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated as mAbA17.

[0100] In some embodiments, antibodies or fragments thereof that bind to 5T4 are described herein, wherein the antibody comprises: (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences depicted in Tables 1-3; and / or (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences depicted in Tables 1-3. In some embodiments, the antibody comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences depicted in Tables 1-3. In some embodiments, the antibody comprises a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences depicted in Tables 1-3.

[0101] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13 and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19 and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15 and 20; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11 and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17 and 23.

[0102] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 2; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0103] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 7; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 8; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 10; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0104] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 12; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 2; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0105] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 13; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 14; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 16; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.

[0106] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23.

[0107] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 24; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0108] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35 and 39; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40 and 43; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37 and 41; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11 and 22 and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 38 and 42.

[0109] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 27; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 28; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 30.

[0110] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 31; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 32; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 33; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 10; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 30.

[0111] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 34; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 28; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 30.

[0112] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 35; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 36; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 37; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 16; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 38.

[0113] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 39; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 40; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 41; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 42.

[0114] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 27; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 43; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 30.

[0115] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 46, 50, 53 and 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 58 and 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 55 and 59; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11 and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 56 and 60.

[0116] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 46; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 47; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 49.

[0117] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 50; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 51; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 52; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 10; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 49.

[0118] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 53; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 47; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 49.

[0119] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 13; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 54; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 55; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 16; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 56.

[0120] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 57; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 58; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 59; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 60.

[0121] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 46; (2) a VHCDR2 having an amino acid sequence of SEQ ID NO: 61; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 49.

[0122] In some embodiments, antibodies comprising the VH and / or VL regions described herein are described herein, wherein the VH and / or VL comprise human framework sequences. In some embodiments, the VH and / or VL regions comprise framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences, e.g., human FR1, human FR2, human FR3, and / or human FR4.

[0123] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized, human or chimeric antibodies. In some embodiments, the antibodies described herein are Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual variable region antibodies, single variable region antibodies, linear antibodies, V regions or multispecific antibodies formed by antibody fragments.

[0124] In some embodiments, described herein are binding agents that bind to substantially the same epitope as an antibody or fragment thereof of any of the antibodies described herein. In some embodiments, described herein are binding agents that compete with an antibody or fragment thereof of any of the antibodies described herein for binding to human 5T4. In some embodiments, the binding agent is an antibody or fragment thereof or an ADC comprising an antibody or fragment thereof.

[0125] In certain aspects, the CDRs of a 5T4 binder (e.g., an antibody or ADC), including a human 5T4 binder, can be determined according to the Kabat system (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0126] In certain aspects, the CDRs of a 5T4 binder (e.g., an antibody or ADC), including a human 5T4 binder, can be determined according to the Chothia system, which will be referred to herein as "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1):175-82; and U.S. Pat. No. 7,709,226).

[0127] In certain aspects, the CDRs of a 5T4 binder (e.g., an antibody or ADC), including a human 5T4 binder, can be prepared according to ImMunoGeneTics The IMGT CDRs can be determined systematically, e.g., as described in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDRs").

[0128] In certain aspects, the CDRs of a 5T4 binder (e.g., an antibody or ADC) (including a human 5T4 binder) can be determined according to the AbM system, which will be referred to herein as "AbM CDRs," e.g., as described in MacCallum et al., 1996, J. Mol. Bipl., 262: 732-745. See also, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0129] In certain aspects, the CDRs of a 5T4 binder (e.g., an antibody or ADC), including a human 5T4 binder, can be determined according to the Contact system, which will be referred to herein as "Contact CDRs" (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5:732-745). Contact CDRs are based on analysis of available complex crystal structures.

[0130] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of a 5T4-binding agent (e.g., antibody or ADC) described herein (including a human 5T4-binding agent) can be varied by one, two, three, four, five, or six amino acid positions, so long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the positions of the CDRs defining any of Table 1, Table 2, or Table 3 can be altered by shifting the N-terminal and / or C-terminal boundaries of the CDRs by one, two, three, four, five, or six amino acids relative to the current CDR positions, as long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In other embodiments, the length of one or more CDRs of the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the 5T4-binding agents (e.g., antibodies or ADCs) described herein (including human 5T4-binding agents) can be varied (e.g., shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described in SEQ ID NOs: 1-24, 27-43 or 46-61, as long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In other embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described in SEQ ID NOs: 1-24, 27-43 or 46-61, as long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%).In some embodiments, the amino terminus of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be shortened or extended by one, two, three, four, five or more amino acids compared to one or more CDRs described in SEQ ID NOs: 1-24, 27-43 or 46-61, as long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). Additionally or alternatively, in some embodiments, the carboxyl terminus of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be shortened or extended by one, two, three, four, five or more amino acids compared to one or more CDRs described in SEQ ID NOs: 1-24, 27-43 or 46-61, as long as binding to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). Whether binding to 5T4 (e.g., human 5T4) is maintained can be determined using any method known in the art, such as the binding assays and conditions described in the "Examples" section described herein. For example, Example 2 described herein describes an assay for measuring binding to 5T4 (e.g., human 5T4).

[0131] In other embodiments, the 5T4 binding agents (e.g., antibodies or ADCs) proposed herein that bind to 5T4, including human 5T4 binding agents, further comprise conservative sequence modifications. For polypeptides as 5T4 binding agents (e.g., antibodies), such as human 5T4 binding agents, conservative sequence modifications include conservative amino acid substitutions, which include those in which the amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. Therefore, in some embodiments, the non-essential amino acid residues predicted in 5T4 are replaced by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions that do not eliminate antigen binding and nucleotides encoding the same are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify the amino acid sequence of a 5T4 binder (e.g., an antibody or ADC) (including a human 5T4 binder) by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, amino acid sequence modification refers to at most 1, 2, 3, 4, 5 or 6 amino acid substitutions to a CDR (such as those described in any one of Tables 1-3). Thus, for example, each such CDR may contain at most 5 conservative amino acid substitutions, such as at most (no more than) 4 conservative amino acid substitutions, such as at most (no more than) 3 conservative amino acid substitutions, such as at most (no more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In some embodiments, a 5T4 binder (e.g., an antibody) (including a human 5T4 binder) contains one or more (including six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a CDR of mAbA4, mAbA15 or mAbA17 (see, e.g., Table 1, Table 2 or Table 3). In some embodiments, 5T4 binding agents (e.g., antibodies), including human 5T4 binding agents, contain VH and VL comprising CDRs identical to the CDRs of mAbA4, mAbA15, or mAbA17 (see, e.g., Table 1, Table 2, or Table 3). In some embodiments, the amino acid sequence modifications do not include any modifications within the SDRs. In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (such as CDR1, CDR2, CDR3, or any combination thereof). In additional embodiments, the amino acid sequence modifications are in the framework region or the constant region.

[0132] In some embodiments, provided herein is an antibody or fragment thereof comprising a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25 and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:26, and the binding of the antibody or fragment thereof to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0133] In some embodiments, provided herein is an antibody or fragment thereof comprising a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:44 and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:45, and binding of the antibody or fragment thereof to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0134] In some embodiments, provided herein is an antibody or fragment thereof comprising a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:62 and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:63, and binding of the antibody or fragment thereof to 5T4 (e.g., human 5T4) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0135] The present disclosure provides 5T4 binding agents (e.g., antibodies) having a masking portion and / or a cleavable portion, wherein one or more 5T4 binding domains of the 5T4 binding agent (e.g., antibody) are masked (e.g., via a masking portion) and / or activatable (e.g., via a cleavable portion). Techniques for masking 5T4 binding agents (e.g., antibodies) are well known in the art, including SAFEbody masking technology (see, e.g., U.S. Patent Application Publication No. 2019 / 0241886) and Probody masking technology (see, e.g., U.S. Patent Application Publication No. 2015 / 0079088). Such techniques can be used to produce masked and / or activatable 5T4 binding agents (e.g., antibodies). Such masked and / or activatable 5T4 binding agents (e.g., antibodies) can be used to prepare conjugates comprising any 5T4 binding agent (e.g., antibody) of the present disclosure, such as human 5T4 binding agents, including immunoconjugates, ADCs, masked ADCs, and activatable antibody-drug conjugates (AADCs), including those directly or indirectly linked to another agent such as a drug. For example, a 5T4 binding agent (e.g., antibody) of the present disclosure, such as a human 5T4 binding agent, can be covalently bound to one or more agents such as a drug via a synthetic linker.

[0136] If desired, the 5T4 binder (e.g., antibody) (including human 5T4 binders) is linked or conjugated (directly or indirectly) to a moiety having an effector function, such as cytotoxic activity (e.g., chemotherapeutic moiety or radioisotope) or immune recruitment activity. The moiety that is linked or conjugated (directly or indirectly) includes a cytotoxic drug (e.g., toxin, such as auristatin (aurostatin)) or a non-cytotoxic drug (e.g., a signal transduction regulator, such as a kinase); or a masking moiety that masks one or more binding domains of the 5T4 binder (e.g., antibody); or a cleavable moiety that allows one or more binding domains of the masked conjugate form of the 5T4 binder (e.g., antibody) to be unmasked in the tumor microenvironment by cleaving the cleavable moiety, thereby activating the 5T4 binder. Moieties that promote immune recruitment may include other antigen binders, such as viral proteins that selectively bind to cells of the innate and / or adaptive immune systems. Alternatively or additionally, 5T4 binders (e.g., antibodies), including human 5T4 binders, are optionally (directly or indirectly) linked or conjugated to a moiety that facilitates separation from a mixture (e.g., a tag) or a moiety with reporting activity (e.g., a detection marker or a reporter protein). It should be understood that the features of the 5T4 binders (e.g., antibodies), including human 5T4 binders, described herein also extend to polypeptides comprising 5T4 binder fragments.

[0137] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein that bind to human 5T4, including 5T4 binding agents, can be (directly or indirectly) connected or conjugated to a polypeptide, which can result in the production of an activatable antibody. In some embodiments, the 5T4 binding agent (e.g., antibody) (including human 5T4 binding agent) is connected or conjugated (directly or indirectly) to another agent. In some embodiments, the other agent is a drug, an ADC is produced, or when the antibody of the ADC comprises a masking portion and a cleavable portion, an AADC is produced.

[0138] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein (including human 5T4 binding agents) are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable agent. The conjugated or recombinantly linked antibodies, including masked or activatable conjugates, can be used, for example, to treat or prevent a disease, disorder, or condition, such as a 5T4-mediated disease, disorder, or condition. The conjugated or recombinantly linked 5T4 binding agents (e.g., antibodies), including masked or activatable conjugates, can be used, for example, to monitor or predict the onset, development, progression, and / or severity of a 5T4-mediated disease, disorder, or condition.

[0139] Such diagnosis and detection can be accomplished, for example, by coupling a 5T4 binding agent (e.g., an antibody) to a detectable substance, including, for example: an enzyme, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; a prosthetic group, including but not limited to streptavidin / biotin or avidin / biotin; a fluorescent material, including but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent material, including but not limited to luminol; a bioluminescent material, including but not limited to luciferase, luciferin, or aequorin; a chemiluminescent material, including but not limited to acridinium-based compounds or halogenated tags; a radioactive material, including but not limited to iodine ( 131 I. 125 I. 123 I and 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In and 111 In), Technetium ( 99 Tc), thallium ( 201 Ti、Ga( 68 Ga and 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133Xe、Fluorine( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Such as 68 Ge, 57 Co. 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, or 117 Sn; positron-emitting metals using various positron emission tomography; and non-radioactive paramagnetic metal ions.

[0140] Also described herein are 5T4-binding agents (e.g., antibodies) recombinantly linked or conjugated (covalently or non-covalently, directly or indirectly) to a heterologous protein or polypeptide (or fragment thereof, such as to a polypeptide (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce a fusion protein and their uses. Specifically, described herein are fusion proteins comprising an antigen binding site of a 5T4-binding agent (e.g., antibody) (including a human 5T4-binding agent) described herein. In some embodiments, the 5T4 binding agent (e.g., an antibody) can be used to target the 5T4 binding agent to specific cells (e.g., cells expressing 5T4, including tumor cells). Other non-limiting heterologous proteins, polypeptides, or peptides linked to 5T4 binding agents (e.g., antibodies) can be used as internalization signals or immune cell engagers.

[0141] In addition, the 5T4 binders (e.g., antibodies) described herein, including human 5T4 binders, can be linked (directly or indirectly) to a marker or "tag" sequence, such as a peptide, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86: 821-24, hexa-histidine provides convenient purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin ("HA") tags (which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37: 767-78)) and "FLAG" tags.

[0142] Methods for linking or conjugating moieties, including polypeptides, to antibodies (directly or indirectly) are well known in the art, any of which can be used to prepare the antibody-drug conjugates or fusion proteins described herein.

[0143] In some embodiments, the 5T4 binding agent (e.g., antibody) described herein is a fusion protein. As used herein, the term "fusion protein" refers to a polypeptide comprising an amino acid sequence of a binding agent (e.g., antibody) and an amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not usually a part of an antibody). In certain embodiments, the fusion protein retains the biological activity of the 5T4 binding agent. In certain embodiments, the fusion protein comprises a 5T4 antibody VH region, a VL region, a VH CDR (one, two or three VH CDRs) and / or a VL CDR (one, two or three VL CDRs), wherein the fusion protein binds to a 5T4 epitope, a 5T4 fragment and / or a 5T4 polypeptide.

[0144] Fusion proteins can be produced, for example, by the techniques of gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of a 5T4 binder (e.g., an antibody), including a human 5T4 binder as described herein, including, for example, a 5T4 binder with higher affinity and lower off-rate (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some embodiments, 5T4 binders (including human 5T4 binders) can be altered by random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. The polynucleotides encoding the 5T4 binders described herein can be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0145] The 5T4 binding agents (e.g., antibodies) described herein (including human 5T4 binding agents) can also be attached to a solid support, which can be used for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0146] The 5T4-binding agents (eg, antibodies) described herein (including human 5T4-binding agents) can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0147] The linker can be a "cleavable moiety" that facilitates release of the linked or conjugated reagent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates (e.g., ADCs or AADCs) of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

[0148] Conjugates of antibodies and agents, including those wherein the agent is a drug for preparing an ADC or an AADC, can be prepared using a variety of bifunctional protein coupling agents, such as N-(β-maleimidopropoxy) succinimide ester (BMPS); N-ε-maleimidocaproyl-oxysuccinimide ester (ECMS); N-γ-maleimidocaproyl-oxysuccinimide ester (GMBS); 1,6-hexane-bis-vinyl sulfone (HBVS); succinimidyl-4-(-N-maleimidomethyl)cyclohexane alkane-1-carboxy-(6-amidohexanoate))(LC-SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH); succinimidyl 3-(bromoacetylamino) propionate (SBAP); succinimidyl iodoacetate (SIA); succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); succinimidyl-4-(N-maleimidomethyl)cyclohexane alkane-1-carboxylate (SMCC); succinimidyl 4-(p-maleimido-phenyl) butyrate (SMPB); succinimidyl-6-(β-maleimidopropionamido) hexanoate (SMPH); N-(ε-maleimidocaproyloxy) sulfosuccinimide ester (sulfo-ECMS); N-(γ-maleimidobutyloxy) sulfosuccinimide ester (sulfo-GMBS); N-(K-maleimidoundecanoyloxy) sulfosuccinimide ester (sulfo-K The following are the sulfosuccinimidyl compounds: m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS); sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB); sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC); sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB); and succinimidyl-(4-vinylsulfone)benzoate) (SVSB).

[0149] The present disclosure also contemplates that conjugates of antibodies and agents, including those wherein the agent is a drug for use in preparing an ADC or AADC, can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2nd ed., 2008)).

[0150] Conventional conjugation strategies for antibodies and agents (including where the agent is a drug for the preparation of an ADC or AADC) are based on random conjugation chemistry involving the ε-amino group of a Lys residue or the thiol group of a Cys residue, which results in heterogeneous conjugates. Recently developed technologies allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These include the engineering of "thiomas", which contain cysteine ​​substitutions that provide reactive thiol groups at positions on the heavy and light chains and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26: 925-32). In another approach, selenocysteine ​​was co-translationally inserted into the antibody sequence by recoding the stop codon UGA from stop to selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105: 12451-56; and Hofer et al., 2009, Biochemistry 48(50): 12047-57).

[0151] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein (including human 5T4 binding agents) are conjugated to cytotoxic agents. In some embodiments, the 5T4 binding agents (e.g., antibodies) disclosed herein (including human 5T4 binding agents) can be optionally conjugated to one or more cytotoxic agents disclosed herein or known in the art to produce ADC or AADC. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including but not limited to methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modicin A chain, α-sarcin, Aleurites fordii protein, dianthus protein, pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, curcin, croton protein, Sapaonaria officinalis inhibitor, gelonin, mitogellin, limitomycin, phenomycin, enomycin, and tricothecenes. In some embodiments, the cytotoxic agent is a radioactive isotope used to produce a radioconjugate or radioconjugate. A variety of radionuclides can be used to prepare radioconjugates, including but not limited to 90 Y. 125 I. 131 I. 123 I. 111 In, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re and 212Bi. Conjugates of polypeptides or molecules with one or more small molecule toxins (eg, calicheamicin, maytansinoids, trichothecenes, and CC1065, and toxinically active derivatives of these toxins) may also be used. Conjugates of polypeptides or molecules with cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0152] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein, including human 5T4 binding agents, are conjugated to drugs such as signal transduction regulators, pro-apoptotic agents, mitotic inhibitors, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormone agents, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors. In some embodiments, the mitotic inhibitor is a sea toxin, auristatin, maytansinoids, and plant alkaloids. In some embodiments, the drug is a sea toxin, auristatin, maytansinoids, and plant alkaloids. An example of auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). Examples of maytansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of actinomycins, anthracyclines, calicheamicins, and duocarmycins. An example of an actinomycin is pyrrolobenzodiazepine (pyrrolobenzodiazepine, PBD). Examples of anthracyclines are PNU-anthracyclines, such as PNU-159682 or derivatives.

[0153] 5T4 binding agents (e.g., antibodies) described herein, including human 5T4 binding agents, may be monospecific, bispecific, trispecific, or have greater multispecificity. Such agents may include monospecific or multispecific antibodies or ADCs. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., bispecific antibodies for 5T4, which have a first binding domain for a first epitope of 5T4 and a second binding domain for a second epitope of 5T4). In some embodiments, monospecific and multispecific (e.g., bispecific) antibodies or ADCs may be constructed based on the sequences of antibodies described herein, such as the CDR sequences listed in Tables 1-3. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse, chimeric, human, or humanized antibodies. In some embodiments, one binding specificity of the multispecific antibody is for 5T4, and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody can comprise more than one target (e.g., antigen) binding domain, wherein different binding domains are specific for different targets (e.g., a first binding domain that binds 5T4 and a second binding domain that binds another target (e.g., antigen) such as an immune checkpoint modulator (e.g., a negative checkpoint modulator). In some embodiments, a multispecific (e.g., bispecific) antibody molecule can bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen). In some embodiments, one of the binding specificities is 5T4 and the other is for one or more of the following: cytotoxic T lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 2 (PD-L2), lymphocyte activation gene-3 (LAG-3; also known as CD223), galectin-3, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3), galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9), V domain Ig VISTA, glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, herpes virus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0154] Methods for preparing multispecific antibodies are known in the art, for example, by co-expressing two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305: 537-40). For further details on producing multispecific antibodies (e.g., bispecific antibodies), see, for example, bispecific antibodies (Kontermann, ed., 2011).

[0155] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9: 2, 182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28: 3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67: 95-106.

[0156] For example, bispecific antibody molecules can be divided into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen binding moieties attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG formats may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assemblies, charge pairs, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ body and / or orthogonal Fab.

[0157] In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, a "knobs-in-holes" strategy, a SEED platform, a common heavy chain (e.g., in a κλ body) and a heterodimer Fc region can be used to engineer a heavy chain for heterodimerization. Strategies to avoid homodimer heavy chain pairing in BsIgG are known in the art, including knobs-in-holes, dimers, asymmetry, charge pairs, HA-TF, SEEDbody and differential protein A affinity.

[0158] Another bispecific antibody form is an IgG with additional antigen binding moieties. For example, it is possible to engineer monospecific IgG to have bispecificity by attaching additional antigen binding units to monospecific IgG, for example, at the N- or C-terminal of heavy or light chains. Exemplary additional antigen binding units include single domain antibodies (e.g., variable heavy chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). The non-limiting examples of additional IgG forms include dual variable domain IgG (DVD-Ig), IgG (H) -scFv, scFv- (H) IgG, IgG (L) -scFv, scFv- (L) IgG, IgG (L, H) -Fv, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four in one). See Spiess et al., Mol. Immunol. 67 (2015): 95-106). In some embodiments, an exemplary antibody format is a B-Body format for monospecific or multispecific (e.g., bispecific antibodies), as described in, for example, International Patent Application Publication No. WO2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0159] Bispecific antibody fragment (BsAb) is the form of bispecific antibody molecule lacking some or all antibody constant domains.For example, some BsAb lack Fc region.In embodiments, bispecific antibody fragment includes heavy chain and light chain region connected by peptide linker allowing BsAb to be effectively expressed in single host cell.Non-limiting examples of bispecific antibody fragment include but are not limited to nanobody, nanobody-HAS, BiTE, double antibody, DART, TandAb, scDiabody, scDiabody-CH3, double antibody-CH3, three bodies (triple body), miniantibody (miniantibody), miniantibody (minibody), TriBi miniantibody, scFv-CH3KIH, Fab-scFv, scFv-CH-CL-scFv, F (ab ') 2, F (ab ') 2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, double antibody-Fc, tandem scFv-Fc and inner antibody.

[0160] Bispecific fusion proteins include antibody fragments connected to other proteins. For example, bispecific fusion proteins can be connected to other proteins to increase additional specificity and / or functionality. In some embodiments, docking lock (dock-and-lock, DNL) method can be used to produce bispecific antibody molecules with higher valence. For example, bispecific antibody fusions with albumin binding protein or human serum albumin can extend the serum half-life of antibody fragments. In embodiments, chemical conjugation, such as chemical conjugation of antibodies and / or antibody fragments, can be used to produce BsAb molecules. Exemplary bispecific antibody conjugates include CovX-body (CovX-body) forms, in which low molecular weight drugs are site-specifically conjugated to a single reactive lysine in each Fab arm or antibody or its fragment. In some embodiments, conjugation improves serum half-life.

[0161] Methods for producing multispecific antibodies (including bispecific antibodies) are known in the art. For example, multispecific antibodies (including bispecific antibodies) can be produced by expressing the component antibodies separately in different host cells and then purifying / assembling or by expressing the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be carried out by various methods known in the art (including affinity chromatography).

[0162] In some embodiments, the 5T4 binders (e.g., antibodies) disclosed herein, including human 5T4 binders, can be provided in any antibody form disclosed herein or known in the art. As a non-limiting example, in some embodiments, the 5T4 binders (e.g., antibodies), including human 5T4 binders, can be selected from: Fabs-in-tandem-Ig (FIT-Ig); DVD-Ig; hybrid hybridoma (quadroma or tetradoma); anticalin platform (Pieris); double antibody; single chain double antibody; tandem single chain Fv fragment; TandAb, trispecific Ab (Affimed); Darts dual affinity retargeting (Macrogenics); bispecific Xmab (Xencor); bispecific T cell engager (Bites; Amgen; 55kDa); triplet (Triplebody); Tribody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs); Duobody platform (Genmab); docking lock (dock and lock) platform; knobs-into-holes (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-Ig = dual variable domain immunoglobulin (Abbott); kappa-lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0163] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a 5T4 binding domain and one or more additional binding domains that bind to one or more non-5T4 targets. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a 5T4 binding domain comprising a VH and / or VL amino acid sequence as disclosed herein, such as those of Table 1, Table 2, or Table 3.

[0164] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies comprising a binding domain that binds 5T4, wherein the binding domain comprises the VH and VL CDRs disclosed herein, such as those shown in Table 1, Table 2, or Table 3.

[0165] Antibodies that bind to 5T4 can be obtained by any suitable method, such as (but not limited to) immunization with whole tumor cells containing 5T4 and collection of antibodies, recombinant technology, or screening libraries of antibodies or antibody fragments using epitopes of the extracellular domain of 5T4. Monoclonal antibodies can be produced using a variety of known techniques (see, e.g., Coligan et al. (eds.), Current Protocols in Immunology, 1: 2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli", DNA Cloning 2: Expression Systems, 2nd ed., Glover et al. (eds.), p. 93 (Oxford University Press 1995)). One exemplary technique for producing monoclonal antibodies involves immunizing an animal with a human 5T4 antigen and generating hybridomas from spleen cells taken from the animal. The hybridoma can produce a monoclonal antibody or antibody fragment that binds to 5T4.

[0166] In another embodiment, monoclonal antibodies or antibody fragments can be separated from antibody phage libraries produced using, for example, the techniques described below: Antibody Phage Display: Methods and Protocols, PMO'Brien and R.Aitken, Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phages displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened for the desired antigen. Clones expressing Fv fragments that can bind to the desired antigen are adsorbed onto the antigen and are therefore separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0167] The variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments (in which VH and VL are covalently linked by a short flexible peptide) or as Fab fragments (in which they are each fused to the constant domains and interact non-covalently), as described, for example, in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).

[0168] The repertoire of VH genes and VL genes can be cloned individually by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be searched for antigen-binding clones, as described in Winter et al. (supra). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self antigens and also self antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described, for example, by Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992).

[0169] Screening of the library can be accomplished by various techniques known in the art. For example, 5T4 (e.g., 5T4 polypeptides, fragments or epitopes) can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate or used for cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning a display library. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by using long washes and monovalent phage display, such as described in Bass et al., Proteins, 8: 309-314 (1990) and WO 92 / 09690; and low coating density of antigen, such as Marks et al., Biotechnol., 10: 779-783 (1992).

[0170] 5T4 binders (e.g., antibodies) can be obtained by designing an appropriate antigen screening program to select phage clones of interest, and then constructing full-length 5T4 binders (e.g., antibodies) clones using VH and / or VL sequences (e.g., Fv sequences) from the phage clones of interest or various CDR sequences from VH and VL sequences and appropriate constant region (e.g., Fc) sequences, as described in Kabat et al., Sequences of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vol. 1-3.

[0171] Likewise, human antibodies that bind to 5T4 can be produced by any of a variety of techniques, including, but not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V region phage library, or other procedures known in the art and based on the disclosure herein. Methods for obtaining human antibodies from transgenic animals are further described in, for example, Bruggemann et al., Curr. Opin. Biotechnol., 8:45558, 1997; Jakobovits et al., Ann. NY Acad. Sci., 764:52535, 1995; Green et al., Nature Genet., 7:13-21, 1994; Lonberg et al., Nature, 368:856-859, 1994; Taylor et al., Int. Immun. 6:579-591, 1994; and U.S. Pat. No. 5,877,397.

[0172] For example, human antibodies in conjunction with 5T4 can be obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigenic attack. For example, International Patent Publication No. WO 98 / 24893 discloses transgenic animals with human Ig loci, wherein due to the inactivation of endogenous heavy and light chain loci, the animals do not produce functional endogenous immunoglobulins. Transgenic non-primate mammal hosts capable of producing immune responses to immunogens are also described, wherein the antibodies have primate constant regions and / or variable regions, and wherein endogenous immunoglobulin encoding loci are replaced or inactivated. International Patent Publication No. WO 96 / 30498 discloses the use of Cre / Lox systems in mammals to modify immunoglobulin loci, such as replacing all or part of constant regions or variable regions to form modified antibody molecules. International Patent Publication No. WO 94 / 02602 discloses non-human mammal hosts with inactivated endogenous Ig loci and functional human Ig loci. U.S. Patent No. 5,939,598 discloses a method for preparing a transgenic mouse, wherein the mouse lacks endogenous heavy chains and expresses an exogenous immunoglobulin locus comprising one or more heterologous constant regions. Using transgenic animals, such as transgenic animals described herein, an immune response to a selected antigen molecule can be generated, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, etc. are known in the art and are used for immunization of transgenic mice described in, for example, International Patent Publication No. WO 96 / 33735. The ability of monoclonal antibodies to inhibit or neutralize the biological activity or physiological effects of the corresponding protein can be tested.

[0173] The present disclosure provides humanized antibodies that bind 5T4 (including human 5T4). The humanized antibodies of the present disclosure may include one or more CDRs from VH and / or VL disclosed herein, such as those shown in Tables 1-3. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from an "import" variable domain. Humanized antibodies that bind 5T4 can be generated using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8):397-402, 2000 and U.S. Pat. Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0174] In some cases, humanized antibodies are constructed by CDR transplantation, in which the amino acid sequences of the six CDRs of the VH and VL of a parent non-human antibody (e.g., a rodent) are transplanted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133-139, 1995) determined that only about one-third of the residues in the CDR actually contact the antigen, and referred to these as "specificity determining residues" or SDRs. In the SDR transplantation technique, only SDR residues are transplanted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36: 25-34, 2005).

[0175] The selection of human variable domains to be used in the production of humanized antibodies (both light and heavy chains) can be important for reducing antigenicity. For example, according to the so-called "best fit" method, the variable domain sequences of non-human (e.g., rodent) antibodies are screened for the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence can be selected as the human framework of the humanized antibody (Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mol. Biol. 196: 901). Another method uses a specific framework derived from the consensus sequence of all human antibodies of the light chain or heavy chain of a specific subgroup. The same framework can be used for several different humanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623). In some cases, the framework was derived from the most abundant human subtype, V L 6Subgroup I(V L 6I) and V H Subgroup III (V H III). In another approach, human germline genes are used as the source of framework regions.

[0176] In an alternative CDR comparison-based paradigm, called superhumanization, framework homology is irrelevant. The approach consists of comparing non-human sequences to a library of functional human germline genes. Those genes encoding canonical structures identical or closely related to the murine sequences are then selected. Next, within the genes that share canonical structures with non-human antibodies, those with the highest homology within the CDRs are selected as framework donors. Finally, non-human CDRs are grafted onto these frameworks (see, e.g., Tan et al., J. Immunol. 169: 1119-1125, 2002).

[0177] In addition, it is generally desired that antibodies be humanized while retaining their affinity for antigens and other favorable biological properties. To achieve this goal, according to a method, humanized antibodies are prepared by analyzing the process of parental sequences and various conceptual humanized products using three-dimensional models of parental sequences and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for one or more target antigens, is achieved. In general, the hypervariable region residues are directly and most involved in influencing antigen binding.

[0178] Another method for antibody humanization is based on a measure of antibody humanness called human string content (HSC). This method compares the mouse sequence to a library of human germline genes and scores the differences as HSC. Multiple different humanized variants are then generated by maximizing their HSC rather than using a global identity measure, thereby humanizing the target sequence. (Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0179] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include those based on the use of enrichment techniques or high-throughput screening techniques to generate large libraries of humanized variants and select the best clones. Antibody variants can be isolated from phage, ribosome and yeast display libraries and by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

[0180] In the framework library method, a collection of residue variants is introduced at specific positions of the framework, and the library is subsequently screened to select the framework that best supports the grafted CDR. The residues to be replaced may include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224: 487-499, 1992), or a more limited set of target residues identified from Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0181] In framework shuffling, the complete framework is combined with non-human CDRs, rather than generating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library can be screened for binding in a two-step selection approach, first humanizing the VL, followed by the VH. Alternatively, a one-step framework shuffling approach can be used. Such an approach has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0182] The "humanization" method is based on the experimental identification of the necessary minimum specific determinant (MSD), and is based on the sequential replacement of non-human fragments into human FR libraries and the evaluation of binding. It starts with the CDR3 region of non-human VH chains and VL chains, and gradually replaces other regions of non-human antibodies into human frameworks, including CDR1 and CDR2 of both VH and VL. This method usually results in epitope retention and identification of antibodies from multiple subclasses with different human V segment CDRs. Humanization allows the separation of antibodies that are 91-96% homologous to human germline gene antibodies. (See, e.g., Alfenito, the third annual PEGS, Protein Engineering Summit of Cambridge Institute of Health Technology, 2007).

[0183] "Human engineering" methods include changing non-human antibodies or antibody fragments, such as mice or chimeric antibodies or antibody fragments, by making specific changes to the amino acid sequence of the antibody, so as to produce antibodies with reduced immunogenicity in humans but still retaining the modified desired binding properties of the original non-human antibodies. Typically, the technology includes classifying the amino acid residues of non-human (e.g., mouse) antibodies as "low risk", "medium risk" or "high risk" residues. Use a global risk / reward calculation to classify, which is calculated for the risk assessment of the replacement of the folding and / or replacement of the resulting antibody by human residues to carry out a specific replacement (e.g., for the immunogenicity in people) prediction benefit. By comparing the amino acid sequence of the variable region from a non-human antibody with the corresponding region of a specific or shared human antibody sequence, it is possible to select a specific human amino acid residue that is replaced at a given position (e.g., low or medium risk) of a non-human (e.g., mouse) antibody sequence. According to the comparison, the amino acid residues at low or medium risk positions in the non-human sequence can be replaced with the corresponding residues in the human antibody sequence. Techniques for making human engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication No. WO 93 / 11794.

[0184] In some embodiments, the 5T4 binders described herein include non-antibody protein scaffolds. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, fynomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz-type inhibitors. Methods for producing such non-antibody protein scaffolds are well known in the art, any of which can be used to produce 5T4 binders comprising non-antibody protein scaffolds (see, e.g., Simeon and Chen, Protein Cell, 9(1): 3-14 (2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1): 293-320 (2017)).

[0185] In addition, nucleic acids encoding 5T4 binders (e.g., antibodies or antibody fragments) or fusion polypeptides disclosed herein, nucleic acids complementary thereto, vectors comprising nucleic acids disclosed herein, and cells comprising nucleic acids or vectors disclosed herein are provided. In some embodiments, the cell expresses the 5T4 binder. In some embodiments, the cell replicates the nucleic acid or vector. In some embodiments, materials for producing 5T4 binders (e.g., human 5T4 binders) and fragments thereof are provided. For example, isolated cells can produce 5T4 binders (e.g., antibodies or antibody fragments). In this regard, cells (e.g., isolated cells) can produce antibodies or fragments thereof comprising VH and VL disclosed herein. In some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding a 5T4 binder (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In various aspects, the isolated polynucleotides comprise nucleotide sequences encoding VH and / or VL, wherein VH and VL comprise complementary determining regions (CDRs) identical to the CDRs disclosed herein.

[0186] In some embodiments, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expressing the one or more polynucleotides in a suitable host cell. Such vectors can be used, for example, to amplify polynucleotides in host cells to produce useful amounts of polynucleotides, and to express binding agents such as antibodies or antibody fragments using recombinant techniques.

[0187] In some embodiments, one or more vectors are expression vectors, wherein one or more polynucleotides are operably connected to one or more polynucleotides comprising expression control sequences. It is particularly contemplated that the recombinant expression construct of autonomous replication, such as plasmids and viral DNA vectors incorporating polynucleotides of one or more coding antibody sequences in conjunction with 5T4. The expression control DNA sequence includes a promoter, an enhancer and an operator, and is usually selected based on the expression system in which the expression construct is to be utilized. Promoter and enhancer sequences are usually selected according to the ability to increase gene expression, and operator sequences are usually selected according to the ability to regulate gene expression. The expression construct can also include a sequence encoding one or more selection markers, and the selection marker allows identification of the host cell carrying the construct. The expression construct can also include a sequence promoting and preferably promoting homologous recombination in the host cell. In some embodiments, the expression construct can also include a sequence necessary for replication in the host cell.

[0188] Exemplary expression control sequences include promoter / enhancer sequences, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985); the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993); the Tie promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995); the simian virus 40 promoter; DRA (downregulated in adenomas; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007); MCT1 (monocarboxylate transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979.2005); and Math1 (mouse atonal homolog 1; Shroyer et al., Gastroenterology, 132:2477-2478, 2007), for expression in mammalian cells, the promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In another variation, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include a suitable polyadenylation sequence (e.g., SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0189] If desired, the one or more polynucleotides may also optionally include a nucleotide sequence encoding a secretion signal peptide, which is fused in frame to the polypeptide sequence. The secretion signal peptide directs cells expressing the one or more polynucleotides to secrete the antibody polypeptide and is cleaved from the secreted polypeptide by the cell. The one or more polynucleotides may further optionally include a sequence whose only intended function is to facilitate large-scale production of the vector. Polynucleotides for gene therapy may be made and administered using methods described in the literature for a variety of transgenics. See, e.g., Isner et al., Circulation, 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy, 7: 989-1011, 1996.

[0190] In some embodiments, the polynucleotide may further comprise additional sequences to facilitate host cell uptake and expression of the antibody or fragment thereof (and / or any other peptide). In some embodiments, a "naked" transgene encoding an antibody or fragment thereof as described herein (e.g., a transgene without a virus, liposome or other carrier to facilitate transfection) is employed.

[0191] Any suitable vector can be used to introduce one or more polynucleotides encoding antibodies or fragments thereof into a host. Exemplary vectors that have been described include replication-defective retroviral vectors, including but not limited to lentiviral vectors (Kim et al., J. Virol., 72(1):811-816, 1998; Kingsman & Johnson, Scrip Magazine, October 1998, pp. 43-46); parvoviral vectors, such as adeno-associated virus (AAV) vectors (U.S. Pat. Nos. 5,474,9351; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnaten Ko et al., J. Invest. Med., 45:87-98, 1997); adenovirus (AV) vectors (U.S. Pat. Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992; Stratford et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992; Perricaudet et al., J. Clin. Invest., 90:626-630, 1992; and Rosenfeld et al., Cell, 68:143-155, 1992); adenovirus adeno-associated virus chimeric (U.S. Pat. No. 5,856,152) or vaccinia virus or herpes virus vectors (U.S. Pat. Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688); liposome (Lipofectin)-mediated gene transfer (BRL); liposome vector (U.S. Pat. No. 5,631,237); and combinations thereof. Any of these expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).Optionally, the viral vector is made replication defective by, for example, deleting or disrupting a selection gene required for viral replication.

[0192] Other non-viral delivery mechanisms contemplated include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), direct microinjection (Harland and Weintraub, J. Cell Biol., 8:7161-7165, 1984), and immunofluorescence staining (Ellison et al., J. Cell Biol., 9:717-718, 1986). Biol., 101: 1094-1099, 1985), liposomes loaded with DNA (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979; Felgner, Sci Am., 276 (6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15):1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene bombardment using high-speed microparticles (Yang et al., Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993).

[0193] The vector (or antibody or fragment thereof or nucleic acid as discussed herein) can be embedded in a liposome. See, e.g., Ghosh and Bachhawat, in: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275(5301):810-814, 1997). Various commercial methods involving "lipofection" technology are also contemplated. In some embodiments, the liposomes can be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with cell membranes and promote entry of liposome-encapsulated DNA into cells (Kaneda et al., Science, 243:375-378, 1989). In some embodiments, the liposomes are complexed or used in combination with a nuclear non-histone chromosomal protein (HMG-1) (Kato et al., J. Biol. Chem., 266: 3361-3364, 1991). In some embodiments, the liposomes are complexed or used in combination with both HVJ and HMG-1. Such expression constructs have been successfully used for transfer and expression of nucleic acids in vitro and in vivo. In some embodiments, 5T4 binding agents (e.g., antibodies) (including human 5T4 binding agents) are contained in the liposomes to target the liposomes to cells (e.g., tumor cells) expressing 5T4 on their surface.

[0194] The cell may comprise one or more polynucleotides or one or more vectors, e.g., the cell is transformed or transfected with one or more polynucleotides encoding a 5T4 binder (e.g., an antibody), including a human 5T4 binder, or one or more vectors comprising the one or more polynucleotides. In some embodiments, the cell expresses a 5T4 binder (e.g., an antibody), including a human 5T4 binder, containing one or more (including six) CDRs with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of mAbA4, mAbA15, or mAbA17 (see, e.g., Tables 1, 2, and / or 3). In some embodiments, the cell expresses a 5T4 binder (e.g., an antibody), including a human 5T4 binder, containing a VH and VL containing the same CDRs as the CDRs of mAbA4, mAbA15, and / or mAbA17 (see, e.g., Tables 1, 2, and / or 3). The cell can be a prokaryotic cell, such as E. coli (see, e.g., Pluckthun et al., Methods Enzymol., 178: 497-515, 1989) or a eukaryotic cell, such as an animal cell (e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell, or a hybridoma cell), a yeast (e.g., Saccharomyces cerevisiae), or a plant cell (e.g., a tobacco, corn, soybean, or rice cell). The use of mammalian host cells can provide translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on the recombinant expression product. Similarly, polypeptides (e.g., 5T4 binders (e.g., antibodies), including human 5T4 binders) can be glycosylated or non-glycosylated and / or have been covalently modified to include one or more water-soluble polymer linkages, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0195] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with vectors such as liposomes, micelles, ghost cells, and protoplasts. Such host cells can be used to amplify polynucleotides and can also be used to express polypeptides encoded by the polynucleotides. In this regard, methods for generating 5T4 binders (e.g., antibodies) can include culturing host cells and isolating the 5T4 binder. Transfer of naked DNA expression constructs into cells can be accomplished using particle bombardment, which relies on the ability to accelerate DNA-coated microparticles to high speeds that allow them to pierce cell membranes and enter cells without killing them (Klein et al., Nature, 327:70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on high voltage discharges to generate an electric current that in turn provides motive force (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). The microparticles used consist of biologically inert materials such as tungsten or gold beads. Host cells can be isolated and / or purified. Host cells can also be cells that are transformed in vivo to cause transient or permanent expression of a polypeptide in vivo. Host cells can also be isolated cells that are transformed ex vivo and introduced, for example, to produce a polypeptide for therapeutic purposes in vivo. The definition of host cells specifically excludes transgenic humans.

[0196] A variety of methods for generating antibodies from polynucleotides are generally well known. For example, Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 describes molecular biology procedures (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). In addition, many publications describe techniques suitable for preparing antibodies by manipulating DNA, generating expression vectors, and transforming and culturing appropriate cells (see, e.g., Mountain and Adair, <<Biotechnology and Genetic Engineering Reviews Chapter 1, Tombs ed., Intercept, Andover, UK, 1992); and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0197] 5T4 binding agents (e.g., antibodies), including human 5T4 binding agents, are produced using any suitable method, such as isolation from an immunized animal, recombinant or synthetic production, or genetic engineering, including as described above. Antibody fragments derived from antibodies are obtained, for example, by proteolysis of the antibody. For example, papain or pepsin digestion of intact antibodies produces a 5S fragment called F(ab')2 or two monovalent Fab fragments and an Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described in, for example, Edelman et al., Methods in Enzymology, 1:422 Academic Press (1967); Nisonoff et al., Arch. Biochem. Biophys., 89:230-244, 1960; Porter, Biochem. J., 73:119-127, 1959; U.S. Pat. No. 4,331,647; and Andrews, SM and Titus JA, Current Protocols in Immunology (Coligan et al., eds.), John Wiley & Sons, New York (2003), pp. 2.8.1, 2.8.10 and 2.10A.1, 2.10A.5.

[0198] 5T4 binding agents (e.g., antibodies), including human 5T4 binding agents, can be genetically engineered. For example, 5T4 binding agents (e.g., antibodies), including human 5T4 binding agents, contain variable region domains, such as produced by recombinant DNA engineering techniques. In this regard, the variable region is optionally modified by insertion, deletion, or alteration in the amino acid sequence of the antibody to produce the antibody of interest, including as described above. Polynucleotides encoding the CDR of interest are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, e.g., Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies", Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (Eds.), p. 166 (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principles and Applications, Birch et al., (Eds.), p. 137 (Wiley Liss, Inc., 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106-110, 1991). Current antibody manipulation techniques allow the construction of engineered variable region domains comprising at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody. Such techniques are used, for example, to humanize an antibody or to improve its affinity for a binding target.

[0199] "Humanized antibody" is an antibody in which the CDRs of the heavy variable chain and light variable chain of a non-human immunoglobulin are transferred to a human variable domain. The constant region does not need to exist, but if it does, in some embodiments, they are optionally substantially identical to human immunoglobulin constant regions, for example, at least about 85-90%, about 95%, 96%, 97%, 98%, 99% or more identical. Therefore, in some cases, all parts of the humanized immunoglobulin, except possible CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. For example, a humanized antibody is a human immunoglobulin (e.g., a host antibody), wherein the hypervariable region residues of the host antibody are replaced by hypervariable region residues with desired specificity, affinity and ability from non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates.

[0200] In some embodiments, the 5T4 binding agents described herein (e.g., antibodies or ADCs) can be used in compositions and methods for treating, preventing, or ameliorating a 5T4-mediated disease, disorder, or condition (including one or more symptoms of the disease, disorder, or condition). 5T4-mediated diseases, disorders, and conditions include cancer, including but not limited to any cancer in which tumor cells overexpress 5T4.

[0201] In some embodiments, described herein is a method for treating tumor immunity in a subject, comprising administering to the subject a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein. In some embodiments, provided herein is a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein, for treating tumor immunity in a subject. In some embodiments, provided herein is a use of a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein in the preparation of a medicament for treating tumor immunity in a subject.

[0202] In some embodiments, described herein is a method for treating a cancer or tumor in a subject, comprising administering to the subject a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein. In some embodiments, provided herein is a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein, for use in treating a cancer or tumor in a subject. In some embodiments, provided herein is a use of a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein in the preparation of a medicament for treating a cancer or tumor in a subject.

[0203] In some embodiments, described herein is a method for alleviating one or more symptoms associated with cancer or a tumor in a subject, comprising administering to the subject a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein. In some embodiments, provided herein is a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein, for alleviating one or more symptoms associated with cancer or a tumor in a subject. In some embodiments, provided herein is a use of a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein in the preparation of a medicament for alleviating one or more symptoms associated with cancer or a tumor in a subject.

[0204] In some embodiments, described herein is a method for reducing the size of a tumor in a subject with a tumor, comprising administering to the subject a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein. In some embodiments, provided herein is a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein, for reducing the size of a tumor in a subject with a tumor. In some embodiments, provided herein is a use of a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein in the preparation of a medicament for reducing the size of a tumor in a subject with a tumor.

[0205] In some embodiments, described herein is a method for enhancing tumor cell removal in a subject with a tumor, comprising administering to the subject a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein. In some embodiments, provided herein is a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein, for enhancing tumor cell removal in a subject with a tumor. In some embodiments, provided herein is a use of a 5T4 binding agent (e.g., an antibody or ADC) or a fragment thereof as described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody or ADC) as described herein in the preparation of a medicament for enhancing tumor cell removal in a subject with a tumor.

[0206] One or more therapeutic agents described herein and a 5T4 binding agent (e.g., antibody or ADC) or fragment thereof described herein or a pharmaceutical composition comprising a binding agent (e.g., antibody or ADC) described herein can be administered to a subject of the methods described herein.

[0207] In some embodiments, the antibody is a human antibody, including but not limited to antibodies having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences, as described, for example, in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. If the antibody contains a constant region, the constant region is also preferably derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, for example to enhance the activity of the antibody, but do not include CDRs derived from other species (e.g., mouse CDRs placed in human variable framework regions).

[0208] In some embodiments, the cancer or tumor cells of the methods described herein express 5T4, for example, on the cell surface. In other embodiments, the cancer or tumor cells overexpress 5T4. In some embodiments, the 5T4 binding agent (e.g., antibody or ADC) is cytotoxic to tumor cells in cell culture. Such cell cultures may include tumor cells expressing or overexpressing 5T4. Tumor cells include, but are not limited to, breast cancer cells, bladder cancer cells, melanoma cells, prostate cancer cells, mesothelioma cells, lung cancer cells, testicular cancer cells, thyroid cancer cells, squamous cell cancer cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, and pancreatic cancer cells.

[0209] In some embodiments, methods for enhancing the removal of tumor cells in a subject are described herein. For example, the method includes administering a 5T4 binding agent (e.g., antibody or ADC) that effectively enhances the removal of tumor cells, such as a human 5T4 binding agent as described herein. In some embodiments, the method includes administering a 5T4 binding agent (e.g., antibody or ADC), including (i) competing with mAbA4, mAbA15 and / or mAbA17 (see, e.g., CDR and VH / VL of Table 1, Table 2 and / or Table 3), (ii) binding to human 5T4 and / or (iii) binding to a 5T4 binding agent in a 5T4 region identified by mAbA4, mAbA15 and / or mAbA17 (see, e.g., CDR and VH / VL of Table 1, Table 2 and / or Table 3), resulting in enhanced removal of tumor cells. In some embodiments, one or more binding agents (e.g., antibodies), polynucleotides, vectors and / or cells as described above can be used to enhance the removal of tumor cells in vivo (e.g., a method for treating cancer in a subject).

[0210] Also provided are methods of regulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject. For example, the method comprises administering to the subject a composition comprising a 5T4 binding agent (e.g., an antibody or ADC) in an amount effective to regulate tumor growth in the subject.

[0211] As used herein, "tumor" refers to any neoplastic cell growth or proliferation, whether malignant or benign, and refers to all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, colon cancer, kidney cancer, lung cancer, squamous cell myeloid leukemia, hemangioma, melanoma, astrocytoma and glioblastoma, as well as other cell proliferative disease states, including, but not limited to: Heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyomas, fibromas, lipomas and teratomas; Lung: bronchial cancer (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma (chondromatous carcinoma), pulmonary fibrosis, ... Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary: kidney (adenocarcinoma, Wilm's tumor), lymphoma, leukemia, renal cell carcinoma ), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma, small cell prostatic carcinoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma); Liver: liver cancer (hepatocellular carcinoma), bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing sarcoma, malignant lymphoma (reticular cell sarcoma), malignant giant cell tumor chordoma, osteochondroma (osteocartilaginous exostosis) exostoses), benign enchondromas, chondroblastomas, chondromyxofibromas, osteoid osteomas, and giant cell tumors; nervous system: skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges (meningiomas, meningosarcomas, gliomatosis), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors (pinealomas), glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas);Gynecology: Uterus (endometrial cancer), Cervix (cervical cancer, preneoplastic cervical atypia), Ovary (ovarian cancer) serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-thecal cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), Vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), Vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), Fallopian tube (carcinoma); Hematology: Blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple sclerosis Myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal glands: neuroblastoma; and thyroid cancer, including medullary thyroid carcinoma. In some embodiments, the tumor or cancer is a recurrent tumor or cancer. In some embodiments, the tumor or cancer is a metastatic tumor or cancer. In some embodiments, the tumor or cancer is a primary tumor or cancer. Additionally or alternatively, the tumor or cancer is resistant to chemotherapy or other non-5T4 targeted anticancer therapies. ;

[0212] Also provided is a method of treating cancer by administering a 5T4-binding agent (eg, an antibody), such as a human 5T4-binding agent, to a subject in need thereof, alone or in combination with another agent.

[0213] "Enhanced" tumor cell removal does not require 100% enhanced removal. Any increase in the removal rate is expected. Similarly, "regulating" tumor growth refers to reducing the size of the tumor, slowing tumor growth, or inhibiting the size increase of an existing tumor. It is not necessary to completely eliminate the tumor; any reduction in tumor size or slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, compared to the removal level observed in the absence of the method (e.g., in a biologically matched control subject or sample not exposed to the agent of the method), tumor cell removal can be enhanced by, for example, at least about 5%, at least about 10%, or at least about 20%. The effect is detected by, for example, a reduction in tumor size, a reduction or maintenance of tumor marker levels, or a reduction or maintenance of tumor cell populations. In some embodiments, compared to the removal of tumor cells in the absence of a 5T4 binding agent (e.g., an antibody) of the method, the removal of tumor cells is enhanced by, for example, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more (about 100%).

[0214] In addition, 5T4 binding agents (such as antibodies or ADC) can be used to alleviate or reduce the side effects associated with cancer, such as bone degeneration, spinal collapse and paralysis. In one aspect, the subject suffers from bone metastasis or is at risk of suffering from bone metastasis, and 5T4 binding agents (such as antibodies or ADC) are administered in an amount that reduces the degeneration of the surrounding bones. Therefore, in some aspects, 5T4 binding agents prevent bone degeneration caused by bone metastasis, wherein tumor cell proliferation is reduced or not reduced. In some aspects, 5T4 binding agents (such as antibodies or ADC) both prevent bone degeneration caused by bone metastasis and reduce tumor cell proliferation. Generally, the impact on tumor cell proliferation (such as, inhibiting proliferation or not affecting proliferation) depends on the microenvironment of specific metastasis. For example, the proliferation of metastases located in a microenvironment with a large amount of type 1 collagen can be suppressed. On the contrary, the proliferation of metastases located in a microenvironment lacking a large amount of type 1 collagen may not be suppressed, but bone degradation near the metastases is reduced or prevented.

[0215] The specific administration regimen of a 5T4 binding agent (e.g., antibody or ADC) for a particular subject will depend in part on the agent used, the amount of the agent administered, the route of administration, and the cause and extent of any side effects. The amount of the agent (e.g., antibody or ADC) administered to a subject (e.g., a mammal such as a human) should be sufficient to achieve the desired response within a reasonable time frame. Therefore, in some embodiments, the amount of a 5T4 binding agent (e.g., antibody or ADC) or pharmaceutical composition described herein administered to a subject is an effective amount.

[0216] Suitable routes for administering compositions comprising a 5T4 binding agent (e.g., antibody or ADC), such as a human 5T4 binding agent (e.g., antibody or ADC), are well known in the art. Although more than one route may be used to administer an agent (e.g., antibody or ADC), a particular route may provide a more direct and more effective response than another route.

[0217] The present disclosure provides a composition, such as a pharmaceutical composition, comprising a 5T4 binding agent (e.g., an antibody or ADC) such as a human 5T4 binding agent and a carrier (e.g., a pharmaceutically acceptable carrier). The specific carrier employed may depend on chemical-physical considerations, such as solubility and lack of reactivity with the binding agent or co-therapy, as well as on the route of administration. Pharmaceutically acceptable carriers are well known in the art, examples of which are described herein. Exemplary drug forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions. Injectable formulations are further described in, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Co., Philadelphia. Pa. Banker and Chalmers. eds. 238-250 pages (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, 622-630 pages (1986)). The preparation of the pharmaceutical compositions provided herein and their various routes of administration can be carried out according to methods well known in the art. Delivery systems useful in the context of the present invention include timed release, delayed release and sustained release delivery systems, so that the delivery of the composition occurs before sensitization of the treated area and has enough time to cause sensitization of the treated area. Many types of release delivery systems are available and known to those of ordinary skill in the art. Suitable release delivery systems include polymer-based systems, such as poly (lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described in, for example, U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems, which are lipids such as sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as monoglycerides, diglycerides and triglycerides; hydrogel release systems; silicone rubber systems; peptide-based systems; wax coatings; compressed tablets using conventional adhesives and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosion systems, in which the active composition is contained in a matrix, as described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) diffusion systems, in which the active component is permeated from a polymer at a controlled rate, as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are suitable for implantation. In one aspect, a pharmaceutical composition comprising a 5T4 binding agent (e.g., an antibody or ADC), such as a human 5T4 binding agent, is placed in a container together with packaging material providing instructions for use of such pharmaceutical composition.Typically, such instructions include tangible expressions describing the concentrations of the agents and, in some embodiments, the relative amounts of excipient ingredients or diluents (eg, water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition.

[0218] In some aspects, the methods described herein further include administering one or more additional agents, including therapeutic agents, which may be present in the composition or may be administered with a 5T4 binding agent (e.g., an antibody or ADC) such as a human 5T4 binding agent, or provided in a separate composition using the same or different routes of administration. One or more additional agents (including therapeutic agents) may be administered (e.g., for combination therapy) together with a 5T4 binding agent (e.g., an antibody or ADC) or separately (e.g., simultaneously, alternately, sequentially). Such additional therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapies and immunotherapeutics, cytotoxic agents, chemotherapeutic agents, and inhibitors.

[0219] Therapeutic antibodies that can be used with a 5T4 binder (e.g., antibody or ADC) as described herein (e.g., for combination therapy) include, but are not limited to, trastuzumab; abciximab; daclizumab; BEC2; IMC-C22; vitaxin; Campath 1H / LDP-03; Smart M195; epratuzumab; betumomab; visilizumab; CM3, a humanized anti-ICAM3 antibody; IDEC-114; ibritumomab tiuxetan; IDEC-131; IDEC-151; IDEC-152; SMART anti-CD3; eculizumab; adalimumab; certolizumab pegol; IDEC-151; MDX-CD4; CD20-streptavidin; CDP571; LDP-02; OrthoClone OKT4A; ruplizumab; natalizumab; and lerudumab; or a biosimilar thereof.

[0220] Immunotherapies and immunotherapeutic agents that can be used with a 5T4 binder (e.g., an antibody or ADC) as described herein (e.g., for combination therapy) include, but are not limited to, cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-α, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factor (including TNF-α), and interferons (including IFN-α, IFN-β, and IFN-γ); aluminum hydroxide (alum); bacillus Calmette-Guérin (BCG); keyhole limpet hemocyanin (KLH); incomplete Freund's adjuvant (IFN-) A); QS-21; DETOX; Levamisole; and dinitrophenol (DNP), and combinations thereof, such as interleukins (eg, IL-2) and other cytokines (eg, IFN-α). In some embodiments, immunotherapy includes immunotherapeutics that regulate immune responses, such as checkpoint inhibitors or checkpoint agonists. In some embodiments, immunotherapeutics are antibody modulators targeting PD-1, 5T4, PD-L2, CEACAM (eg, CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFβ, OX40, 41BB, LIGHT, CD40, GITR, TGF-β, TIM-3, SIRP-α, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, etc., known in the art. In some embodiments, the immunotherapeutic agent is an agent that increases the activity of natural killer (NK) cells. In some embodiments, the immunotherapeutic agent is an agent that suppresses immune response suppression. In some embodiments, the immunotherapeutic agent is an agent that suppresses suppressor cells or suppresses cell activity. In some embodiments, the immunotherapeutic agent is an agent or therapy that suppresses Treg activity. In some embodiments, the immunotherapeutic agent is an agent that suppresses the activity of inhibitory immune checkpoint receptors.

[0221] In some embodiments, immunotherapeutic agent includes T or NK or NKT cell regulators selected from the agonist or activator of costimulatory molecules. In one embodiment, the agonist of costimulatory molecules is selected from GITR, OX40, ICOS, SLAM (eg, SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3 or CD83 ligand agonist (eg, agonistic antibodies or their antigen-binding fragments or soluble fusions). In other embodiments, effector cell combination includes bispecific T cell adapter (eg, with CD3 and tumor antigen (eg, EGFR, PSCA, PSMA, EpCAM, HER2, etc.) bispecific antibody molecules) or bispecific NK cell adapter.

[0222] Cytotoxic agents that can be used (e.g., for combination therapy) with 5T4 binding agents as described herein (e.g., antibodies or ADCs) include substances that inhibit or prevent cell function and / or cause cell death or destruction. Exemplary cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); growth inhibitors; enzymes and fragments thereof, such as nuclear lytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Other exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-A inhibitors; fatty acid biosynthesis inhibitors; cell cycle signaling inhibitors; HDAC inhibitors, proteasome inhibitors; and cancer metabolism inhibitors.

[0223] Chemotherapeutic agents that can be used with a 5T4 binding agent (e.g., antibody or ADC) as described herein (e.g., for combination therapy) include compounds that can be used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, erlotinib, bortezomib, disulfiram, epigallocatechin gallate, salt spore amine A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant, sunitinib, letrozole, imatinib mesylate, fmasunate, oxaliplatin, 5-FET (5-fluorouracil), leucovorin, rapamycin, lapatinib, Lonafamib (SCH66336), sorafenib (Bayer Labs), gefitinib, AG1478; alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trishydroxymethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callylin statin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talcum duocarcin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratis tatin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, chlorpromazine, clofosamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g. calicheamicins, particularly calicheamicin gamma II and calicheamicin omega I (Angew Chem. Inti. Ed. Engl. 1994 33: 183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; espamycins;and neocanstatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, elarubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, quelamyc in), rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folate analogs, such as leucovorin, methotrexate, and pteropterin , trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calutosterone, drostanolone propionate, epithioandrostrol, melastosane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid (folinic acid acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Ore.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes, particularly T-2 toxin, verrucarin A, roridin A, and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel, (without Cremophor), albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and docetaxel (docetaxel / doxetaxel); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; etoposide (VP-16); ifosfamide; mitoxantrone; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid (retinoic acid); acid); and pharmaceutically acceptable salts, acids and derivatives of any of the above. Chemotherapeutic agents also include (i) antihormonal agents used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, troxifene, keoxifene, LY117018, onapristone and toremifene citrate; (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; (i ii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, triptorelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf and H-Ras; (vii) ribozymes, such as inhibitors of VEGF expression (e.g., ) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, e.g. and rIL-2; topoisomerase 1 inhibitors, such as and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0224] Chemotherapeutic agents also include antibodies as described above, including alemtuzumab, bevacizumab; cetuximab; panitumumab, rituximab, pertuzumab, tositumomab, and the antibody drug conjugate gemtuzumab ozogamicin.Additional humanized monoclonal antibodies with therapeutic potential as agents for combination with a 5T4-binding agent (e.g., antibody) as described herein include: apolizumab, aselizumab, atlizumab, bapilizumab, bivacizumab maytansine, cantuzumab maytansine, becelizumab, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab ... zumab), felvizumab, fontolizumab, gemtuzumab ozogamicin, ituzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab Monoclonal antibody, nimotuzumab, nivolumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, rulizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-8744695, Wyeth Research and Abbott Laboratories), which is a recombinant, pure human sequence, full-length IgG1λ antibody genetically modified to recognize the interleukin-12p40 protein.Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live bacillus Calmette-Guérin, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, Interferon alpha-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0225] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinolone acetonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasone-17-propionate, fluocortolone caproate, fluocortolone pivalate, and flupredniidine acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN BioTherapeutics, LLC); antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept, infliximab, adalimumab, certolizumab pegol, golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra, T cell costimulation blockers such as abatacept, interleukin 6 (IL-6) blockers such as tocilizumab; interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; β7 integrin blockers such as rhuMAbβ7; IgE pathway blockers such as anti-Ml prime; blockers of secreted homotrimer LTa3 and membrane-bound heterotrimer LTal / l32, such as anti-lymphotoxin alpha (LTa); miscellaneous research agents, such as thioplatin, PS-341, phenyl butyrate, Et-I8-OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid, and their derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolamine, and 9-aminocamptothecin; podophyllotoxin; tegafur; bexarotene; bisphosphonates, such as clodronate, etidronate, NE-58095, zoledronic acid / zoledronate, alendronate, pamidronate, tiludronate, or risedronate; and epidermal growth factor receptor (EGF-R); vaccines, such as Vaccines; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium, pixantrone; farnesyl transferase inhibitors, such as lonafarnib (SCH 6636); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (abbreviation for a regimen of oxaliplatin combined with 5-FU and folinic acid). Chemotherapeutic agents also include poly ADP ribose polymerase (PARP) inhibitors: olaparib, rucaprib, niraparib, talzoparib.

[0226] Inhibitors that can be used with a 5T4-binding agent (e.g., an antibody) as described herein (e.g., for combination therapy) include, but are not limited to, kinase inhibitors such as imatinib, baricitinib, gefitinib, erlotinib, sorafenib, dasatinib, sunitinib, lapatinib, nilotinib, pirfenidone, pazopanib, crizotinib, velotinib, crottinib, vandetanib, ruxolitinib, axitinib, bosutinib, regorafenib, tofacitinib, cabozantinib, ponatinib, trametinib, dabrafenib, afatinib, ibrutinib, ceritinib, idelalisib, nintedanib, palbociclib, lenvatinib, cobimetinib, abemaciclib, acalabrutinib, alectinib, binimetinib, inib), brigatinib, encorafenib, erdafitinib, everolimus, fostamatinib, gilter, larotrectinib, lorlatinib, netarsudil, osimertinib, pemitinib, pexidartinib, ribociclib, temsirolimus, XL-092, XL-147, XL-765, XL-499, and XL-880. In some embodiments, the compounds as described herein can be used in combination with HSP90 inhibitors (e.g., XL888), liver X receptor (LXR) modulators, retinoid-related orphan receptor gamma (RORy) modulators, checkpoint inhibitors such as CK1 inhibitors or CK1α inhibitors, Wnt pathway inhibitors (e.g., SST-215) or mineralocorticoid receptor inhibitors (e.g., ixacetolone) or XL-888 for the treatment of diseases disclosed herein such as cancer.In some embodiments, a 5T4-binding agent (e.g., an antibody) as disclosed herein can be used in combination with an inhibitor of one or more of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, 1NS-R, IGF-1R, IR-R, PDGFαR, PDGFβ / R, CSFIR, KIT, FLK-II, KDR / FL K-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphAl, EphA2, Eph A3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYR, FRK, JAK (JAK1 and / or JAK2), ABL, ALK, CDK7, CDK12, KRAS and B-Raf.

[0227] Other non-limiting examples of inhibitors that can be used with a 5T4 binding agent (e.g., an antibody or ADC) as described herein, e.g., for treating cancer (e.g., for combination therapy), include FGFR inhibitors (FGFR1, FGFR2, FGFR3, or FGFR4, e.g., pemigatinib), EGFR inhibitors (also known as ErB-1 or HER-1; e.g., erlotinib, gefitinib, vandetanib, osimertinib, cetuximab, necitumumab, or panitumumab), VEGFR inhibitors or pathway blockers (e.g., bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, vandetanib, radama murucumab, lenvatinib, ziv-aflibercept), PARP inhibitors (e.g., olaparib, rucaparib, veliparib, or niraparib), and JAK inhibitors (e.g., ruxolitinib, baricitinib, itatinib), IDO inhibitors (e.g., epacadostat, NLG919 or BMS-986205, MK7162), LSD1 inhibitors, TDO inhibitors, P13K-δ inhibitors (e.g., parsaclisib), PI3K-γ inhibitors such as PI3K-γ selective inhibitors, Pim inhibitors, CS F1R inhibitors, TAM receptor tyrosine kinases (Tyro-3, Axl and Mer), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, chemokine receptor inhibitors (e.g., CCR2 or CCR5 inhibitors), SHP1 / 2 phosphatase inhibitors, histone deacetylase (HDAC) such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extra terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors), or a combination thereof.

[0228] In some embodiments, a 5T4-binding agent (e.g., an antibody or ADC) as disclosed herein can be used in combination with a PD-1 inhibitor or a 5T4 inhibitor, such as an anti-PD-1 monoclonal antibody or an anti-5T4 monoclonal antibody, such as nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, avelumab, cemiplimab, spartalizumab, camrelizumab, or relizumab), cetrelimab, toripalimab, sintilimab, AB122, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, and TSR-042, AMP-224, AMP-514, PDR001, durvalumab, pidilizumab, CT-011), CK-301, BMS936559, MPDL3280A, tislelizumab, BMS-935559, MEDI4736, FAZ053, KN035, CS1001, CBT-502, A167, STI-A101, BGB-A333, MSB-2311, HLX20, AUNP12, CA-170, BMS-986189, LY3300054, and MSB0010718C.

[0229] In some embodiments, a multispecific binding agent as disclosed herein can be used in combination with a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody, e.g., ipilimumab (Yervoy), tremelimumab, and AGEN1884, or with a phosphatidylserine inhibitor, such as bavituximab (PGN401), or with antibodies to cytokines (IL-10, TGF-b, etc.), or with a bispecific antibody that binds 5T4 and CTLA-4 (e.g., AK104) or PD-1 and CTLA-4, or with other anticancer agents such as cemiplimab.

[0230] The additional agent may be a pharmaceutically acceptable salt, ester, amide, hydrate and / or prodrug of any of these therapeutic agents or other agents described above.

[0231] It is to be understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein.Accordingly, the following examples are intended to illustrate but not to limit the present disclosure. Example

[0232] Example 1. Antibody Generation

[0233] To obtain binders to human 5T4, antibody discovery was performed by phage display.

[0234] A. Phage Display

[0235] To obtain binders to human 5T4, antibody discovery was performed by phage display of a human Fab library and was performed using standard protocols. The extracellular domain of human 5T4 was purchased from Acro Biosystems (biotinylated human 5T4 His-Avitag Acro, catalog number TPG-H83Eb) or was generated. The generated antigens consisted of human 5T4, cynomolgus monkey 5T4, and mouse 5T4, all cloned into a vector containing a C-terminal 6-his-Avi tag and biotinylated in vitro using a co-expressed biotin ligase using standard procedures where possible.

[0236] Phage clones are screened for the ability to bind biotinylated human 5T4. In brief, the phage library of Fab format is constructed using an expression vector (also referred to as phagemid) that can be replicated and expressed in phage. Heavy chain and light chain are encoded in identical expression vectors, wherein the heavy chain merges with the truncated variant of phage coat protein pill. Light chain and heavy chain-pIII fusions are expressed as independent polypeptides and assembled in bacterial periplasm, wherein redox potential enables the formation of disulfide bonds, to form the antigen-binding domains (Fab) of candidate antibodies.

[0237] The library was created using sequences derived from specific human heavy chain variable domains and specific human light chain variable domains. The light chain variable domains within the screening library were generated, diversity was introduced into the VL CDR3 (L3), and wherein the light chain VL CDR1 (L1) and CDR2 (L2) retained human germline sequences. For the screened library, all three CDRs of the VH domain were diversified to match the position amino acid frequency according to the CDR length found in the human antibody repertoire. The phage display heavy chain (SEQ ID NO: 72) and light chain (SEQ ID NO: 73) scaffolds used in the library are listed below, wherein "x" represents the CDR amino acids that were changed to create the library, and bold italics represent constant CDR sequences.

[0238] The sequence of SEQ ID NO: 72 is

[0239] EVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKGLEWVAXXXXXXXXXXXXXXXXXRFTISADTSKNTAYLQMNSLRAEDTAVYYCARXXXXXXXXXXXXXXW GQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.

[0240] The sequence of SEQ ID NO: 73 is

[0241]

[0242] Diversity was generated by mutagenesis using degenerate DNA oligonucleotide primers to introduce diversity into VL CDR3 (L3) and VH CDR1 (H1), CDR2 (H2) and CDR3 (H3) to mimic the diversity found in natural antibody repertoires, as described in Kunkel, TA (PNAS January 1, 1985, 82 (2) 488-492), the entire contents of which are incorporated herein by reference. Briefly, single-stranded circular DNA incorporating uracil was prepared from isolated phage using standard procedures and Kunkel mutagenesis was performed to introduce diversity into the four CDRs. The chemically synthesized DNA was then electroporated into TG1 cells and subsequently recovered. The recovered cells were subcultured and infected with M13K07 helper phage to generate a phage library.

[0243] Phage panning was performed using standard procedures. Briefly, the first round of phage panning was performed with targets immobilized on streptavidin magnetic beads subjected to approximately 1 × 10 12 Phage from the prepared library in a volume of 1 ml in PBST-2% BSA. After 1 h of incubation, bead-bound phage were separated from the supernatant using a magnetic stand. The beads were washed three times to remove nonspecifically bound phage and then counted by OD 600 After incubation at room temperature for 20 minutes, the infected cells were incubated in 25 ml of 2xYT + ampicillin and M13K07 helper phage (final concentration of about 1 × 10 10pfu / ml) in subculture, and allow to grow overnight at 37 ℃ with vigorous shaking. The next day, phage was prepared by PEG precipitation using standard procedures. The pre-clearance of phages with specificity to the beads of SAV coating was performed before panning. The second round of panning used KingFisher magnetic bead processor and 50 or 100nM beads to fix the 5T4 antigen, using standard procedures (100nM 5T4 was used for the 3rd round, and 50nM 5T4 was used for the 4th round). Carry out 3-4 rounds of phage panning in total to enrich the phages showing the Fab specific to the 5T4 antigen. Use polyclonal ELISA to confirm the 5T4 specific enrichment, separate single clones and further verify by performing monoclonal phage ELISA. DNA sequencing is used to determine the sequence of the CDR of the Fab clone containing the separation of candidate antibodies.

[0244] The genes encoding the heavy and light chain variable domains of the candidate antibodies were cloned separately into mammalian expression vectors for expression as full-length IgG in mammalian cells.

[0245] For full-length IgG, the heavy chain constant region (e.g., CH1 = normal text; hinge = italic text; CH2 = bold text; and CH3 = underlined text) includes the following amino acid sequence:

[0246]

[0247] For full-length IgG, the light chain constant region (e.g., CL) includes the following amino acid sequence:

[0248] RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 75).

[0249] IgG antibodies were purified from the culture supernatant using protein A resin.

[0250] Example 2. Screening assay

[0251] A bivalent binding assay was used to provide data on whether antibodies with VH / VL obtained by phage display in Example 1 bind to immobilized 5T4 from all three clinically relevant species: human, cynomolgus monkey (cyno), and mouse. For the bivalent binding and cross-reactivity assays, 5T4 human, cynomolgus monkey, and mouse antigens were synthesized using sequences from a protein database and expressed as biotinylated proteins using a his-avi tag and in vivo biotinylation with a co-expressed BirA enzyme.

[0252] Standard Octet-based assays were used to qualitatively determine whether each antibody bound to each antigen. For these assays, 200 nM biotinylated antigen was immobilized on a streptavidin biosensor in 1X kinetic buffer (ForteBio). The loaded sensor was then immersed in a well containing 500 nM of the antibody of interest and the association binding signal was monitored. An increase in signal above the negative control during the association step was considered to be binding to the antigen.

[0253] For qualitative binding affinity using Octet, all 27 antibodies tested showed binding to human 5T4, 13 antibodies also showed binding to cynomolgus 5T4, and 13 antibodies also showed binding to mouse 5T4. The data for exemplary antibodies are shown in Table 4 below.

[0254] Table 4. Qualitative bivalent binding affinity by Octet

[0255]

[0256] The relative affinity of each 5T4 binder obtained by phage display in Example 1 to human 5T4 was determined using a monovalent binding assay. Human 5T4 was purchased from Acro Biosystems (Acro catalog number TPGH52E) and was also produced separately. The monovalent interaction of the binder with human 5T4 was monitored using an Octet (Pall ForteBio) instrument. For these assays, 36nM antibody was immobilized on an anti-human Fc (AHC) sensor in 1X kinetic buffer (ForteBio). The sensor was then immersed in a well containing 3X serial dilutions of human 5T4 ranging from 1 μM to 1.37nM. The association was allowed to reach equilibrium. The sensor was then moved to a well containing only 1X kinetic buffer and allowed to dissociate to equilibrium. The association and dissociation rates were measured, and the monovalent K was calculated from these using the onboard analysis software (ForteBio). D .

[0257] Of all 27 tested antibodies, most showed weak binding in the monovalent binding affinity-internal (Lot 20-019-62) assay. mAbA4, mAbA15, and mAbA17 showed strong or moderate binding in all three batches. The data are shown in Table 5 below.

[0258] Table 5. Qualitative monovalent binding affinity by Octet

[0259]

[0260] Example 3: Additional screening and selection

[0261] Antibodies selected for binding to 5T4, eg, such as those described in Example 2, are evaluated for binding to cells expressing 5T4.

[0262] Cells at 70%-90% confluence were harvested on the day of the assay. In a v-bottom 96-well plate (Costar 3897), 100,000 cells per well were incubated at +4°C for 30 minutes with 50 μL per well, 8-point or 12-point antibody dilution series. After incubation, the cells were washed once with PBS by centrifugation at 200 x g for 5 minutes. The cells were then incubated at +4°C for 30 minutes with 50 μL per well of Alexa Fluor488 goat anti-human IgG Fab (Jackson Immuno Research 109-547-003) diluted 1:100. The cells were washed twice with PBS by centrifugation at 200 x g for 5 minutes and collected on iQue Screener Plus (Sartorius). The mean fluorescence intensity of the BL1 (Alexa-Fluor-488) channel was plotted against the antibody concentration to generate a dose response curve and EC50 value.

[0263] Among the 27 antibodies tested, 26 antibodies showed binding affinity to HEK-5T4 cells, 16 antibodies showed binding affinity to MCF-7 cells, and 26 antibodies showed binding affinity to HEK-cyno-5T4 cells.

[0264] Selected antibodies that bound to all three HEK-5T4, MCF-7, and HEK-cyno-5T4 cell lines (including mAbA4, mAbA15, and mAbA17) were further evaluated in internalization assays in HEK-5T4 and MCF-7 cells.

[0265] Adherent cells at 70%-90% confluence were harvested and plated overnight at 20,000 cells / well in 50 μL of culture medium in a 96-well black transparent / flat bottom half-area plate (Corning 3882) at 37°C. For suspension cell lines, 96-well plates were coated with poly-L-ornithine solution (Millipore-Sigma) at 37°C for 1 hour, and cells were seeded in 50 μL of culture medium at a density of 40,000 cells / well approximately 1-2 hours before the assay. The antibodies were diluted in cell culture medium, mixed with 3X molar concentration of Incucyte Human FabFluor-pH Red Antibody Labeling Reagent, and incubated at room temperature for 15 minutes. All dilutions of the antibody / FabFluor mixture were prepared at 2X final concentration. Then 50 μL of the 2X antibody / Fab-Fluor mixture was added directly to each well with pre-seeded cells in duplicate, and the plate was quickly transferred to Incucyte. The plate was then scanned every 30 minutes for up to 24 hours. Note that the first scan (0 hours) was started within 2-3 minutes of adding the antibody / FabFluor mixture to the cells. The plates were analyzed by reading the red signal generated over time, normalized to the % confluence of the cells in that well. A small amount of antibody was tested at different concentrations on the target cell line, and the optimal concentration was selected based on a good signal-to-noise ratio.

[0266] In the internalization assay, 6 antibodies (including mAbA4 and mAbA15) showed strong internalization in HEK-5T4 cells, one antibody showed moderate internalization in HEK-5T4 cells, and 7 antibodies showed no internalization or non-specific internalization in HEK-5T4 cells. In another assay, 2 antibodies showed strong internalization in MCF 7 cells, 4 antibodies showed moderate internalization in HEK-5T4 cells, and 8 antibodies showed no internalization or non-specific internalization in MCF-7 cells.

[0267] Example 4: Developability Assay

[0268] Antibodies selected for binding to 5T4 are tested in various developability methods, e.g., as described in Examples 2 and 3. For example, various chromatography methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and vertical monolayer adsorption chromatography (SMAC), are employed to assess developability factors such as percent monomer, solubility, and antibody aggregation or precipitation.

[0269] Size exclusion chromatography (SEC) analysis was performed on an Agilent 1100 HPLC using a 7.8 mm ID x 30 cm TSKgel G3000SWXL column (Tosoh Bioscience LLC, PN 08541) and a 6 mm x 4 cm guard column (PN 08543). Antibodies were diluted in Dulbecco's PBS (pH 7.4, without Ca 2+ / Mg 2+ ) and clarified by centrifugation to precipitate particles while still retaining soluble aggregates. The mobile phase buffer was 2X concentration of Dulbecco's PBS (pH 7.4, without Ca) diluted from the 10X stock concentration. 2+ / Mg 2+ ). For each sample, 10 μL was loaded and eluted isocratically at 1.0 mL / min for 20 minutes at ambient temperature. The absorbance was monitored at 280 nm. The chromatographic peaks were integrated to determine the homogeneity percentage and retention time. In addition to molecular size analysis (sizing), the column stationary phase and the choice of mobile phase also support hydrophobic and electrostatic interactions (secondary interactions are much milder than SMAC). Data analysis was performed using Agilent ChemStation B.04.03S P1.

[0270] The results showed that mAbA4, mAbA15, and mAbA17 (as well as other antibodies tested) had strong developability based on SEC analysis.

[0271] Hydrophobic interaction chromatography (HIC) analysis was performed on an Agilent 1100 HPLC using a 4.6 mm ID x 3.5 cm TSKgel Butyl-NPR column (Tosoh Bioscience LLC, PN 14947). The antibody was diluted in Dulbecco's PBS (pH 7.4, without calcium Ca) and lysate. 2+ / Mg 2+) was standardized to 1 mg / mL concentration. At ambient temperature, the column was balanced with 100% mobile phase buffer A (2M ammonium sulfate / 20mM sodium phosphate, pH 7.0) at a flow rate of 1mL / min. For each sample, 10 μL was loaded, and a gradient from 100% mobile phase buffer A to 100% mobile phase buffer B (20mM sodium phosphate, pH 7.0) was used to elute for 15 minutes at 1.0mL / min, kept at 100% B for 3 minutes to wash the column, and returned to 100% A for 2 minutes to balance. Monitor absorbance at 280nm. Calculate sample retention time and compare with a set of standard controls to identify the presence of antibodies and various substances with increased retention time (increased hydrophobicity). Data analysis was performed using Agilent ChemStation B.04.03S P1.

[0272] The results of hydrophobic interaction chromatography (HIC) analysis showed that some antibodies had strong developability, while others had moderate developability. Based on HIC analysis, mAbA4, mAbA15, and mAbA17 were those with strong developability. Antibody hydrophobicity can affect antibody aggregation, solubility, and viscosity. The results showed that these antibodies had a low tendency to aggregate and precipitate.

[0273] Vertical monolayer adsorption chromatography (SMAC) analysis was performed on Agilent 1100HPLC using a 4.6mm ID x 300mm Zenix SEC 300 column (iSepax Technologies, PN 213300P-4630) and a 4.6mm x 50mm guard column (PN 213300P-4605). The antibody was standardized to a concentration of 1 mg / mL in Dulbecco PBS (pH 7.4, without Ca2+ / Mg2+), and clarified by centrifugation to precipitate particles while still retaining soluble aggregates. The mobile phase buffer was Dulbecco PBS (pH 7.4, without Ca2+ / Mg2+), and the concentration was 2X (diluted from 10X stock solution concentration). For each sample, 10 μL was loaded and eluted isocratically at ambient temperature for 32 minutes at 0.4 mL / min. Absorbance was monitored at 280 nm. Sample retention times were calculated and compared to a set of standard controls to identify antibodies with increased retention times (increased tendency to form aggregates and / or increased hydrophobic / electrostatic interactions) since column stationary phase in conjunction with mobile phase selection promotes secondary interactions in addition to molecular size analysis. Data analysis was performed using an Agilent ChemStation B.04.03SP1.

[0274] Results from the vertical monolayer adsorption chromatography (SMAC) analysis showed that some antibodies had strong developability, while others had moderate developability. Based on the SMAC analysis, mAbA4, mAbA15, and mAbA17 were those with strong developability. The assay was based on good retention times, which indicated colloidal stability and low aggregation tendency.

[0275] Example 5: Epitope binning assay

[0276] Epitope binning assays were used to determine which epitopes of 5T4 each antibody obtained by phage display in Example 1 bound.

[0277] In order to maintain the overall structure of 5T4 protein, while still being able to carry out epitope binding determination, each of the seven continuous sequences (epitopes 1-7) of cloned human 5T4 is replaced by each of the corresponding mouse sequences to produce mouse-human chimeras. In order to produce chimeras, full-length human and mouse 5T4 genes are synthesized by GenScript and cloned into a mammalian vector with a 6-His-Avi tag. Then each of the seven sequences (epitopes 1-7) as described herein is taken out from the human gene, and appropriate primers are used to clone into the mouse gene respectively. Epitope 1 (H1) of human 5T4 corresponds to SSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAART (SEQ ID NO: 76); epitope 2 (H2) of human 5T4 corresponds to VKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVR (SEQ ID NO: 77); epitope 3 (H3) of human 5T4 corresponds to AGAFEHLPSLRQLDLSHNPLADLSPFAFSG (SEQ ID NO: 78); epitope 4 (H4) of human 5T4 corresponds to SNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLE (SEQ ID NO: 79); epitope 5 (H5) of human 5T4 corresponds to LASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESL (SEQ ID NO: 80); NO: 80); epitope 6 (H6) of human 5T4 corresponds to HLEDNALKVLHNGTLAELQGLPHIRVFL (SEQ ID NO: 81); and epitope 7 (H7) of human 5T4 corresponds to DNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTS (SEQ ID NO: 82). Antigens were transiently expressed using the Expi293 protein expression system (Thermo Fisher), biotinylated in vitro using co-expressed BirA biotin ligase, and purified using Ni-NTA affinity chromatography.As shown in Table 6, seven chimeric constructs of human (H) and mouse (M) sequences were generated: (1) H1 M2 M3 M4 M5 M6 M7 (SEQ ID NO: 83); (2) M1 H2 M3 M4 M5 M6 M7 (SEQ ID NO: 84); (3) M1 M2 H3 M4 M5 M6 M7 (SEQ ID NO: 85); (4) M1 M2 M3 H4 M5 M6 M7 (SEQ ID NO: 86); (5) M1 M2 M3 M4 H5 M6 M7 (SEQ ID NO: 87); (6) M1 M2 M3 M4 M5 H6 M7 (SEQ ID NO: 88); and (7) M1 M2 M3 M4 M5 M6 H7 (SEQ ID NO: 89). Each antibody was then tested for binding to each chimeric construct and the full-length human protein. Any significant binding signal to the chimera indicates the region to which the antibody specifically binds.

[0278] Table 6. 5T4 chimeric constructs

[0279]

[0280]

[0281] Standard Octet-based assays were used for epitope binning assays to establish epitope binning for each antibody. For these assays, 50 nM biotinylated antigen was immobilized on a streptavidin sensor in 1X kinetic buffer (ForteBio). The sensor was then immersed in a well containing 100 nM of the antibody in question and the association signal was monitored. The chimera that effectively knocked out antibody binding corresponded to the specific epitope bound by the antibody. Full-length human 5T4 was included in the assay for reference.

[0282] The results showed that 9 antibodies specifically bound to epitope 4 of 5T4, 3 antibodies specifically bound to epitope 7 of 5T4, 3 antibodies specifically bound to epitope 2 of 5T4, 1 antibody specifically bound to epitope 4 and epitope 6 of 5T4, and 11 antibodies bound to full-length 5T4 (FL). Table 7 summarizes exemplary epitope binning results (for mAbA4, mAbA15, and mAbA17).

[0283] Table 7. Epitope binning assay

[0284] Antibody Epitope binning mAbA4 4 mAbA15 2 mAbA17 7

[0285] Example 6: Antibody selection

[0286] As described above, among all the antibodies screened from the phage library as described in Example 1, 27 antibodies that were still able to bind to 5T4 after being formatted as IgG were further tested as described in Examples 2-5. Three antibodies named mAbA4, mAbA15 and mAbA17 were selected based on a variety of activities, including their specificity for binding to human 5T4, their binding to cynomolgus monkey 5T4, their cell surface binding signals, and their strong developability evaluated by a variety of methods such as SEC, HIC and SMAC. The VH, VL and CDR sequences of these antibodies are shown in Tables 1-3.

[0287] Example 7. Binding of Antibodies to Human 5T4 (ELISA)

[0288] A. Normal binding

[0289] The selected antibodies were tested for their ability to bind to hu5T4 using ELISA. TM) Unconjugated human TPBG / 5T4 protein (AcroBiosystems (Newark, DE, USA), catalog number TPG-H52E5) was diluted to 2 μg / mL in PBS, mixed thoroughly, and plated in 96-well plates (Nunc ) and incubated at 4°C overnight.

[0290] On day 2, wash with 0.1% The plates were washed four times with 200 μL of casein in PBS (Blocker TM The wells were blocked with 5% casein (purchased from ThermoFisher Scientific) and incubated with shaking at room temperature for 2 hours.

[0291] Prepare a 1:3 dilution series of IgG-ADC in PBS and add to a 96-well plate at a volume of 100 μL per well. Incubate the plate with shaking at room temperature for 1 hour.

[0292] Goat anti-human IgG Fcγ specific HRP conjugate (Jackson Immunoresearch, catalog number 109-035-098) was diluted to a factor of 1:15000 in PBS. The ELISA plate was washed six times with PBS-T using a plate washer (BioTek ELx405), after which 100 μL of goat anti-human IgG Fcγ specific HRP conjugate was added to each well. The plate was then incubated at room temperature for 30 minutes while shaking.

[0293] The ELISA plate was washed six times with PBS-T using a plate washer (BioTek ELx405), followed by the addition of 100 μL 3,3',5,5'-tetramethylbenzidine (TMB) to each well. The plate was developed until a dark blue color became visible. H2SO4 (2N, 100 μL) was then added to quench the reaction, and the absorbance of each well was read on a plate reader (SpetraMax M5 equipped with SoftMaxPro software, available from Molecular Devices).

[0294] B. Stress Test

[0295] 1. High pH stress test. The ability of the antibodies to bind 5T4 was tested using an ELISA under alkaline (high pH) conditions. 1 mg / mL of the antibody in 0.1% w / w H2O2 / 1x PBS was stored at room temperature in the dark for 24-48 hours, after which 50 mM methionine was added. The samples were stored frozen prior to analysis, which was performed by ELISA as described in Section A above. Table 8 below reports the EC values ​​calculated for each antibody tested. 50 .

[0296] Table 8. Binding of antibodies to hu5T4 at high pH

[0297] mAbs condition <![CDATA[EC 50 (nM)]]> mAbA4 pH 8.5 Day 0 0.120 mAbA4 pH 8.5 Day 1 0.127 mAbA4 pH 8.5 Day 4 0.114 mAbA4 pH 8.5 Day 7 0.128 mAbA4 pH 8.5 Day 14 0.136 mAbA15 pH 8.5 Day 0 0.134 mAbA15 pH 8.5 Day 1 0.128 mAbA15 pH 8.5 Day 4 0.133 mAbA15 pH 8.5 Day 7 0.143 mAbA15 pH 8.5 Day 14 0.131 mAbA17 pH 8.5 Day 0 0.153 mAbA17 pH 8.5 Day 1 0.174 mAbA17 pH 8.5 Day 4 0.150 mAbA17 pH 8.5 Day 7 0.158 mAbA17 pH 8.5 Day 14 0.167

[0298] 2. Low pH stress test. The ability of the antibodies to bind 5T4 was also tested using ELISA under acidic (low pH) conditions. Solutions of 1 mg / mL to 10 mg / mL of the antibodies were prepared in 20 mM Tris / 10 mM EDTA at pH 8.5 and stored at 37°C for 1 to 2 weeks. The samples were stored frozen prior to analysis, which was performed by ELISA as described in Section A above. Table 9 below reports the EC values ​​calculated for each test antibody. 50 .

[0299] Table 9. Binding of antibodies to hu5T4 at low pH

[0300] mAbs condition <![CDATA[EC 50 (nM)]]> mAbA4 pH 5.5 Day 0 0.133 mAbA4 pH 5.5 Day 1 0.132 mAbA4 pH 5.5 Day 4 0.125 mAbA4 pH 5.5 Day 7 0.124 mAbA4 pH 5.5 Day 14 0.122 mAbA15 pH 5.5 Day 0 0.124 mAbA15 pH 5.5 Day 1 0.134 mAbA15 pH 5.5 Day 4 0.123 mAbA15 pH 5.5 Day 7 0.132 mAbA15 pH 5.5 Day 14 0.138 mAbA17 pH 5.5 Day 0 0.164 mAbA17 pH 5.5 Day 1 0.159 mAbA17 pH 5.5 Day 4 0.157 mAbA17 pH 5.5 Day 7 0.190 mAbA17 pH 5.5 Day 14 0.153

[0301] 3. Oxidative stress. The antibodies were tested for their ability to bind to 5T4 using an ELISA in an oxidative environment. Solutions of 1 mg / mL to 10 mg / mL of antibody were prepared in 20 mM citrate / 50 mM NaCl at pH 5.5 and stored at 37°C for 1 to 2 weeks. Samples were stored frozen prior to analysis by ELISA as described in Section A above. Table 10 below reports the EC values ​​calculated for each antibody tested. 50 .

[0302] Table 10. Binding of antibodies to hu5T4 under oxidative stress

[0303] mAbs condition <![CDATA[EC 50 (nM)]]> mAbA4 PBS Time 0 0.182 mAbA4 PBS time 24 hours 0.200 mAbA4 PBS time 48 hours 0.189 mAbA4 <![CDATA[PBS + H2O2 Time 0]]> 0.225 mAbA4 <![CDATA[PBS + H2O2 for 24 hours]]> 0.393 mAbA4 <![CDATA[PBS + H2O2 for 48 hours]]> 0.386 mAbA15 PBS Time 0 0.229 mAbA15 PBS time 24 hours 0.212 mAbA15 PBS time 48 hours 0.203 mAbA15 <![CDATA[PBS + H2O2 time 0]]> 0.191 mAbA15 <![CDATA[PBS + H2O2 for 24 hours]]> 0.190 mAbA15 <![CDATA[PBS + H2O2 for 48 hours]]> 0.189 mAbA17 PBS Time 0 0.198 mAbA17 PBS time 24 hours 0.186 mAbA17 PBS time 48 hours 0.185 mAbA17 <![CDATA[PBS + H2O2 time 0]]> 0.188 mAbA17 <![CDATA[PBS + H2O2 for 24 hours]]> 0.183 mAbA17 <![CDATA[PBS + H2O2 for 48 hours]]> 0.207

[0304] Example 8. Cross-reactivity of antibodies with cynomolgus monkey, rat and mouse 5T4 (ELISA and Octet)

[0305] The ability of the selected antibodies to bind to non-human 5T4-Ts was tested using ELISA and Octet. Using the ELISA protocol described in Example 7, the human 5T4 antigen was replaced with: recombinant cynomolgus monkey 5T4-Fc (derived from R&D Systems (Minneapolis, Minnesota, USA), catalog number 2280-TG-100 or LSBio (Seattle, Washington, USA), catalog number LS-G137131), recombinant mouse 5T4-Fc (R&D Systems, catalog number 5049-TG-100), recombinant rat TPBG / 5T4 (LSBio catalog number G56592) with 6His-SUMO, N-terminal tags, recombinant mouse TPBG / 5T4 (LSBio, catalog number G12168) antigen with His, N-terminal tags. For Octet analysis, protein A biochips were coated with antibodies, washed with PBS, and then reacted with antigens as described above. All antibodies tested bound to human, cynomolgus monkey and rat 5T4. No significant binding cross-reactivity was observed with mouse 5T4.

[0306] Example 9. Cell Binding of Antibodies to 5T4-Transfected CHO and HEK293 Cells

[0307] The selected antibodies were tested for their ability to bind to HEK293 and CHO cells transfected to express human, cynomolgus monkey, rat or mouse 5T4. Untransfected cells were used as controls to observe nonspecific binding. mAbA4, mAbA15 and mAbA17 reacted to HEK293 and CHO cells transfected to express human and cynomolgus monkey 5T4, but not to cells transfected to express rat or mouse 5T4.

[0308] *****

[0309] Throughout this application, reference is made to various publications, patents, patent applications and other documents. For all purposes, including to more fully describe the prior art related to the subject matter disclosed herein, the disclosures of these publications, patents, patent applications and other documents are all incorporated by reference into this application. Although the disclosed subject matter has been described with reference to the examples provided above, it should be understood that various modifications may be made without departing from the spirit of the disclosed subject matter. After reviewing this specification, many variations of the present invention will become apparent to those skilled in the art.

Claims

1. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (i) VH CDR1, VH CDR2 and VH CDR3 shown in VH comprising the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2 and VL CDR3 shown in VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) VH CDR1, VH CDR2 and VH CDR3 set forth in VH comprising the amino acid sequence of SEQ ID NO: 44, and VL CDR1, VL CDR2 and VL CDR3 set forth in VL comprising the amino acid sequence of SEQ ID NO: 45; or (iii) VH CDR1, VH CDR2 and VH CDR3 shown in VH comprising the amino acid sequence of SEQ ID NO:62, and VL CDR1, VL CDR2 and VL CDR3 shown in VL comprising the amino acid sequence of SEQ ID NO:

63.

2. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13 and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19 and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; as well as (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17 and 23.

3. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

4. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35 and 39; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40, and 43; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37, and 41; as well as (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 38 and 42.

5. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 41; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

30.

6. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50, 53, 13, and 57; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 58 and 61; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 55 and 59; as well as (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16 and 21; (2) having a VL CDR2 selected from the group consisting of an amino acid sequence of SEQ ID NOs: 5, 11, and 22; and (3) A VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 56 and 60.

7. An antibody or fragment thereof that binds to 5T4, wherein the antibody or fragment thereof comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 52; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 59; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

49.

8. The antibody or fragment thereof according to any one of claims 1 to 7, wherein the antibody or fragment thereof further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as shown in any one of SEQ ID NOs: 25, 26, 44, 45, 62 and 63.

9. The antibody or fragment thereof according to any one of claims 1 to 7, wherein the antibody or fragment thereof further comprises a human framework sequence.

10. The antibody or fragment thereof according to any one of claims 1 to 8, wherein the antibody or fragment thereof comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 45; or (iii) VH comprising the amino acid sequence of SEQ ID NO: 62 and VL comprising the amino acid sequence of SEQ ID NO:

63.

11. The antibody or fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.

12. The antibody or fragment thereof according to any one of claims 1 to 11, wherein the antibody is a humanized antibody, a human antibody or a chimeric antibody.

13. The antibody or fragment thereof according to any one of claims 1-12, wherein the antibody or fragment thereof is Fab, Fab′, F(ab′)2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a dual variable region antibody, a single variable region antibody, a linear antibody, a V region, or a multispecific antibody formed by an antibody fragment.

14. The antibody or fragment thereof according to any one of claims 1 to 13, conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent.

15. The antibody or fragment thereof of claim 14, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin or a drug.

16. A binding agent that binds to substantially the same epitope as the antibody or fragment thereof of any one of claims 1-15. The binding agent according to claim 16 , which is an antibody or a fragment thereof.

18. The binding agent of claim 16, comprising a non-antibody protein scaffold.

19. The binding agent of claim 18, wherein the non-antibody protein scaffold comprises a fibronectin scaffold, anticalin, adnectin, affibody, DARPin, fynomer, affitin, affilin, avimer, cysteine-rich desmin peptide, or an engineered Kunitz-type inhibitor.

20. A binding agent that competes with the antibody or fragment thereof of any one of claims 1 to 15 for binding to human 5T4.

21. The binding agent of claim 20, wherein the binding agent is an antibody or a fragment thereof.

22. One or more vectors comprising one or more polynucleotides or complementary polynucleotides encoding the antibody or fragment thereof according to any one of claims 1-15.

23. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 15 or the binding agent according to any one of claims 16 to 21, and a pharmaceutically acceptable carrier.

24. A method of treating cancer or a tumor in a subject, comprising administering to the subject the antibody or fragment thereof of any one of claims 1-15 or the pharmaceutical composition of claim 23.

25. A method for alleviating one or more symptoms associated with cancer or a tumor in a subject, comprising administering to the subject the antibody or fragment thereof of any one of claims 1-15 or the pharmaceutical composition of claim 23.

26. A method for reducing tumor size in a subject having a tumor, comprising administering to the subject the antibody or fragment thereof of any one of claims 1-15 or the pharmaceutical composition of claim 23.

27. A method for enhancing tumor cell removal in a subject having a tumor, comprising administering to the subject the antibody or fragment thereof of any one of claims 1-15 or the pharmaceutical composition of claim 23.

28. A method of treating a 5T4-related disease, disorder or condition in a subject, comprising administering to the subject the antibody or fragment thereof of any one of claims 1-15 or the pharmaceutical composition of claim 23.

29. The method of any one of claims 24-28, wherein one or more therapeutic agents in combination with the antibody or fragment thereof or the pharmaceutical composition are administered to the subject.

30. The method of any one of claims 24-29, wherein the subject is a human subject.

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