5T4 binder and its use

Abstract

The present disclosure provides 5T4 binders (including antibodies such as multispecific antibodies like bispecific antibodies, and antibody-drug conjugates) and their use. The present disclosure also provides compositions comprising a 5T4 binder. Such compositions include, in some embodiments, antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies and ADCs that bind to 5T4. Such compositions include, in some embodiments, antibodies and ADCs that bind to essentially the same human 5T4 epitope as the antibodies comprising the CDRs described herein (e.g., Tables 1-3).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 341,944, filed May 13, 2022. The entire disclosure of the provisional application is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer - readable sequence listing submitted in XML file format together with this application, the entire contents of which are incorporated herein by reference. The sequence listing of the XML file submitted together with this application is named "14529 - 107 - 228_SEQ_LISTING.xml", was created on May 8, 2023, and is 102,425 bytes in size.

[0003] Field The present disclosure generally relates to binding agents such as antibodies or fragments thereof that bind to 5T4, including human 5T4, and methods of using the same.

Background Art

[0004] 5T4 is an N - glycosylated transmembrane 72 kDa glycoprotein containing eight leucine - rich repeats. 5T4, also known as Wnt - activated inhibitor factor 1 or WAIF1, is often called a cancer - fetal antigen because it is expressed in fetal trophoblast cells (the place where it was first discovered) or as trophoblast glycoprotein (TPBG). 5T4 is found in tumors including colorectal, ovarian, and gastric. Its expression has been used as a prognostic adjunct. 5T4 has very limited expression in normal tissues but is widely distributed throughout its occurrence in malignant tumors. Its limited expression seems to give 5T4 the potential to be a target for cancer treatment, but the success of treatment with binding agents and vaccines targeting 5T4 has not yet been achieved.

[0005] Accordingly, there is still a need in the art for agents that can treat, prevent, or alleviate 5T4-mediated diseases, disorders, or conditions, including those involving targeting 5T4 and tumor cells expressing 5T4. SUMMARY OF THE INVENTION

[0006] 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 that bind to 5T4 and antibody-drug conjugates (ADCs). In some embodiments, such binding agents bind to the same epitope of human 5T4 as the antibodies comprising the CDRs (e.g., Tables 1-3) described herein. In some embodiments, such binding agents bind to the same epitope of human 5T4 as the antibodies comprising the heavy chain variable region and the light chain variable region described herein (e.g., Tables 1-3).

[0007] The present disclosure also provides compositions comprising 5T4-binding agents. Such compositions include, in some embodiments, antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to 5T4 and ADCs. Such compositions include, in some embodiments, antibodies and ADCs that bind to essentially the same epitope of human 5T4 as the antibodies comprising the CDRs described herein (e.g., Tables 1-3). Such compositions include, in some embodiments, antibodies and ADCs that bind to essentially the same epitope of human 5T4 as the antibodies comprising the heavy chain variable region and the light chain variable region described herein (e.g., Tables 1-3).

[0008] The present disclosure also includes methods of treating, preventing, or alleviating a 5T4-mediated disease, disorder, or condition, including one or more symptoms thereof, with a 5T4-binding agent or a composition comprising a 5T4-binding agent. Such compositions include antibodies that bind to 5T4, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to 5T4 and ADCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

Figure 1A

Figure 1B

[0010] The present disclosure provides 5T4 binders. Such agents include antibodies and ADCs that bind to human 5T4, including antibodies (e.g., multispecific including monospecific or bispecific) and ADCs that bind to 5T4. Such binders are useful in compositions and methods for treating, preventing, or alleviating a disease, disorder, or condition, including one or more symptoms of a 5T4-mediated disease, disorder, or condition. 5T4-mediated diseases, disorders, or conditions include, but are not limited to, various cancers in which tumor cells express or overexpress 5T4. As used herein, the term "overexpress" means to transcribe and translate more gene product than normal (such as in normal cells), and the process is often characteristic of cancer cells. Further, the 5T4 binders described herein, such as 5T4-binding antibodies (e.g., multispecific antibodies including monospecific or bispecific antibodies) and 5T4-binding ADCs, are useful for killing and / or removing tumor cells. 5T4 binders described herein, such as 5T4-binding antibodies (e.g., multispecific antibodies including monospecific or bispecific antibodies) and 5T4-binding ADCs, are useful in compositions and methods for treating cancer.

[0011] Unless otherwise indicated, the term "5T4" refers to any natural 5T4 derived from any vertebrate source, including a polypeptide (the terms "polypeptide" and "protein" are used interchangeably herein), or any mammal such as a primate (e.g., human, cynomolgus monkey (cyno), dog), and rodent (e.g., mouse and rat). 5T4, also known as "5T4 tumor trophoblast glycoprotein" or "Wnt activation inhibitory factor 1" or "5T4 tumor trophoblast glycoprotein" or "5T4 fetal cancer 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 resulting from intracellular processing, or any fragment thereof. In some embodiments, 5T4 includes 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 includes the 5T4 extracellular domain. The term 5T4 also encompasses naturally occurring variants of 5T4, such as SNPs, splice variants, and allelic variants. The full-length amino acid sequence of human 5T4 is shown below (exemplary signal sequence = italicized, exemplary extracellular domain = underlined).

Chemical formula

Chemical formula

Chemical formula

[0012] As used herein, the term "binding agent" or grammatical equivalents thereof refers to a molecule (e.g., an antibody) having one or more antigen-binding sites that bind to an antigen. In some embodiments, the 5T4 binding agents described herein are antibodies, antibody fragments, or other peptide-based molecules, and conjugates of antibodies, antibody fragments, or peptide-based molecules (e.g., ADCs) that bind to 5T4, e.g., human 5T4.

[0013] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and in the broadest sense, and specifically include, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions having polyepitope or monoepitope specificity, recombinantly produced antibodies, single-domain (e.g., VHH) antibodies, single-characteristic antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or humanized versions of antibodies having full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of an antibody consisting only of a heavy chain. Examples of single-domain antibodies include antibodies that are naturally lacking a light chain, such as those derived from camelid species (e.g., llama), single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies, but are not limited thereto. Single-domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, and cow. VHH can also be derived from species other than camelid animals that can produce heavy-chain antibodies that are naturally lacking a light chain. Antibodies also include antibody fragments (and / or polypeptides containing antibody fragments) that retain 5T4 binding properties. Non-limiting examples of antibody fragments include the antigen-binding region and / or effector region of an antibody, such as Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single-chain antibody molecules, bispecific variable domain antibodies, single variable domains, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, bisdiabodies, disulfide-bonded Fv (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments consisting of non-covalently associated variable regions of heavy and light chains). Generally speaking, the variable (V) region domain can be any suitable arrangement of the variable domains of immunoglobulin heavy chain (VH) and / or light chain (VL). For example, antibodies also include tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers.Thus, for example, the V-region domain can be a dimer and can include a VHH-VHH, VH-VH, VH-VL, or VL-VL dimer that binds to 5T4. Optionally, VH and VL may be covalently linked either directly or via a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are considered herein to be included in the category of "antibody fragments". Another form of antibody fragment is a peptide that includes one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also referred to as "minimal recognition units" or "hypervariable regions") can be obtained by constructing a polynucleotide that encodes one or more CDRs of interest. Such polynucleotides are prepared, for example, by using 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.), page 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.), page 137, Wiley-Liss, Inc. (1995)).Antibody fragments include, for example, single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetra-bodies, variable domains of novel antigen receptors (v-NARs), and bis single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, an antibody comprising VH and / or VL contains one or more constant regions, such as one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions, including light chain and / or heavy chain constant regions. In some embodiments, an antibody may comprise any of the above epitope-binding fragments. The antibodies described herein can be of 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.

[0014] As used herein with respect to a binder (e.g., an antibody or an ADC), the term "monospecific" refers to a binder having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0015] The term "bispecific", when used with respect to a binding agent (e.g., an antibody or an ADC), means that the binding agent is capable of specifically binding to at least two distinct antigenic determinants, e.g., formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or VHH domains that bind to different antigens or different epitopes on the same antigen. Such bispecific binding agents (e.g., antibodies or ADCs) can 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 can 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 an ADC) contains two antigen-binding sites, each can bind to a different antigenic determinant. Such bispecific binding agents (e.g., antibodies or ADCs) can bind to two different epitopes on the same antigen (e.g., an epitope on 5T4).

[0016] The terms "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that, when compared and aligned to obtain a maximum correspondence (introducing gaps if necessary) without considering any conservative amino acid substitutions as part of sequence identity, are the same or have the same nucleotide or amino acid residues at a specified percentage. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining an alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variants. Two nucleic acids or polypeptides are substantially identical if, when measured using a sequence comparison algorithm or by visual inspection and compared and aligned for maximum correspondence, in some embodiments, they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% nucleic acid or amino acid residue identity, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity. In some embodiments, identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40 - 60 residues, at least about 60 - 80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60 - 80 residues, for example, at least about 80 - 100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding region of a target protein or antibody. In some embodiments, identity exists over a region of the nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40 - 60 bases, at least about 60 - 80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60 - 80 bases, for example, over at least about 80 - 1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the nucleotide sequence encoding the protein of interest.

[0017] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues having similar side chains are generally defined in the art and include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β - branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substituting phenylalanine with tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not inhibit binding to the target binding site of the polypeptide, soluble protein, or antibody containing the amino acid sequence. Methods for identifying amino acid conservative substitutions that do not eliminate binding are well known in the art.

[0018] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may contain non-amino acids (e.g., interrupted thereby). These terms also include amino acid polymers that have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage or conjugation (directly or indirectly) with a moiety such as a labeling component or a drug (e.g., a toxin), etc. Also included in this definition are, for example, polypeptides containing one or more analogs of amino acids (including, e.g., non-natural amino acids, etc.), and other modifications known in the art. Since the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in some embodiments, the polypeptide can occur as a single chain or a single-chain dimer.

[0019] As used herein, an "antigen" is a moiety or molecule that includes an epitope to which a binding agent (e.g., an antibody or an ADC) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen to which the binding agents (e.g., antibodies or ADCs) described herein bind is 5T4 (e.g., human 5T4), or a fragment thereof, e.g., one or more domains of 5T4.

[0020] As used herein, "epitope" is a term in the art and refers to the localized 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. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of the polypeptide ("linear" epitope), or the epitope can be amino acids in two or more non-contiguous regions of the polypeptide ("conformational", "non-linear", or "discontinuous" epitope), for example including human 5T4. In general, it will be understood by those skilled in the art that linear epitopes may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether the amino acids are folded into a native three-dimensional protein structure. In other embodiments, an antibody requires that the amino acid residues that make up the epitope exhibit a particular conformation (e.g., bend, twist, rotation, or fold) in order to recognize and bind the epitope.

[0021] If two antibodies recognize the same, overlapping, or adjacent epitope in three-dimensional space, the antibodies bind to the "epitope" or "essentially the same epitope" or "same epitope". The most widely used rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitope in three-dimensional space is a competition assay, which can be configured in various different formats using, for example, either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on the cell surface, and the ability of an unlabeled antibody to block the binding of a labeled antibody is measured using a radioactive, fluorescent, or enzyme label.

[0022] "Epitope binning" is a process of classifying antibodies based on the epitopes recognized by the antibodies. More specifically, epitope binning involves clustering antibodies based on their epitope recognition characteristics and using a competitive assay combined with a computational process to identify antibodies with different binding specificities, and includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies. Further details regarding the determination methods of antibody epitope binning and epitope binding are described herein, as shown in Example 5.

[0023] As used herein, the terms "specifically binds," "specifically recognizes," "immunologically specifically binds," "selectively binds," "immunologically specifically recognizes," and "immunologically specific" are similar terms with respect to antibodies and such binding refers to a molecule (e.g., an epitope) that binds to an antigen, as would be understood by one of ordinary skill in the art. In some embodiments, "specifically binds" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in any combination of the above, than when using alternative substances including related and unrelated proteins. For example, a molecule that specifically binds to an antigen generally binds to other peptides or polypeptides with a lower affinity, as determined by, for example, an immunoassay, Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with a higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction is at least two-fold the background signal or noise and may exceed ten-fold the background. For considerations regarding binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). 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 the antibody or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA.In some embodiments, the molecule that specifically binds to an antigen binds to the antigen with a Ka that is at least 2 log, 2.5 log, 3 log, 4 log, or greater than that, compared to the Ka when these molecules bind to another antigen. In some embodiments, the molecule that specifically binds to an antigen does not cross-react with other proteins. In another particular embodiment, the molecule that specifically binds to an antigen does not cross-react with other non-5T4 proteins. In some embodiments, "specifically binds" means, for example, that a polypeptide or molecule binds to a protein or target with a K of about 0.1 mM or less, but more typically less than about 1 μM. D means binding. In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a 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. D means binding. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize a protein or target in multiple species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize multiple proteins or targets. In some embodiments, it is understood that a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require, but can include, exclusive binding, e.g., binding to a single target. Thus, in some embodiments, a polypeptide or molecule can specifically bind to multiple targets. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, in certain cases, an antibody can contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In another particular embodiment, an antibody can be bispecific and can contain at least two antigen-binding sites with different specificities. Generally, although not necessarily, reference to "binding" means "specific binding".

[0024] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., a binder such as an antibody or ADC) and its binding partner (e.g., an antigen such as 5T4). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 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 partner Y can generally be represented by 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 to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present disclosure. In one embodiment, the "K D " or "K D value" can be measured, for example, by biolayer interferometry (BLI) using an OctetQK384 system (ForteBio, Menlo Park, CA). As another method, K D can also be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881), or, for example, using a surface plasmon resonance (SPR) assay by BIACORE™ using a BIACORE™-2000 or BIACORE™-3000 (BIACORE, Inc., Piscataway, NJ). "On-rate" or "rate of binding" or "binding rate" or "k on ", as well as "off-rate" or "rate of dissociation" or "dissociation rate" or "k off" can also be determined, for example, by the same SPR or BLI techniques described above using, respectively, an OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ).

[0025] When used with respect to a 5T4 binder (e.g., an antibody or an ADC), the term “compete” means binders that compete for the same epitope or binding site on the target, and this includes competition between such binders as determined by an assay in which the binder being tested prevents or inhibits the 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). To determine whether a test binder competes with a reference molecule for binding to 5T4 (e.g., human 5T4), a number of types of competitive binding assays can be used. Examples of assays that can be used include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (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 labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeled RIA using I-125 labeling (e.g., Morel et al., (1988) Molec. Immunol. 25:7-15), and direct labeled RIA (see, e.g., Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of a purified antigen (e.g., 5T4 such as human 5T4) bound to a solid-phase surface or cell having either an unlabeled test antigen-binding protein (e.g., a test 5T4 antibody or ADC) or a labeled antigen-binding protein (e.g., a reference 5T4 antibody or ADC). Competitive inhibition can be measured by determining the amount of label that binds to the solid-phase surface or cell in the presence of the test antigen-binding protein.Typically, the test antigen-binding protein is present in excess. The antibodies identified by a competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to an adjacent epitope that is close enough to the epitope bound by reference to cause steric hindrance of the antibody (e.g., epitopes with overlapping similar epitopes). Typically, when competitive antibodies are present in excess, the specific binding of the reference antibody to the common antigen is inhibited by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, the binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0026] As used herein, the terms "constant region" or "constant domain" are terms of antibodies well known in the art and refer to the carboxy-terminal portions of the light chain and / or heavy chain that do not directly participate in the binding of the antibody to an antigen, for example, but exhibit various effector functions such as interaction with Fc receptors. This term includes portions of immunoglobulin molecules that generally have a more conserved amino acid sequence compared to immunoglobulin variable domains.

[0027] The "effector function" of an antibody refers to the biological activity attributable to the Fc region of the antibody (e.g., the Fc region of a native sequence or an amino acid sequence variant Fc region) and varies depending on the isotype of the antibody. Examples of effector functions of antibodies include C1q 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.

[0028] The term "Fc region" as used 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. The boundaries of the Fc region of an immunoglobulin heavy chain can vary widely, but the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are provided below (CH2 domain = bold, CH3 domain = underlined). [Chemical Formula]

[0029] 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 (e.g., B cell receptor, BCR), and the like. 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 evaluated using various assays as disclosed herein.

[0030] A "native sequence Fc region" is identical to the amino acid sequence of an Fc region found in nature and includes an amino acid sequence in which no artificial manipulation, modification, and / or alteration (e.g., isolation, purification, selection, inclusion or combination with other sequences such as variable region sequences) has been performed. Native sequence human Fc regions include the Fc region of native sequence human IgG1 (non-A and A allotypes), the Fc region of native sequence human IgG2, the Fc region of native sequence human IgG3, and the Fc region of native sequence human IgG4, as well as their natural variants.

[0031] The "variant Fc region" contains an amino acid sequence different from that of the native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions (multiple possible). In some embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein can have at least about 80% homology, or at least about 90% homology, e.g., at least about 95% homology, with the native sequence Fc region and / or the Fc region of the parent polypeptide. The variant Fc region described herein can have a loss of effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are provided below (CH2 domain = amino acid changes in bold and underlined, CH3 domain = underlined text).

Chemical formula

[0032] As used herein, the term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50 - 70 kDa, the amino-terminal portion of which contains a variable region of about 120 - 130 or more amino acids, and the carboxy-terminal portion of which contains one or more constant regions. "Heavy chain" can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), for example, based on the amino acid sequence of the constant domain. These give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, and include subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0033] As used herein, the term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are, for example, two different types, kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0034] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that interacts with an antigen and contains the amino acid residues that confer on the binding fragment, domain, or region its specificity and affinity for the antigen (e.g., CDR). As used herein, "antigen-binding fragment" includes "antibody fragments" that include portions of an antibody that contain one or more CDRs, such as the antigen-binding or variable region of an antibody.

[0035] 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, intrabodies, single-chain Fv (scFv) (e.g., including single-specificity, bispecific, etc.), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-bonded Fv (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0036] In some embodiments, antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules that contain one or more antigen-binding sites that bind to the 5T4 antigen.

[0037] The antibody can be of 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 antibodies described herein are IgG antibodies (e.g., human IgG), or of that class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass thereof.

[0038] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H)-chain / light (L)-chain pairs, the amino acid sequences of the H chains are identical, and the amino acid sequences of the L chains are identical. In some embodiments, the antibody is a two-chain antibody unit comprising a VHH-VHH pair, and the amino acid sequences of the VHHs are identical. In some embodiments, the H and / or L chains comprise a constant region, e.g., a human constant region. In some embodiments, the L-chain constant region of such an antibody is a kappa or lambda light-chain constant region, e.g., a human kappa or lambda light-chain constant region. In some embodiments, the H-chain constant region of such an antibody comprises a gamma heavy-chain constant region, e.g., a human gamma heavy-chain constant region. In some embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0039] 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 higher affinity than it binds to an irrelevant control protein, and / or binds to human 5T4 with a higher affinity than it binds to an irrelevant control protein. For example, the antibody or fragment thereof can specifically recognize and bind to 5T4 or a part thereof. "Specific binding" means that the antibody or fragment thereof binds to 5T4 with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than its affinity for an irrelevant control protein (e.g., chicken egg white lysozyme). In some embodiments, the antibody or fragment thereof can bind substantially exclusively to 5T4 (e.g., distinguishable from other known polypeptides by a measurable difference in binding affinity). In some embodiments, the 5T4 binder (e.g., an antibody or ADC) can react with 5T4 sequences other than the human 5T4 sequence (e.g., cynomolgus monkey 5T4 sequence).

[0040] The terms "variable region" or "variable domain" generally refer to the portions of the light or heavy chains that are located at the amino termini of the light or heavy chains, respectively, of an antibody having a length of approximately 120 - 130 amino acids for the heavy chain and approximately 100 - 110 amino acids for the light chain, and are used for the binding and specificity of a particular antibody to its particular 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 regions vary widely between antibodies. The V regions mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, the variability is not uniformly distributed throughout the entire 110 - amino acid span of the variable region. Rather, the V region consists of stretches of relatively low variability (e.g., relatively invariant), called framework regions (FRs), of about 15 - 30 amino acids, separated by shorter regions of greater variability (e.g., extreme variability), called "hypervariable regions" or "complementarity - determining regions". The variable regions of the heavy and light chains each contain four framework regions (FR1, FR2, FR3, and FR4), which predominantly adopt a β - sheet conformation and are connected by three hypervariable regions, which form loops connecting the β - sheet structures and, in some cases, form part of the β - sheet structure. The hypervariable regions in each chain are held together in close proximity by the framework and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen - binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions such as the antibody's involvement in antibody - dependent cell - mediated cytotoxicity (ADCC) and complement - dependent cytotoxicity (CDC). The variable regions have widely different sequences between different antibodies. The sequence variability is concentrated in the CDRs, while the less variable portions of the variable regions are called the framework regions (FRs). The CDRs of the light and heavy chains are mainly responsible for the interaction between the antibody and the antigen.In a specific embodiment, the variable region is a human variable region.

[0041] As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region", or "CDR" refer to regions of the antibody variable region where the sequences are hypervariable and / or form structurally defined loops. Generally, an antibody contains 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). Several hypervariable region notations are used and are included herein. Kabat CDRs are based on sequence variability and are 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)). Chothia, instead, refers to the positions of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using Kabat numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B, and when neither 35A nor 35B is present, the loop ends at 32, when only 35A is present, the loop ends at 33, and when both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering Vol. 2, Chapter 3, Springer Verlag). The "Contact" hypervariable regions are based on analysis of available complex crystal structures. The residues derived from each of these hypervariable regions are shown below.

[0042] The Universal Numbering System has been developed and widely adopted (ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system specialized for human and other vertebrate immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complex (MHC). In this specification, CDRs are referred to both in terms of the amino acid sequence and their position within the light or heavy chain. The "position" of the CDRs within the structure of the immunoglobulin variable domain is conserved across species and exists in a structure called a loop, so that by using a numbering system that aligns the variable domain sequences according to structural features, CDRs and framework residues can be readily identified. This information can be used when grafting and substituting CDR residues derived from a certain immunoglobulin into an acceptor framework typically derived from a human antibody. A further numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). For example, the correspondence between numbering systems, including Kabat numbering and the IMGT proprietary 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 described below. The various systems known in the art or described herein represent different ways of defining CDRs and are often considered equivalent when they are used to define the same antibody. The exemplary system shown herein is a combination of Kabat and Chothia. [Table 11]

[0043] The hypervariable region may include the following "extended hypervariable regions": 24 - 36 or 24 - 34 (L1), 46 - 56 or 50 - 56 (L2), and 89 - 97 or 89 - 96 (L3) for VL, and 26 - 35 or 26 - 35A (H1), 50 - 65 or 49 - 65 (H2), and 93 - 102, 94 - 102, or 95 - 102 (H3) for VH. The terms "hypervariable region", "HVR", "HV", "complementary determining region", or "CDR" as used herein are used interchangeably.

[0044] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, for example, for introducing the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, and may contain selectable sequences or selectable markers for stable integration into the chromosome of the host cell. Further, the vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients not present in the medium. Expression control sequences may include constitutive and / or inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more nucleic acid molecules (e.g., both the antibody heavy and light chains or both antibody VH and VL) are co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or separate expression vectors. In the case of single vector expression, the coding nucleic acids can be operably linked to one common expression control sequence or to different expression control sequences such as one inducible promoter and one constitutive promoter. Introduction of the nucleic acid molecule into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting for expression of the gene product, or other analytical methods suitable for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art understand that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., the 5T4 binder described herein), and further understand that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0045] "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 5T4-expressing cells, such as 5T4-expressing tumor cells. 5T4-mediated diseases include, but are not limited to, cancers that express or overexpress 5T4.

[0046] "Effective amount" generally refers to an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or the underlying cause, prevent the occurrence of symptoms and / or their underlying cause, and / or improve or repair damage resulting from or associated with a disease, disorder, or condition. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0047] 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) that is sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or condition, and / or associated symptoms. The therapeutically effective amount of an agent comprising a therapeutic agent can be an amount necessary for (i) reducing or improving the progression or exacerbation of a given disease, disorder, or condition, (ii) reducing or improving the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) enhancing or augmenting the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein). The "therapeutically effective amount" of a substance / molecule / agent of the present disclosure (e.g., a 5T4 antibody or ADC) can vary based on several factors, such as the individual's medical condition, age, gender, and weight, and the ability of the substance / molecule / agent to elicit the desired response in the individual. A therapeutically effective amount encompasses an amount where any toxic or adverse effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term "therapeutically effective amount" refers to the amount of an antibody or other agent (e.g., an ADC or drug) that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0048] "Preventive effective amount" means an amount of a drug that, when administered to a subject, will have the intended preventive effect, such as prevention or delay of the onset (or recurrence) of a disease, disorder or condition, or reduction in the likelihood of onset (or recurrence) of a disease, disorder or condition, or related symptom(s).

[0049] A complete therapeutic or preventive effect is not necessarily achieved by administration of a single dose and may occur only after administration of a series of doses. Thus, a therapeutically or preventively effective amount may be administered in one or more administrations.

[0050] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0051] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, stabilizer, or preservative that is non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. In many cases, the carrier is an aqueous pH buffered solution. Examples of carriers include buffers such as phosphoric acid, citric acid, 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 carbohydrates including glucose, mannose, or dextrin; 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™, polyethylene glycol (PEG), and PLURONICS™. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic agent is administered. Such carriers can be well-applied sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Physiological saline as well as aqueous dextrose 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition can also optionally contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can have the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.Oral compositions containing the formulation can include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions containing a pharmaceutical compound can include, for example, a prophylactically or therapeutically effective amount of a 5T4 binder (e.g., an antibody or ADC) in an isolated or purified form, together with an amount of carrier suitable to provide a form for proper administration to a subject (e.g., a patient). The formulation needs to be suitable for the mode of administration.

[0052] In some embodiments, the present disclosure provides a 5T4 binder that can be used herein as a therapeutic agent. Such agents include antibodies (e.g., multispecific including monospecific or bispecific) and ADCs that bind to human 5T4. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, and variants thereof having increased or decreased affinity or other properties.

[0053] In some embodiments, described herein is a 5T4 binder (e.g., an antibody or an ADC) that binds to 5T4 and comprises a 5T4 polypeptide, a 5T4 polypeptide fragment, a 5T4 peptide, or a 5T4 epitope. In some embodiments, the 5T4 binder is a human or humanized antibody (e.g., comprising a human constant region) that binds to 5T4 and comprises a 5T4 polypeptide, a 5T4 polypeptide fragment, a 5T4 peptide, or a 5T4 epitope. In some embodiments, a 5T4 binder such as a human 5T4 binder (e.g., an antibody or an ADC) can bind to 5T4 expressed on the surface of mammalian (e.g., human) cells including 5T4-expressing tumor cells. In some embodiments, the 5T4 binder (e.g., an antibody or an ADC) binds to a 5T4 extracellular epitope (e.g., a 5T4 epitope) expressed on cells such as tumor cells. In some embodiments, described herein is a 5T4 binder (e.g., an antibody or an ADC) that binds to 5T4 such as human 5T4 or a portion thereof. In some embodiments, 5T4 is human 5T4. In some embodiments, the 5T4 binder is a human 5T4 binder (e.g., an antibody or an ADC that binds to human 5T4). Exemplary amino acid sequences of human 5T4 are described herein.

[0054] In some embodiments, the 5T4 binding agents (e.g., antibodies or ADCs) described herein compete for binding to 5T4, such as human 5T4, with a 5T4 binding agent (e.g., antibody or ADC) comprising the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 amino acid sequences shown in Tables 1-3 of any one of the antibodies described herein. 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 a 5T4 binding agent (e.g., antibody or ADC) comprising 1, 2, and / or 3 VH CDRs and / or 1, 2, and / or 3 VL CDRs from the antibodies identified as (a) mAbA4, (b) mAbA15, and (c) mAbA17 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 a 5T4 binding agent (e.g., antibody or ADC) comprising 1, 2, and / or 3 VH CDRs and 1, 2, and / or 3 VL CDRs from the antibodies identified as (a) mAbA4, (b) mAbA15, and (c) mAbA17 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 a 5T4 binding agent (e.g., antibody or ADC) comprising the VH region and VL region from the antibodies identified as (a) mAbA4, (b) mAbA15, and (c) mAbA17.In some embodiments, the 5T4 binder (e.g., an antibody or an ADC) described herein competes with a 5T4 binder (e.g., an antibody or an ADC) 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, with respect to binding to 5T4 such as human 5T4.

[0055] In some embodiments, the 5T4 binder (e.g., an antibody) described herein comprises a VH region, a VL region, a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of any one of the antibodies 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 shown in Tables 1-3. Thus, in some embodiments, the 5T4 binder (e.g., an antibody or an ADC) described herein comprises 1, 2, and / or 3 heavy chain CDRs and / or 1, 2, and / or 3 light chain CDRs from the antibodies identified as (a) the antibody identified as mAbA4, (b) the antibody identified as mAbA15, and (c) the antibody identified as mAbA17 shown in Tables 1-3. In some embodiments, the 5T4 binder (e.g., an antibody or an ADC) described herein comprises 1, 2, and / or 3 heavy chain CDRs and 1, 2, and / or 3 light chain CDRs from the antibodies identified as (a) the antibody identified as mAbA4, (b) the antibody identified as mAbA15, and (c) the antibody identified as mAbA17.

[0056] In some embodiments, the 5T4 binder (e.g., an antibody or ADC) comprises a VH region comprising VH CDR1, VH CDR2, and / or VH CDR3 of any one of the binders described herein (e.g., see any one of Tables 1-3), and / or a VL region comprising VL CDR1, VL CDR2, and / or VL CDR3. Thus, in some embodiments, the 5T4 binder (e.g., an antibody or ADC) described herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs of Table 1. In some embodiments, the 5T4 binder (e.g., an antibody or ADC) described herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs of Table 2. In some embodiments, the 5T4 binder (e.g., an antibody or ADC) described herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs of Table 3.

[0057] The antibody identified as mAbA4 comprises the VH sequence of SEQ ID NO: 25 and the VL sequence of SEQ ID NO: 26.

[0058] The antibody identified as mAbA15 comprises the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 45.

[0059] The antibody identified as mAbA17 comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

Table 1-1

Table 1-2

Table 2-1

Table 2-2

Table 3-1

Table 3-2

[0060] In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises a VH region or VH domain. Additionally or alternatively, in some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises a VL region or VL domain. In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein has a combination of (i) a VH domain or VH region, and / or (ii) a VL domain or VL region.

[0061] In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises a heavy chain having a combination of (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 described herein as well as the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69). Other exemplary IgG heavy chains include any VH sequence described herein as well as the following CH1, hinge, CH2, and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70).

[0062] In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises (i) a VL domain described in any one of Tables 1-3, and (ii) a light chain constant domain (CL). Exemplary light chains (e.g., a light chain that pairs with an IgG heavy chain) include any VL sequence described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 71).

[0063] In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises a heavy chain having a combination of (a)(i) a VH described in any one of Tables 1-3 and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3), and a light chain having a combination of (b)(i) a VL described in any one of Tables 1-3 and (ii) a light chain constant domain in IgG format (CL or CL1). Exemplary 5T4 binders (e.g., antibodies or ADCs) comprise an IgG heavy chain comprising any VH sequence described herein and the amino acid sequence of SEQ ID NO: 69 or 70, and a light chain comprising any VL sequence described herein and the amino acid sequence of SEQ ID NO: 71.

[0064] In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises one or more CDRs, such as six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 specified in Table 1. In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising a human 5T4 binder described herein comprises one or more CDRs, such as six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 specified in Table 2. In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs, such as six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 specified in Table 3. In some embodiments, a 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs, such as six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 specified in Tables 1, 2, and / or 3.

[0065] In some embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more VH CDRs comprising three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 as set forth in Table 1. In other embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more VL CDRs comprising three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 as set forth in Table 1. In still other embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs comprising the three VH CDRs listed in Table 1, e.g., VH CDR1, VH CDR2, VH CDR3, and one or more CDRs comprising the three VL CDRs listed in Table 1, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0066] In some embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs comprising three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 as listed in Table 2. In other embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs comprising three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 as listed in Table 2. In some further embodiments, a 5T4 binder comprising a human 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more CDRs comprising the three VH CDRs listed in Table 2, e.g., VH CDR1, VH CDR2, VH CDR3, and one or more CDRs comprising the three VL CDRs listed in Table 2, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0067] In some embodiments, the 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising the human 5T4 binder described herein comprises one or more CDRs comprising three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 3. In other embodiments, the 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising the human 5T4 binder described herein comprises one or more CDRs comprising three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 3. In yet some other embodiments, the 5T4 binder (e.g., an antibody such as a bispecific antibody or an ADC) comprising the human 5T4 binder described herein comprises one or more CDRs comprising three VH CDRs, e.g., VH CDR1, VH CDR2, VH CDR3 listed in Table 3, and one or more CDRs comprising three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 3.

[0068] In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody) comprises 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 binder described herein (e.g., an antibody or ADC such as a bispecific antibody) comprises 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 binder described herein (e.g., an antibody or ADC such as a bispecific antibody) comprises 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 binder described herein (e.g., an antibody or ADC such as a bispecific antibody) comprises 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 binder described herein (e.g., an antibody or ADC such as a bispecific antibody) comprises 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 binder described herein (e.g., an antibody or ADC such as a bispecific antibody) comprises 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.

[0069] In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3. In other embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3. In still other embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) 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 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VH CDR1 comprising an amino acid sequence comprising any one of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 31, 34, 35, 39, 46, 50, 53, and 57. In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VH CDR2 comprising an amino acid sequence comprising 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, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VH CDR3 comprising an amino acid sequence comprising any one of SEQ ID NOs: 3, 9, 15, 20, 29, 33, 37, 41, 48, 52, 55, and 59. In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from VH CDR1, VH CDR2, VH CDR3 shown in any one of the amino acid sequences shown in Tables 1-3. In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VL CDR1 comprising an amino acid sequence comprising any one of SEQ ID NOs: 4, 10, 16, and 21.In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 11, and 22. In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 6, 17, 23, 30, 38, 42, 49, 56, and 60. In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a VL CDR1 and / or a VL CDR2 and / or a VL CDR3 independently selected from the VL CDR1, VL CDR2, VL CDR3 shown in any one of the amino acid sequences shown in Tables 1-3.

[0070] In some embodiments, the 5T4 binding agent described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a heavy chain variable (VH) region comprising a VH CDR1 comprising an amino acid sequence selected from the group consisting of (1)(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 comprising 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 CDR3 comprising 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 a VL CDR1 comprising an amino acid sequence selected from the group consisting of (1)(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 comprising 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 comprising 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.

[0071] In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising 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 comprising 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 CDR3 comprising 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.

[0072] In some embodiments, the 5T4 binder described herein (e.g., an antibody such as a bispecific antibody or an ADC) comprises a light chain variable (VL) region comprising (1) a VL CDR1 comprising 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 comprising 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 comprising 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.

[0073] In some embodiments, the present specification describes an antibody or a fragment thereof that binds to 5T4, and the antibody or the fragment thereof comprises a heavy chain variable (VH) region comprising (a) (1) (i) an amino acid sequence selected from the group consisting of 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 for VH CDR1, (2) (i) an amino acid sequence selected from the group consisting of 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 for VH CDR2, and (3) (i) an amino acid sequence selected from the group consisting of 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 for VH CDR3, and / or a light chain variable (VL) region comprising (1) (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 4, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 16 and (iv) SEQ ID NO: 21 for VL CDR1, (2) (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 5, (ii) SEQ ID NO: 11, and (iii) SEQ ID NO: 22 for VL CDR2, and (3) (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 30, or 49, (ii) SEQ ID NO: 17, 38, or 56, and (iii) SEQ ID NO: 23, 42, or 60 for VL CDR3.

[0074] In some embodiments, the present specification describes an antibody or a fragment thereof that binds to 5T4, and the antibody or the fragment thereof comprises a heavy chain variable (VH) region comprising (1) a VH CDR1 comprising 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 comprising 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 CDR3 comprising 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.

[0075] In some embodiments, the present specification describes an antibody or a fragment thereof that binds to 5T4, and the antibody or the fragment thereof comprises a light chain variable (VL) region comprising (1) a VL CDR1 comprising 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 comprising 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 comprising 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.

[0076] In some embodiments, described herein is an antibody or fragment thereof that binds to 5T4 and that comprises all three heavy chain complementarity determining regions (CDRs) and / or all three light chain CDRs derived from the antibody identified as mAbA4 comprising the VH sequence of SEQ ID NO: 25 and the VL sequence of SEQ ID NO: 26, the antibody identified as mAbA15 comprising the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 45, or the antibody identified as mAbA17 comprising the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs derived from the antibody identified as mAbA4. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs derived from the antibody identified as mAbA15. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs derived from the antibody identified as mAbA17.

[0077] In some embodiments, described herein is an antibody or fragment thereof that binds to 5T4, the antibody comprising (a) a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in Tables 1-3, and / or (b) a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 shown in Tables 1-3. In some embodiments, the antibody comprises a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in Tables 1-3. In some embodiments, the antibody comprises a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 shown in Tables 1-3.

[0078] In some embodiments, described herein is an antibody comprising a heavy chain variable (VH) region comprising (a) (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 a light chain variable (VL) region comprising (b) (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.

[0079] In some embodiments, described herein is an antibody comprising a heavy chain variable (VH) region comprising (a) (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable (VL) region comprising (b) (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0080] In some embodiments, described herein is an antibody comprising a heavy chain variable (VH) region comprising (a) (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 9, and a light chain variable (VL) region comprising (b) (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0081] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 12, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0082] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0083] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 18, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 20; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 23.

[0084] In some embodiments, described herein are antibodies comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 24, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0085] In some embodiments, described herein are antibodies comprising: (a) a variable heavy (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 variable light (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.

[0086] In some embodiments, described herein are antibodies comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 30.

[0087] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 31, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 32, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 33; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 30.

[0088] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 34, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 30.

[0089] In some embodiments, described herein is an antibody comprising: (a) a variable heavy (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 35, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 36, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 37; and (b) a variable light (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0090] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 39, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 40, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 41; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 42.

[0091] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 43, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 30.

[0092] 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.

[0093] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 46, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 47, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0094] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 50, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 51, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 52; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0095] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 53, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 47, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0096] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 54, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 55; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 56.

[0097] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 57, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 58, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 59; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 60.

[0098] In some embodiments, described herein is an antibody comprising: (a) a heavy chain variable (VH) region comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 46, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 61, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 48; and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0099] In some embodiments, provided herein are VH regions and / or VL regions as described herein, where the VH and / or VL include a human framework sequence. In some embodiments, the VH region and / or VL region include a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence, e.g., a human FR1, human FR2, human FR3, and / or human FR4.

[0100] In some embodiments, the antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibody is a humanized antibody, a human antibody, or a chimeric antibody. In some embodiments, the antibodies described herein are multispecific antibodies formed from Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single-chain antibodies, bispecific variable region antibodies, single variable region antibodies, linear antibodies, V regions or antibody fragments.

[0101] In some embodiments, provided herein is a binder that binds to essentially the same epitope as any one of the antibodies or fragments thereof described herein. In some embodiments, provided herein is a binder that competes with any one of the antibodies or fragments thereof described herein for binding to human 5T4. In some embodiments, the binder is an antibody or fragment thereof, or an ADC comprising an antibody or fragment thereof.

[0102] In certain embodiments, a 5T4 binder (e.g., an antibody or an ADC) comprising a human 5T4 binder can be determined by 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, U.S. Department of Health and Human Services, NIH Pulication No. 91-3242).

[0103] In certain embodiments, a 5T4 binder (e.g., an antibody or an ADC) comprising a human 5T4 binder can be determined by the Chothia system, which is referred to herein as "Chothia CDR" (e.g., see 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. Patent No. 7,709,226).

[0104] In certain embodiments, the CDRs of a 5T4 binder (e.g., an antibody or an ADC) comprising a human 5T4 binder can be determined by the ImMunoGeneTics (IMGT®) system as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDR").

[0105] In certain embodiments, the CDRs of a 5T4 binder (e.g., an antibody or an ADC) that comprises a human 5T4 binder can be determined by the AbM system, which is referred to herein as "AbM CDR" and is described, for example, in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See also, for example, Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0106] In certain embodiments, the CDRs of a 5T4 binder (e.g., an antibody or an ADC) that comprises a human 5T4 binder can be determined by the Contact system, which is referred to herein as "Contact CDR" (see, for example, MacCallum RM et al., 1996, J Mol Biol 5:732-745). Contact CDR is based on the analysis of available complex crystal structures.

[0107] In some embodiments, the positions 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 binder (e.g., an antibody or ADC) comprising a human 5T4 binder as described herein can differ by 1, 2, 3, 4, 5, or 6 amino acid positions, provided that binding 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%). For example, in some embodiments, the positions defining any of the CDRs in Table 1, 2, or 3 can differ from the current CDR positions by shifting the N-terminal and / or C-terminal boundaries of the CDR by 1, 2, 3, 4, 5, or 6 amino acids, provided that binding 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%). In other embodiments, the length 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 binder (e.g., an antibody or ADC) comprising a human 5T4 binder as described herein can differ by 1, 2, 3, 4, 5, or more amino acids (e.g., be shorter or longer), provided that binding 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%).For example, in some embodiments, the CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be 1, 2, 3, 4, 5 or more amino acids shorter compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding 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%). In other embodiments, the CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be 1, 2, 3, 4, 5, or more amino acids longer compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding 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%). In some embodiments, the amino terminus of the CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be shortened or extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding 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%). Additionally or alternatively, in some embodiments, the carboxy terminus of the CDR1, CDR2, and / or CDR3 of the VH and / or VL can be shortened or extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-43, or 46-61, provided that binding 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%).To confirm whether binding to 5T4 (e.g., human 5T4) is maintained, any method known in the art can be used, such as the binding assays and conditions described in the "Examples" section of this specification. For example, in Example 2 described in this specification, an assay for measuring binding to 5T4 (e.g., human 5T4) is described.

[0108] In other embodiments, the 5T4 binding agent (e.g., an antibody or ADC) that binds to 5T4 and is presented herein further comprises conservative sequence modifications. For polypeptides that are 5T4 binding agents such as human 5T4 binding agents (e.g., antibodies), conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Thus, in some embodiments, predicted non-essential amino acid residues in 5T4 are replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids that do not eliminate antigen binding and its nucleotide coding are well known in the art (see, for example, 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 the 5T4 binding agent (e.g., an antibody or ADC) comprising a human 5T4 binding agent 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, the amino acid sequence modification refers to up to 1, 2, 3, 4, 5, or 6 amino acid substitutions with respect to the CDR, such as those described in any one of Tables 1-3. Thus, for example, each such CDR can include up to 5 conservative amino acid substitutions, such as up to 4 (or fewer) conservative amino acid substitutions, such as up to 3 (or fewer) conservative amino acid substitutions, such as up to 2 (or fewer) conservative amino acid substitutions, or 1 or fewer conservative amino acid substitutions.In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder comprises one or more CDRs (e.g., see Tables 1, 2, or 3) having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of mAbA4, mAbA15, or mAbA17. In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder contains a VH and a VL containing CDRs identical to those of mAbA4, mAbA15, or mAbA17 (e.g., see Tables 1, 2, or 3). In some embodiments, the amino acid sequence modification does not include any modification within the SDR. In some embodiments, the amino acid sequence modification does not include any modification within the CDRs (such as CDR1, CDR2, CDR3, or any combination thereof). In further embodiments, the amino acid sequence modification is within the framework or constant region.

[0109] In some embodiments, provided herein is an antibody or a fragment thereof comprising a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26, wherein 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%).

[0110] In some embodiments, provided herein is an antibody or a fragment thereof comprising a VH amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 44, and a VL amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 45, wherein the binding of the antibody or the 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%).

[0111] In some embodiments, provided herein is an antibody or a fragment thereof comprising a VH amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62, and a VL amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63, wherein the binding of the antibody or the 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%).

[0112] The present disclosure also provides a 5T4 binder (e.g., an antibody) having a masking moiety and / or a cleavable moiety, wherein one or more 5T4 binding domains of the 5T4 binder (e.g., an antibody) are masked (e.g., via the masking moiety) and / or activatable (e.g., via the cleavable moiety). Techniques for masking a 5T4 binder (e.g., an antibody) are well known in the art and include the SAFEbody masking technique (see, e.g., US Patent Application Publication No. 2019 / 0241886) and the Probody masking technique (see, e.g., US Patent Application Publication No. 2015 / 0079088). Such techniques can be used to produce masked and / or activatable 5T4 binders (e.g., antibodies). Such masked and / or activatable 5T4 binders (e.g., antibodies) are useful for the preparation of complexes including immune complexes, ADCs, masked ADCs, and activatable antibody-drug conjugates (AADCs), including any one of the 5T4 binders (e.g., antibodies) such as the human 5T4 binders of the present disclosure, which include those directly or indirectly conjugated to another agent such as a drug. For example, a 5T4 binder (e.g., an antibody) such as the human 5T4 binder of the present disclosure can be conjugated to one or more agents such as a drug via a covalent bond by a synthetic linker.

[0113] Optionally, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder is (directly or indirectly) linked or conjugated to a moiety having an effector function such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or immunomobilizing activity. Moieties that are (directly or indirectly) linked or conjugated include cytotoxic drugs (e.g., toxins such as auristatin), or non-cytotoxic (e.g., signal transduction regulators such as kinases, or masking moieties that mask one or more binding domains of a 5T4 binder (e.g., an antibody), or cleavable moieties that activate a 5T4 binder (e.g., an antibody) by cleaving to remove the masking of one or more binding domains of the 5T4 binder in the form of a masked complex in the tumor microenvironment. Moieties that promote immunomobilization may include other antigen-binding agents such as viral proteins that selectively bind to cells of the innate and / or adaptive immune system. Alternatively, or in addition, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder may be (directly or indirectly) linked or conjugated to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety having reporter activity (e.g., a detection label or a reporter protein). It will be understood that the features of the 5T4 binders (e.g., antibodies) comprising a human 5T4 binder described herein extend to polypeptides comprising 5T4 binder fragments.

[0114] In some embodiments, a 5T4 binder (e.g., an antibody) comprising a 5T4 binder described herein that binds to human 5T4 can be (directly or indirectly) linked or conjugated to a polypeptide, thereby generating an activatable antibody. In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder is (directly or indirectly) linked or conjugated to an additional agent. In some embodiments, the additional substance is a drug, and when the antibody of the ADC comprises a masking moiety and a cleavable moiety, an ADC or an AADC is provided.

[0115] In some embodiments, the 5T4 binding agent (e.g., an antibody) comprising the human 5T4 binding agent described herein is (directly or indirectly) conjugated or recombinantly linked to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable agent. The conjugated or recombinantly linked antibody comprising a masked or activatable complex can be useful, for example, in the treatment or prevention of a disease, disorder or condition such as a 5T4-mediated disease, disorder or condition. The conjugated or recombinantly linked 5T4 binding agent (e.g., an antibody) comprising a masked or activatable complex can be useful, for example, in monitoring or predicting the onset, development, progression, and / or severity of a 5T4-mediated disease, disorder, or condition.

[0116] Such diagnosis and detection can be accomplished, for example, by conjugating a 5T4 binding agent (e.g., an antibody) to a detectable substance such as, but not limited to, an enzyme including horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; a moiety including streptavidin / biotin or avidin / biotin (but not limited thereto); a fluorescent substance including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent substance including, but not limited to, luminol; a bioluminescent substance including, but not limited to, luciferase, luciferin, or aequorin; a chemiluminescent material including, but not limited to, an acridinium-based compound or HALOTAG; 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 ( 99Technetium (Tc), thallium ( 201 Titanium (Ti), gallium ( 68 Ga and 67 Gallium (Ga), palladium ( 103 Palladium (Pd), molybdenum ( 99 Molybdenum (Mo), xenon ( 133 Xenon (Xe), fluorine ( 18 Fluorine (F), 153 Samarium (Sm), 177 Lutetium (Lu), 159 Gadolinium (Gd), 149 Promethium (Pm), 140 Lanthanum (La), 175 Ytterbium (Yb), 166 Holmium (Ho), 90 Yttrium (Y), 47 Scandium (Sc), 186 Rhenium (Re), 188 Rhenium (Re), 142 Praseodymium (Pr), 105 Rhodium (Rh), 97 Ruthenium (Ru), 68 Germanium (Ge), 57 Cobalt (Co), 65 Zinc (Zn), 85 Strontium (Sr), 32 Phosphorus (P), 153 Gadolinium (Gd), 169 Ytterbium (Yb), 51 Chromium (Cr), 54 Manganese (Mn), 75 Selenium (Se), 113 Tin (Sn), or 117 Tin (Sn) (including, but not limited to, these) radioactive substances; positron-emitting metals using various positron emission tomography methods; and can be carried out by binding to non-radioactive paramagnetic metal ions.

[0117] Further provided 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 a fragment thereof, e.g., a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) for generating a fusion protein, and uses thereof. In particular, described herein are fusion proteins comprising an antigen-binding fragment of a 5T4 binding agent (e.g., an antibody) comprising a human 5T4 binding agent (e.g., CDR1, CDR2, and / or CDR3 of VH and / or VL) described herein and a heterologous protein, polypeptide, or peptide. In some embodiments, the heterologous protein, polypeptide, or peptide to which the 5T4 binding agent (e.g., an antibody) is linked is useful for targeting the 5T4 binding agent to a particular cell (e.g., a 5T4-expressing cell including a tumor cell). Other non-limiting heterologous proteins, polypeptides, or peptides to which the 5T4 binding agent (e.g., an antibody) may be linked may be useful as internalization signals or immune cell engagers.

[0118] Furthermore, a 5T4 binding agent (e.g., an antibody) comprising a human 5T4 binding agent described herein may be linked (directly or indirectly) to a marker or “tag” sequence, e.g., a peptide, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is, inter alia, a hexahistidine peptide, e.g., a tag provided by a 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, hexahistidine provides facile purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag corresponding to an epitope derived from the hemagglutinin protein of influenza (Wilson et al., 1984, Cell 37:767-78), and the “FLAG” tag.

[0119] Methods for linking or conjugating a moiety (including a polypeptide) to an antibody (either directly or indirectly) are well known in the art, and any of these can be used to make the antibody-drug conjugates or fusion proteins described herein.

[0120] In some embodiments, the 5T4 binding agents (e.g., antibodies) described herein are fusion proteins. As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of a binding agent (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not normally part of the antibody). In certain embodiments, the fusion protein retains the biological activity of the 5T4 binding agent. In certain embodiments, the fusion protein comprises the VH region, VL region, VH CDR (1, 2, or all 3 VH CDRs), and / or VL CDR (1, 2, or all 3 VL CDRs) of a 5T4 antibody, and the fusion protein binds to a 5T4 epitope, 5T4 fragment, and / or 5T4 polypeptide.

[0121] The fusion protein can be generated, for example, by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be employed, for example, to modify the activity of a 5T4 binder, including the human 5T4 binders described herein, such as a 5T4 binder (e.g., an antibody) that includes a 5T4 binder with higher affinity and lower dissociation rate (see, for example, U.S. Patent 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, a 5T4 binder that includes a human 5T4 binder can be modified by performing error-prone PCR, random nucleotide insertion, or introduction of random mutations by other methods prior to recombination. The polynucleotide encoding the 5T4 binder described herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[0122] A 5T4 binder (e.g., an antibody) that includes the human 5T4 binder described herein may also be bound to a solid support useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0123] A 5T4 binder (e.g., an antibody) that includes the human 5T4 binder described herein may also be linked or conjugated (directly or indirectly) to a secondary antibody to form an antibody heterocomplex.

[0124] The linker can be a "cleavable moiety" that facilitates the release of the linked or conjugated agent intracellularly, although 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 containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photo-labile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid multi-drug transporter-mediated resistance.

[0125] Complexes of antibodies and agents (where the agent includes those which are drugs for the preparation of ADCs or AADCs) can be prepared using various bifunctional protein coupling agents such as N-(β-maleimidopropyl)oxysuccinimide ester (BMPS), N-ε-maleimidocaproyl-oxysuccinimide ester (ECMS), N-γ-maleimidocaproyl-oxysuccinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4(-N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 3-(bromoacetamido)propionic acid succinimidyl (SBAP), succinimidyl iodoacetate (SIA), (4-iodoacetyl)aminobenzoic acid succinimidyl (SIAB), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(p-maleimidophenyl)butyric acid succinimidyl (SMPB), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-(ε-maleimidocaproyl)oxysulfosuccinimide ester (sulfo-ECMS), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS), N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl (sulfo-SMCC), 4-(p-maleimidophenyl)butyric acid sulfosuccinimidyl (sulfo-SMPB), and succinimidyl-(4-vinylsulfone)benzoic acid succinimidyl) (SVSB).

[0126] The present disclosure further contemplates that conjugates of antibodies and agents (including cases where the agent is a drug for the preparation of an ADC or AADC) can be prepared using any suitable method disclosed in the art (e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0127] Conventional conjugation strategies of antibodies and agents (including cases where the agent is a drug for preparing an ADC or AADC) are based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, and result in heterogeneous conjugates. Recently developed techniques enable site-specific binding to antibodies, providing uniform loading and avoiding subpopulations of complexes with altered antigen-binding or pharmacokinetics. These techniques include the manipulation of "thioMabs" that introduce reactive thiol groups and contain cysteine substitutions at positions on the heavy and light chains that 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 method, site-specific covalent bonding at the nucleophilic selenol group of selenocysteine in the presence of other natural amino acids is enabled by recoding the stop codon UGA from termination to selenocysteine insertion, thereby inserting selenocysteine into the antibody sequence during translation (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).

[0128] In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder described herein is conjugated to a cytotoxic agent. In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder disclosed herein may be conjugated, optionally, with one or more cytotoxic agent(s) disclosed herein to produce an 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 fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the cytotoxic agent is a radioisotope for generating a radiolabeled complex or radiolabeling agent. 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 212A variety of radionuclides, including but not limited to Bi, are available for the production of radioconjugates. Complexes of a polypeptide or molecule with one or more small molecule toxins such as calicheamicin, maytansinoid, trichothecene, and CC1065, and derivatives of these toxins having toxin activity can also be used. Complexes of a polypeptide or molecule with a cytotoxic drug are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (dimethyl adipimidate HCL etc.), active esters (disuccinimidyl suberate etc.), aldehydes (glutaraldehyde etc.), bis-azide compounds (bis(p-azidobenzoyl)hexanediamine etc.), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine etc.), diisocyanates (toluene 2,6-diisocyanate etc.), and bis-active fluorine compounds (1,5-difluoro-2,4-dinitrobenzene etc.).

[0129] In some embodiments, the 5T4 binding agent (e.g., an antibody) comprising the human 5T4 binding agent described herein is conjugated to a drug such as a signal transduction regulator, apoptosis promoter, mitotic inhibitor, antitumor antibiotic, immunomodulator, nucleic acid for gene therapy, alkylating agent, anti-angiogenic agent, antimetabolite, boron-containing agent, chemoprotective agent, hormonal agent, antihormonal agent, corticosteroid, photoactive therapeutic agent, oligonucleotide, radionuclide agent, radiosensitizer, topoisomerase inhibitor, and tyrosine kinase inhibitor. In some embodiments, the mitotic inhibitor is dolastatin, auristatin, maytansinoid, and plant alkaloid. In some embodiments, the drug is dolastatin, auristatin, maytansinoid, and plant alkaloid. Examples of auristatin are 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 actinomycin, anthracycline, calicheamicin, and duocarmycin. An example of actinomycin is pyrrolobenzodiazepine (PBD). An example of anthracycline is PNU-anthracycline such as PNU-159682 or a derivative.

[0130] 5T4 binders (e.g., antibodies) that include the human 5T4 binders described herein may be monospecific, bispecific, trispecific, or of higher-order multispecificity. Such agents may include monospecific or multispecific antibodies or ADCs. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody directed to 5T4 having 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 can be constructed based on the sequences of the 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 murine, chimeric, human, or humanized antibodies. In some embodiments, one of the binding specificities of the multispecific antibody is for 5T4 and the other is for any other target (e.g., antigen). In some embodiments, the multispecific (e.g., bispecific) antibody can include multiple target (e.g., antigen) binding domains, and the different binding domains are specific for different targets (e.g., a first binding domain that binds to 5T4 and a second binding domain that binds to another target (e.g., antigen), such as an immune checkpoint regulator (e.g., a negative checkpoint regulator)). In some embodiments, the multispecific (e.g., bispecific) antibody molecule can bind to multiple (e.g., two or more) epitopes on the same target (e.g., antigen).In some embodiments, one of the binding specificities is for 5T4 and the other is for one or more of 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 suppressor of T cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0131] Methods for making multispecific antibodies are well known in the art and include, for example, co-expression of two immunoglobulin heavy chain-light chain pairs where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0132] Exemplary structures of multispecific antibodies are known in the art and are 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.

[0133] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety added, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody complexes. As non-limiting examples, BsIgG formats can include crossMab, DAF(2-in-1), DAF(4-in-1), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and / or orthogonal Fab.

[0134] In some embodiments, BslgG comprises heavy chains engineered for heterodimerization. For example, the heavy chains can be engineered for heterodimerization using the "knob-into-hole" strategy, SEED platform, common heavy chains (e.g., in κλ-body), and use of a heterodimeric Fc region. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art and include knob-into-hole, duo-body, asymmetric, charge pair, HA-TF, SEEDbody, and differences in protein A affinity.

[0135] Another format of bispecific antibodies is IgG with an additional antigen-binding moiety. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at the N-terminus or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and diabody variable domains (e.g., single chain variable fragments or variable fragments). Non-limiting examples of the added IgG formats include bispecific 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, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (4-in-1). See Spiess et al. Mol Immunol. 67(2015):95-106. In some embodiments, exemplary antibody formats of monospecific or multispecific (e.g., bispecific antibodies) are in the B-Body format, as described, for example, in International Patent Application Publication No. WO2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0136] Bispecific antibody fragments (BsAbs) are formats of bispecific antibody molecules that lack some or all of the constant domains of antibodies. For example, some BsAbs lack the Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions connected by a peptide linker that enables efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, sc diabodies, sc diabody-CH3, diabody-CH3, triabodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies, but are not limited thereto.

[0137] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate higher-valency bispecific antibody molecules. For example, fusion of a bispecific antibody to an albumin-binding protein or human serum albumin can extend the serum half-life of the antibody fragment. In some embodiments, chemical conjugation, such as chemical conjugation of an antibody and / or an antibody fragment, can be used to create BsAb molecules. Exemplary bispecific antibody conjugates include the CovX-body format in which a low molecular weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine in the antibody or its fragment. In some embodiments, conjugation improves the serum half-life.

[0138] Methods for producing multispecific antibodies, including bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecific antibodies, can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly, or by expression of the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by various methods known in the art, including affinity chromatography.

[0139] In some embodiments, 5T4 binders (e.g., antibodies) comprising the human 5T4 binders disclosed herein can be provided in any antibody format disclosed herein or known in the art. By way of non-limiting example, in some embodiments, 5T4 binders (e.g., antibodies) comprising a human 5T4 binder can be Fab-in-tandem-lg (FIT-lg), DVD-lg, hybrid hybridoma (quadroma or tetroma), anticalin platform (Pieris), diabody, single-chain diabody, tandem single-chain Fv fragment, TandAb, trispecific Ab (Affimed), DARTS dual affinity retargeting (Macrogenics), bispecific Xmab (Xencor), bispecific T cell engager (Bites, Amgen, 55 kDa), Triplebodies, Tribody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs), DuoBody platform (Genmab), dock and lock platform, knob-into-hole (KIH) platform, humanized bispecific IgG antibody (REGN1979) (Regeneron), Mab2 bispecific antibody (F-Star), DVD-lg = dual variable domain immunoglobulin (Abbott), kappa-lambda body, TBTI = tetravalent bispecific tandem Ig, and CrossMab (Roche).

[0140] 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 targets other than 5T4. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise a 5T4 binding domain comprising VH and / or VL amino acid sequences disclosed herein, such as those in Table 1, Table 2, or Table 3.

[0141] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies comprising a binding domain that binds to 5T4, comprising VH and VL CDRs disclosed herein and those described in, for example, Table 1, Table 2, or Table 3.

[0142] Antibodies that bind to 5T4 may be obtained by any suitable method, including but not limited to immunization with all tumor cells containing 5T4 and antibody harvesting, recombinant techniques, or screening libraries of antibodies or antibody fragments using 5T4 extracellular domain epitopes. Monoclonal antibodies can be generated using a variety of known techniques (see, for example, 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,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for generating monoclonal antibodies involves immunizing an animal with the human 5T4 antigen and generating hybridomas from spleen cells harvested from the animal. The hybridomas may produce monoclonal antibodies or antibody fragments that bind to 5T4.

[0143] In a further embodiment, the monoclonal antibody or antibody fragment can be isolated from an antibody phage library generated using, for example, the techniques described in Antibody Phage Display: Methods and Protocols, P.M. O’Brien and R.Aitken, eds, Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various fragments of the antibody variable region (Fv) fused to the phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and are thus separated from non-binding clones within the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0144] The variable domains can be functionally displayed on the phage either as single-chain Fv (scFv) fragments in which VH and VL are covalently linked via a short flexible peptide, as described, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994), or as Fab fragments in which they are each fused to a constant domain and interact non-covalently.

[0145] The repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then this can be screened for antigen-binding clones as described by Winter et al. (supra). Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned, as described by Griffiths et al., EMBO J, 12:725-734 (1993), to provide a single human antibody source against a wide range of non-self and self antigens without any immunization. Finally, naive libraries can also be made synthetically, for example, by cloning unrearranged V gene segments derived from stem cells, encoding the highly variable CDR3 region using PCR primers containing random sequences, and achieving rearrangement in vitro.

[0146] Screening of libraries can be accomplished by various techniques well known in the art. For example, 5T4 (e.g., 5T4 polypeptide, fragment, or epitope) can be used to coat the wells of adsorption plates, can be expressed or used in cell sorting on host cells attached to adsorption plates, can be conjugated to biotin for capture with streptavidin-coated beads, or can be used in any other method for panning display libraries. Selection of antibodies with slow dissociation rates (e.g., good binding affinity) can be facilitated by long washing times and the use of monovalent phage display, as described by Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by low antigen coating density, as described by Marks et al., Biotechnol., 10:779-783 (1992).

[0147] The 5T4 binding agent (e.g., antibody) can be obtained by designing an antigen screening procedure suitable for selecting the desired phage clone, and subsequently constructing a full-length 5T4 binding agent (e.g., antibody) clone using the VH and / or VL sequences (e.g., Fv sequences) derived from the desired phage clone and suitable constant region (e.g., Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD. (1991), vols. 1-3, or various CDR sequences derived from the VH and VL sequences.

[0148] Similarly, human antibodies that bind to 5T4 can be generated by any of several techniques including, but not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., including B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice having 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, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8:455-58, 1997; Jakobovits et al., Ann. N.Y. Acad. Sci., 764:525-35, 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. Patent No. 5,877,397.

[0149] For example, a human antibody that binds to 5T4 can be obtained from a transgenic animal engineered to produce a specific human antibody in response to antigen administration. For example, International Patent Publication No. WO98 / 24893 discloses transgenic animals having the human Ig locus, which do not produce functional endogenous immunoglobulins due to inactivation of the endogenous heavy and light chain loci. Transgenic non-human mammalian hosts that can initiate an immune response to an immunogen, wherein the antibody has primate constant and / or variable regions and the locus encoding the endogenous immunoglobulin is replaced or inactivated, are also described. International Patent Publication No. WO96 / 30498 discloses the use of the Cre / Lox system to modify immunoglobulin loci in mammals such that all or part of the constant or variable region is replaced to form a modified antibody molecule. International Patent Publication No. WO94 / 02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Patent No. 5,939,598 discloses a method for producing a transgenic mouse in which the mouse lacks an endogenous heavy chain and expresses an exogenous immunoglobulin locus containing one or more heterologous constant regions. Transgenic animals such as the transgenic animals described herein can be used to elicit an immune response against a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human-derived monoclonal antibodies. Immunization protocols, adjuvants, etc. are known in the art and are used, for example, for immunization of transgenic mice as described in International Patent Publication No. WO96 / 33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.

[0150] The present disclosure provides humanized antibodies that bind to 5T4, including human 5T4. The humanized antibodies of the present disclosure may include one or more CDRs derived from VH and / or VL disclosed herein, such as those shown in Tables 1-3. A variety of 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 from a non-human source. These non-human amino acid residues are often referred to as "import" residues and typically are derived from an "import" variable domain. Humanized antibodies that bind to 5T4 can be made using techniques well 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. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120, and 4,816,567).

[0151] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the VH and VL of a parental non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9:133-139, 1995) demonstrated that only about one-third of the residues in the CDRs actually contact the antigen, and named these "specificity-determining residues" or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., Methods 36:25-34, 2005).

[0152] The selection of both light and heavy human variable domains used to generate a humanized antibody can be important for reducing antigenicity. For example, in the so-called "best fit" method, the sequences of the variable domains of non-human (e.g., rodent) antibodies are screened against a library of all known human variable domain sequences. The human sequence most closely related to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of the light or heavy chain. The same framework may 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 is the most abundant human subclass V L subgroup I (V L 6I) and V H subgroup III (V H III) derived from the consensus sequence. In another method, human germline genes are used as the source of the framework region.

[0153] In an alternative paradigm based on comparison of CDRs called hyperhumanization, framework homology is not relevant. This method consists of a comparison of non-human sequences with the functional human germline gene repertoire. Then, genes encoding canonical structures identical or closely related to the mouse sequence are selected. Next, among the genes sharing the canonical structure with the non-human antibody, the gene with the highest homology to the CDR is selected as the framework donor. Finally, the non-human CDRs are grafted onto these frameworks (see, e.g., Tan et al., J. Immunol. 169:1119-1125, 2002).

[0154] Furthermore, it is generally desirable that the antibody be humanized while maintaining its affinity for the antigen and other favorable biological properties. To achieve this goal, in one method, a humanized antibody is prepared by a process of analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the three-dimensional higher-order structure deduced from a selected immunoglobulin candidate sequence. 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). By examining these displays, it becomes possible to analyze the role that the residues play in the function of the immunoglobulin candidate sequence, for example, to analyze the residues that affect the ability of the immunoglobulin candidate to bind to the antigen. Thus, framework residues can be selected and combined from the recipient and grafted sequences such that desired antibody properties, such as an increase in affinity for the target antigen(s), are achieved. Generally, the hypervariable region residues are most directly and substantially involved in affecting antigen binding.

[0155] Another method of antibody humanization is based on a measure of antibody humanization called the human string content (HSC). This method compares the mouse sequence to the repertoire of human germline genes and scores the differences as the HSC. Then, instead of using a global identity metric, the target sequence is humanized by maximizing its HSC, generating multiple diverse humanized variants (Lazar et al., Mol. Immunol. 44:1986-1998, 2007).

[0156] In addition to the above methods, empirical methods can be used to generate and select humanized antibodies. These methods include those based on the generation of large libraries of humanized variants and the selection of the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, for example, 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).

[0157] In the framework library approach, a collection of residue variants is introduced at specific positions in the framework, and subsequently, a library is selected such that the framework that best supports the grafted CDRs is chosen. The residues to be substituted may include some or all of the "Vernier" residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, J. Mol. Biol. 224:487-499, 1992) or may be from a more restricted set of target residues identified by Baca et al. (J. Biol. Chem. 272:10678-10684, 1997).

[0158] In framework shuffling, rather than creating a combinatorial library of selected residue variants, the entire framework is combined with non-human CDRs (see, e.g., Dall’Acqua et al., Methods 36:43-60, 2005). The library may be screened for binding in a two-step selection process that first humanizes VL and subsequently humanizes VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than two-step screening since the resulting antibodies exhibit improvements in biochemical and physicochemical properties including increased expression, affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0159] The “humanization” method is based on the experimental identification of essential minimal specificity determinants (MSDs) and on the sequential replacement of non-human fragments into a library of human frameworks and the evaluation of binding. This starts with the CDR3 regions of the non-human VH and VL chains and progressively replaces other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, with human frameworks. By this method, typically, the epitope is retained and multiple subclasses of antibodies with different human V segment CDRs are identified. Humanization enables the isolation of antibodies with 91-96% homology to human germline antibodies. (See, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007).

[0160] The "human engineering" method involves modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by introducing specific changes into the amino acid sequence of the antibody so as to generate modified antibodies that have reduced immunogenicity in humans but still retain the desired binding properties of the original non-human antibody. Generally, this technique involves classifying the amino acid residues of the non-human (e.g., murine) antibody as "low-risk", "medium-risk", or "high-risk" residues. The classification is performed using a global risk / reward calculation that assesses the impact of the substitution on the folding of the resulting antibody (e.g., due to immunogenicity in humans) and / or the expected benefit relative to the risk of being replaced by a human residue. The specific human amino acid residues to be substituted at a given position (e.g., low or medium risk) in the non-human (e.g., murine) antibody sequence can be selected by aligning the amino acid sequence derived from the variable region of the non-human antibody with the corresponding region of a specific human antibody sequence or a consensus human antibody sequence. 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 according to the alignment. Techniques for making human engineered proteins are described in more detail in Studnicka et al., Protein Engineering 7:805-814 (1994); U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication No. WO93 / 11794.

[0161] In some embodiments, the 5T4 binder described herein comprises a scaffold of a non-antibody protein. Non-limiting examples of such scaffolds of non-antibody proteins include fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, finomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz-type inhibitors. Methods for generating such scaffolds of non-antibody proteins are well known in the art, and any one of them can be used to generate a 5T4 binder comprising a non-antibody protein scaffold (see, for example, 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)).

[0162] In addition, provided are nucleic acids encoding the 5T4 binders (e.g., antibodies or antibody fragments) or fusion polypeptides disclosed herein, nucleic acids complementary thereto, vectors comprising the nucleic acids disclosed herein, and cells comprising the nucleic acids or vectors disclosed herein. In some embodiments, the cells express a 5T4 binder. In some embodiments, the cells replicate the nucleic acid or vector. In some embodiments, provided are materials for generating a 5T4 binder, e.g., a human 5T4 binder, and fragments thereof. For example, isolated cells can produce a 5T4 binder (e.g., an antibody or antibody fragment). In this regard, cells (e.g., isolated cells) can produce an antibody or fragment thereof comprising the VH and VL disclosed herein. In some embodiments, the polynucleotides described herein can comprise one or more nucleic acid sequences encoding a 5T4 binder (e.g., an antibody, or antibody fragment). In some embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In various aspects, the isolated polynucleotide comprises nucleotide sequences encoding VH and / or VL, and the VH and VL thereof comprise complementarity determining regions (CDRs) identical to the CDRs disclosed herein.

[0163] In some embodiments, one or more vectors (e.g., expression vectors) can contain one or more polynucleotides for expressing one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying polynucleotides in a host cell to create useful amounts and for expressing binding agents, such as antibodies or antibody fragments, using recombinant techniques.

[0164] In some embodiments, one or more vectors are expression vectors in which one or more polynucleotides are operably linked to one or more polynucleotides comprising expression control sequences. Specifically contemplated are self-replicating recombinant expression constructs such as plasmids and viral DNA vectors incorporating one or more polynucleotides encoding antibody sequences that bind to 5T4. Expression control DNA sequences include promoters, enhancers, and operators and are generally selected based on the expression system in which the expression construct is to be utilized. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, while operator sequences are generally selected for their ability to regulate gene expression. The expression construct may also include sequences encoding one or more selectable markers that enable the identification of host cells carrying the construct. The expression construct can also include sequences that facilitate, preferably promote, homologous recombination in the host cell. In some embodiments, the expression construct may also include sequences necessary for replication in the host cell.

[0165] Exemplary expression control sequences include promoter / enhancer sequences such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshhart et al., Cell, 41:521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993), type promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995), simian virus 40 promoter, DRA (downregulated in adenoma; Alrefai et al., Am. J. 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 homolog1, 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 other variations, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0166] Optionally, one or more polynucleotides can further optionally include a nucleotide sequence encoding a secretion signal peptide fused in-frame with the polypeptide sequence. The secretion signal peptide directs the secretion of the antibody polypeptide by the cell expressing the one or more polynucleotides and is cleaved from the secreted polypeptide by the cell. The one or more polynucleotides can further optionally include sequences that function only to facilitate the bulk production of the vector. Polynucleotides for gene therapy can be produced and administered using procedures described in the literature for various transgenes. See, for example, Isner et al., Circulation, 91:2687-2692, 1995, and Isner et al., Human Gene Therapy, 7:989-1011, 1996.

[0167] In some embodiments, the polynucleotide can further include additional sequences to facilitate uptake by the host cell 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 described herein (e.g., a transgene that does not include a virus, liposome, or other vector to facilitate transfection) is used.

[0168] One or more polynucleotides encoding an antibody or a fragment thereof can be introduced into a host using any suitable vector. Exemplary vectors described include lentiviral vectors (Kim et al., J. Virol., 72(1):811-816, 1998, Kingsman & Johnson, Scrip Magazine, October 1998, pp. 43-46), parvovirus vectors such as adeno-associated virus (AAV) vectors (U.S. Patent Nos. 5,474,935; 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; Gnatenko et al., J. Invest. Med., 45:87-98, 1997), adenovirus (AV) vectors (U.S. Patent 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 Perricaudet et al., J. Clin. Invest., 90:626-630, 1992, and Rosenfeld et al., Cell, 68:143-155, 1992), adenovirus-adeno-associated virus chimeras (U.S. Patent No. 5,856,152), or vaccinia virus or herpesvirus vectors (U.S. Patent Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688), lipofectin-mediated gene transfer (BRL), liposome vectors (U.S. Patent No. 5,631,237), and combinations thereof.Any of these expression vectors can be prepared using standard recombinant DNA techniques such as those described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994). Optionally, the viral vector is made replication-deficient by deleting or disrupting a selectable gene required for viral replication.

[0169] Other contemplated non-viral delivery mechanisms 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., 101:1094-1099, 1985), DNA-loaded liposomes (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 a high-velocity microprojectile (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990), and receptor-mediated transfection (see 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).

[0170] The vector (or the antibody or its fragment or nucleic acid discussed herein) may be encapsulated in liposomes. See, for example, 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 approaches involving "lipofection" technology are also contemplated. In some embodiments, the liposomes can be complexed with hemagglutinating virus (HVJ). This has been shown to promote fusion with the cell membrane and facilitate cellular entry of liposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989). In some embodiments, the liposomes form a complex with or are used in conjunction with the nuclear non-histone chromosomal protein (HMG-1) (Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In some embodiments, the liposomes are complexed with or used in combination with both HVJ and HMG-1. Such expression constructs have been successfully used in nucleic acid transfer and expression in vitro and in vivo. In some embodiments, a 5T4 binder (e.g., an antibody) comprising a human 5T4 binder is included in the liposomes to target the liposomes to cells (such as tumor cells) expressing 5T4 on their surface.

[0171] The cell may contain one or more polynucleotides or one or more vectors. For example, the cell is transformed or transfected with a 5T4 binder (e.g., an antibody) containing a human 5T4 binder or one or more polynucleotides encoding a vector containing one or more polynucleotides. In some embodiments, the cell expresses a 5T4 binder (e.g., an antibody) containing a human 5T4 binder that contains one or more CDRs having 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) containing a human 5T4 binder that contains VH and VL containing CDRs identical to the CDRs of mAbA4, mAbA15, and / or mAbA17 (see, e.g., Tables 1, 2, and / or 3). The cell can be a prokaryotic cell, e.g., Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or a eukaryotic cell, e.g., an animal cell (e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell, or a hybridoma cell), 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, the polypeptide (e.g., a 5T4 binder (e.g., an antibody) containing a human 5T4 binder) may be glycosylated or non-glycosylated and / or may be covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0172] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, shadow cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and for expressing polypeptides encoded by polynucleotides. In this regard, a process for the production of a 5T4 binder (e.g., an antibody) can include culturing a host cell and isolating the 5T4 binder. Introduction of a naked DNA expression construct into cells can be achieved using particle bombardment, which relies on the ability to accelerate DNA-coated microprojectiles at high speed, thereby enabling them to penetrate the cell membrane and enter the cell without killing the cell (see Klein et al., Nature, 327:70-73, 1987). Several devices have been developed for accelerating small particles. One such device relies on a high-voltage discharge to generate an electric current, which in turn provides the power (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). The microprojectiles used are composed of biologically inert materials such as tungsten or gold beads. Host cells can be isolated and / or purified. Host cells can also be cells transformed in vivo to cause transient or permanent expression of a polypeptide in vivo. Host cells can also be isolated cells transformed ex vivo and introduced after transformation, for example, to produce a polypeptide in vivo for therapeutic purposes. The definition of host cells expressly excludes transgenic humans.

[0173] Various methods for producing antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). Furthermore, numerous publications describe techniques suitable for the manipulation of DNA, the construction of expression vectors, and the preparation of antibodies by the transformation and culture of appropriate cells (see, for example, Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992), and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0174] 5T4 binders (e.g., antibodies) that include a human 5T4 binder are produced using any suitable method such as being isolated from an immunized animal, produced recombinantly or synthetically, or genetically engineered, including the above. Antibody fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. For example, papain or pepsin digestion of whole antibodies results in 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 generate 3.5S Fab monovalent fragments. Methods for generating antibody fragments are described, for example, in 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. Patent No. 4,331,647, and Andrews, S.M. and Titus, J.A. in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1 - 2.8.10 and 2.10A.1 - 2.10A.5.

[0175] A 5T4 binder (e.g., an antibody) containing a human 5T4 binder can be genetically engineered. For example, a 5T4 binder (e.g., an antibody) containing a human 5T4 binder includes a variable region domain produced, for example, by recombinant DNA engineering techniques. In this regard, the variable region is optionally modified by insertions, deletions, or changes in the amino acid sequence of the antibody to produce the antibody of interest, including the above. The polynucleotide encoding the CDR of interest is prepared, for example, by using the polymerase chain reaction to synthesize the variable region using the mRNA of antibody-producing cells as a template (see, for example, Courtenay Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106-110, 1991). Current antibody engineering techniques enable the construction of engineered variable region domains containing 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.

[0176] A "humanized antibody" is an antibody in which the CDRs of the heavy and light variable chains of a non-human immunoglobulin have been transferred to human variable domains. Constant regions need not be present, but if present, they are optionally substantially identical to human immunoglobulin constant regions and in some embodiments are, for example, at least about 85-90%, about 95%, 96%, 97%, 98%, 99% or more identical. Thus, in some cases, all parts of the humanized immunoglobulin (possibly excluding the CDRs) are substantially identical to the corresponding parts of the native human immunoglobulin sequence. For example, a humanized antibody is a human immunoglobulin (e.g., a host antibody) in which residues from the hypervariable regions of the host antibody have been replaced by residues from the hypervariable regions of a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) that have the desired antibody specificity, affinity, and ability.

[0177] In some embodiments, the 5T4 binding agents (e.g., antibodies or ADCs) described herein are useful in compositions and methods for treating, preventing, or alleviating a disease, disorder, or condition, including one or more symptoms of a 5T4-mediated disease, disorder, or condition. 5T4-mediated diseases, disorders, and conditions include, but are not limited to, any cancer in which tumor cells overexpress 5T4.

[0178] In some embodiments, described herein is a method for treating tumor immunity in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein. In some embodiments, provided herein is a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein for use in treating tumor immunity in a subject. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein in the production of a medicament for treating tumor immunity in a subject.

[0179] In some embodiments, described herein is a method for treating cancer or a tumor in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein. In some embodiments, provided herein is a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein for use in treating cancer or a tumor in a subject. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or ADC) described herein or a fragment thereof or a binder (e.g., an antibody or ADC) described herein in the production of a medicament for treating cancer or a tumor in a subject.

[0180] In some embodiments, described herein is a method for alleviating one or more symptoms associated with cancer or a tumor in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein. In some embodiments, provided herein is a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein for use in alleviating one or more symptoms associated with cancer or a tumor in a subject. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein in the production of a medicament for alleviating one or more symptoms associated with cancer or a tumor in a subject.

[0181] In some embodiments, described herein is a method for reducing the size of a tumor in a subject having a tumor, the method comprising administering to the subject a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein. In some embodiments, provided herein is a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein for use in reducing the size of a tumor in a subject having a tumor. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) described herein or a fragment thereof or a binder (e.g., an antibody or an ADC) described herein in the production of a medicament for reducing the size of a tumor in a subject having a tumor.

[0182] In some embodiments, described herein is a method for enhancing the removal of tumor cells in a subject having a tumor, the method comprising administering to the subject a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) or a fragment thereof described herein or a binder (e.g., an antibody or an ADC) described herein. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) or a fragment thereof described herein or a binder (e.g., an antibody or an ADC) described herein for enhancing the removal of tumor cells in a subject having a tumor. In some embodiments, provided herein is the use of a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) or a fragment thereof described herein or a binder (e.g., an antibody or an ADC) described herein in the production of a medicament for enhancing the removal of tumor cells in a subject having a tumor.

[0183] The subject of the methods described herein can be administered one or more therapeutic agents described herein in combination with a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) or a fragment thereof described herein or a binder (e.g., an antibody or an ADC) described herein.

[0184] In some embodiments, the antibody is a human antibody, including but not limited to an antibody having a variable region in which both the framework and CDRs are derived from human germline immunoglobulin sequences, such as those described 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. When the antibody contains a constant region, the constant region is also preferably derived from human germline immunoglobulin sequences. A human antibody may contain amino acid residues not encoded by human germline immunoglobulin sequences, for example, to enhance the activity of the antibody, but does not contain CDRs derived from other species (e.g., mouse CDRs placed within a human variable framework region).

[0185] In some embodiments, the cancer cells or tumor cells of the methods described herein express 5T4, such as on the cell surface. In some further embodiments, the cancer cells or tumor cells overexpress 5T4. In some embodiments, a 5T4 binding agent (e.g., an antibody or ADC) is cytotoxic to tumor cells in cell culture. Such cell culture may include tumor cells that express or overexpress 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 carcinoma cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, and pancreatic cancer cells.

[0186] In some embodiments, described herein is a method of enhancing the removal of tumor cells in a subject. For example, the method includes administering an amount of a 5T4 binder (e.g., an antibody or an ADC), such as a human 5T4 binder described herein, effective to enhance the removal of tumor cells. In some embodiments, the method includes administering a 5T4 binder (e.g., an antibody or an ADC), wherein the 5T4 binder (i) competes for binding with mAbA4, mAbA15, and / or mAbA17 (see, e.g., Tables 1, 2, and / or 3), (ii) binds to human 5T4, and / or (iii) binds to a region of 5T4 recognized by the CDRs and VH / VL of mAbA4, mAbA15, and / or mAbA17 (see, e.g., Tables 1, 2, and / or 3), resulting in enhanced removal of tumor cells. In some embodiments, one or more of the above binders (e.g., antibodies), polynucleotides, vectors, and / or cells can be used in a method of enhancing the removal of tumor cells in vivo (e.g., a method of treating cancer in a subject).

[0187] Also provided are methods of modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject. For example, the method includes administering to the subject a composition comprising a 5T4 binder (e.g., an antibody or an ADC) in an amount effective to modulate tumor growth in the subject.

[0188] "Tumor", as used herein, refers to any tumor cell growth or proliferation, and any pre-cancerous and cancerous cells and tissues, whether malignant or benign. The terms "cancer" and "cancerous" refer to or describe a physiological state in mammals typically characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, breast cancer, colon cancer, kidney cancer, lung cancer, squamous cell myelogenous 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, rhabdomyoma, fibroma, lipoma, and teratoma; Lung: bronchogenic carcinoma (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Digestive system: esophageal cancer (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, leiomyosarcoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyosarcoma); Genitourinary tract: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia, renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma of the prostate, sarcoma, small cell carcinoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma); Liver: liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), malignant giant cell tumor of bone, myeloid sarcoma (osteomyeloid sarcoma), benign chondroma, chondroblastoma, chondromyxoid fibroma, multiple myeloma and giant cell tumor;Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteomyelitis), meninges (meningioma, meningosarcoma, gliosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal nerve fibroma, meningioma, glioma, sarcoma); Gynecology: uterus (endometrial cancer), cervix (cervical cancer, pre-tumor cervical dysplasia), ovary (ovarian cancer) serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa cell tumor, Sertoli-Leydig cell tumor, germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, diffuse sarcoma (embryonal rhabdomyosarcoma)), fallopian tube (carcinoma); Hematology: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin lymphoma (malignant lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and Adrenals: neuroblastoma, and cancers of the thyroid including medullary thyroid cancer. 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 anti-cancer therapies.;

[0189] Also provided is a method of treating cancer by administering a 5T4 binder (e.g., an antibody) such as a human 5T4 binder to a subject in need thereof, alone or in combination with another agent.

[0190] "Enhancing" the removal of tumor cells does not require enhancing the removal by 100%. Some enhancement in the rate of removal is contemplated. Similarly, "modulating" tumor growth refers to reducing the tumor size, slowing tumor growth, or inhibiting an increase in the size of an existing tumor. It is not necessary to completely eliminate the tumor. Any decrease in tumor size or slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, the removal of tumor cells can be enhanced, for example, by at least about 5%, at least about 10%, or at least about 20% compared to the degree of removal observed in the absence of using the method (e.g., a subject or specimen that is a biologically matched control not exposed to the agent of the method). This effect is detected, for example, by a reduction in tumor size, a decrease or maintenance of the level of a tumor marker, or a reduction or maintenance of the tumor cell population. In some embodiments, the removal of tumor cells is enhanced, for example, by 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%) compared to the removal of tumor cells in the absence of a 5T4 binder (e.g., an antibody).

[0191] Furthermore, for example, a 5T4 binder (e.g., an antibody or an ADC) can be used to alleviate or reduce cancer-related side effects such as bone deterioration, spinal collapse, and paralysis. In one aspect, the subject has or is at risk of having bone metastases, and the 5T4 binder (e.g., an antibody or an ADC) is administered in an amount that reduces the deterioration of the surrounding bone. Thus, in some aspects, the 5T4 binder prevents bone deterioration due to bone metastases, and tumor cell growth is reduced or not reduced. In some aspects, the 5T4 binder (e.g., an antibody or an ADC) prevents bone deterioration due to bone metastases and reduces tumor cell growth. Generally, the effect on tumor cell growth (e.g., inhibition of growth or no effect on growth) depends on the specific metastatic microenvironment. For example, the growth of metastases located in a microenvironment having a substantial amount of type I collagen can be inhibited. In contrast, the growth of metastases located in a microenvironment lacking a substantial amount of type I collagen cannot be inhibited, but the deterioration of the bone near the metastases is reduced or prevented.

[0192] The specific dosing regimen of a 5T4 binder (e.g., an antibody or an ADC) for a particular subject depends 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., an antibody or an ADC) administered to a subject (e.g., a mammal such as a human) should be sufficient to produce the desired response over a reasonable time frame. Thus, in some embodiments, the amount of the 5T4 binder (e.g., an antibody or an ADC) or pharmaceutical composition described herein administered to a subject is an effective amount.

[0193] Suitable routes for administering a composition comprising a 5T4 binder (e.g., an antibody or an ADC) such as a human 5T4 binder (e.g., an antibody or an ADC) are well known in the art. Multiple routes can be used to administer the agent (e.g., an antibody or an ADC), but a particular route may provide a more immediate and effective response than another route.

[0194] The present disclosure provides a composition such as a pharmaceutical composition comprising a 5T4 binder (e.g., an antibody or an ADC) such as a human 5T4 binder and a carrier (e.g., a pharmaceutically acceptable carrier). The particular carrier used may depend on physicochemical considerations such as lack of solubility and reactivity with the binder or co-therapy, as well as the route of administration. Pharmaceutically acceptable carriers are well known in the art and examples thereof are described herein. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. For injectable formulations, see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincot Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (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 sustained release, delayed release, and extended release delivery systems, whereby the delivery of the composition is carried out before and for a sufficient time to cause sensitization of the site to be treated. Many types of release delivery systems are available and are known to those skilled in the art. Suitable release delivery systems include polymeric bases such as poly(lactide-glycolide), copolyesters, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymeric systems that are lipids such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as monoglycerides and triglycerides, hydrogel release systems, cyrotic systems, peptide-based systems, wax coatings, compressed tablets using conventional binders 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 matrix form, such as those 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 ingredient permeates at a rate controlled from a polymer, as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. Further, pump-based hardware delivery systems can be used, some of which are implant compatible. Pharmaceutical compositions containing a 5T4 binder (e.g., an antibody or ADC) such as a human 5T4 binder are, in one aspect, placed in a container together with a packaging material providing instructions for use of such pharmaceutical composition. Generally, such instructions include a tangible representation describing the reagent concentration and, in some embodiments, the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition.

[0195] In some aspects, the methods described herein further comprise administering one or more additional agents comprising a therapeutic agent, which may be present in the composition, may be administered with a 5T4 binder (e.g., an antibody or ADC) such as a human 5T4 binder, or may be provided as a separate composition using the same or different routes of administration. The one or more additional agents comprising a therapeutic agent may be administered together with or separately from the 5T4 binder (e.g., an antibody or ADC) (e.g., for combination therapy). Such additional therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapies and immunotherapy agents, cytotoxic agents, chemotherapeutic agents, and inhibitors.

[0196] Therapeutic antibodies that can be used together with the 5T4 binding agents (e.g., antibodies or ADCs) described in this specification (e.g., for combination therapy) include trastuzumab, abciximab, daclizumab, BEC2, IMC-C22, vitaxin, Campath 1H / LDP-03, Smart M195, epratuzumab, bevacizumab, visilizumab, CM3, humanized anti-ICAM3 antibody, IDEC-114, ibritumomab tiuxetan, IDEC-131, IDEC-151, IDEC-152, SMART anti-CD3, eculizumab, adalimumab, certolizumab, IDEC-151, MDX-CD4, CD20-treponema, CDP571, LDP-02, OrthoClone OKT4A, lulizumab, natalizumab, and rilonacept, or their biosimilars, but are not limited thereto.

[0197] Immunotherapies and immunotherapeutic agents that can be used with a 5T4 binder (e.g., an antibody or ADC) described herein (e.g., for combination therapy) include granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), cytokines such as macrophage inflammatory protein (MIP)-1-alpha, 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-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma), aluminum hydroxide (alum), Bacillus Calmette-Guerin (BCG), keyhole limpet hemocyanin (KLH), incomplete Freund's adjuvant (IFA), QS-21, DETOX, levamisole, and dinitrophenyl (DNP), and combinations thereof, such as combinations of interleukins, e.g., IL-2, with other cytokines, e.g., IFN-alpha, but are not limited thereto. In some embodiments, immunotherapy includes an immunotherapeutic agent that modulates the immune response, e.g., a checkpoint inhibitor or a checkpoint agonist. In some embodiments, the immunotherapeutic agent is an antibody modulator that targets, among others well-known in the art, PD-1, 5T4, PD-L2, CEACAM (e.g., CEACAM-1, -3 and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF beta, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2. In some embodiments, the immunotherapeutic agent is an agent that enhances the activity of natural killer (NK) cells. In some embodiments, the immunotherapeutic agent is an agent that inhibits the suppression of the immune response. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or the activity of suppressor cells. In some embodiments, the immunotherapeutic agent is an agent or therapy that inhibits Treg activity.In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of an inhibitory immune checkpoint receptor.

[0198] In some embodiments, the immunotherapeutic agent includes a T cell or NK cell or NKT cell regulator selected from an agonist or activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from an agonist of GITR, OX40, ICOS, SLAM (e.g., 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 a CD83 ligand (e.g., an agonist antibody or an antigen-binding fragment thereof, or a soluble fusion). In other embodiments, the combination of effector cells includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds CD3 and a tumor antigen (e.g., especially EGFR, PSCA, PSMA, EpCAM, HER2)) or a bispecific NK cell engager.

[0199] Cytotoxic agents that can be used with a 5T4 binder (e.g., an antibody or ADC) described herein (e.g., for combination therapy) include substances that inhibit or prevent cell function and / or cause cell death or destruction. Exemplary cytotoxic agents include radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu), growth inhibitors, enzymes such as nucleolytic enzymes and fragments thereof, and toxins such as small molecule toxins or enzymatically active toxins (including fragments and / or variants thereof) of bacterial, fungal, plant, or animal origin, but are not limited thereto. Other exemplary cytotoxic agents can be selected from antimicrotubule 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, immunotherapy agents, apoptosis promoters, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signal inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors.

[0200] Chemotherapeutic agents that can be used with the 5T4 binding agents (e.g., antibodies or ADCs) described herein (e.g., for combination therapy) include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib, bortezomib, disulfiram, epigallocatechin gallate, salinosporamide 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, lonafarnib (SCH 66336), sorafenib, Bayer Labs), gefitinib, AG1478, alkylating agents such as thiotepa and CYTOXAN®, cyclophosphamide, alkyl sulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carbocone, meturedopa, and uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine, ethyleneimine and methylamelamine including acetogenin (especially, bratasin and bratasinone), camptothecin (including topotecan and irinotecan), bryostatin, calistatin, CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs), cryptophycin (especially, cryptophycin 1 and cryptophycin 8), corticosteroids (including prednisone and prednisolone), cyproterone acetate, 5α-reductase including finasteride and dutasteride, vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, drastatin, aldosterone, duocarmycin talc (including synthetic analogs KW-2189 and CB1-TM1), eribulin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide,Uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine, antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γII and calicheamicin ωI (Angew Chem. Inti. Ed. Engl. 1994 33:183 - 186), dynemicin including dynemicin A, bisphosphonates such as clodronate, esperamicin, and neocarzinostatin chromophore and related pigment protein enediyne antibiotic chromophores), aclacinomycin, actinomycin, aclarubicin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardifilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6 - diazo - 5 - oxo - L - norleucine, doxorubicin, morpholino - doxorubicin, cyanomorpholino - doxorubicin, 2 - pyrrolino - doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin, antimetabolites such as methotrexate and 5 - fluorouracil (5 - FU), folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, purine analogs such as fludarabine, 6 - mercaptopurine, thiampurine, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone, anti - adrenal agents such as aminoglutethimide, mitotane, trilostane, folic acid supplements such as folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, demeclocycline, diaziquone,Elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitansineoids such as mitansine and ansamitocin, mitoguazone, mitoxantrone, mopidanol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, Ore.), razoxane, lysoxine, sizofuran, spirogermanium, tenuazonic acid, triazicone, 2,2’,2’’-trichloroethylamine, trichothecene (especially T-2 toxin, verrucarin A, roridin A, and anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gasitocin, arabinoside (“Ara-C”), cyclophosphamide, thiotepa, taxoids such as paclitaxel, ABRAXANE® (chromophore-free), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and docetaxel / doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, capecitabine, ibandronate, CPT-II; topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing. Chemotherapeutic agents include (i) antihormonal agents that act to control or inhibit the hormonal action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and tremifene citrate, (ii) aromatase inhibitors that inhibit the aromatase enzyme that controls estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole, (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin, buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all transretionic acid, fenretinide, and troxacitabine (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-alpha, Ralf, and H-Ras, (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors, (viii) vaccines such as gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®, PROLEUKIN®, rIL-2, topoisomerase 1 inhibitors, such as LEIRTOTECAN®, ABARELIX®, and (ix) also include any pharmaceutically acceptable salts, acids, and derivatives of the above.

[0201] Also included as chemotherapeutic agents are alemtuzumab, bevacizumab, cetuximab, panitumumab, rituximab, pertuzumab, tositumomab, and the above-mentioned antibodies including antibody-drug conjugates, gemtuzumab ozogamicin. Additional humanized monoclonal antibodies having therapeutic potential as agents in combination with a 5T4 binder (e.g., an antibody) described herein include apolizumab, aselizumab, atorizumab, bapineuzumab, bivatuzumab mertansine, canertuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eclizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labeuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nivolumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumabAnti-interleukin-12 (ABT-8744695, Wyeth Research and Abbott Laboratories), which is a recombinant of a full-length IgG1λ antibody with an exclusive human sequence genetically modified to recognize celmoleukin, tucusituzumab, umavizumab, ultoxizumab, ustekinumab, visilizumab, and interleukin-12 p40 protein, is included. Chemotherapeutic agents include dexamethasone, interferon, colchicine, methotrexate, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, interferon alpha-2a, interferon alpha-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nolfetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, tamoxifen, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and their pharmaceutically acceptable salts are also included.

[0202] Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivolate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fludrocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, acrometasone propionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasone-17-propionate, fludrocortolone caproate, fludrocortolone pivalate and fluprednidene acetate, immune-selective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-form (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 lonafarnib, beta7 integrin blockers, such as rhuMAbBeta7, IgE pathway blockers, such as anti-M1 prime, secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTal / l32 blockers, such as anti-lymphotoxin alpha (LTa), various investigational agents, such as thio-platin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832), polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanol, procyanidin, betulinic acid and its derivatives, autophagy inhibitors, such as chloroquine, delta-9-tetrahydrocannabinol (dronabinol), beta-lapachone, lapachol, cortisone, betulinic acid, acetylcamptothecin, scoplectin, 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 THERATOPE® vaccine, perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341), CCI-779, tipifarnib (R11577), olaphenib, ABT510, Bcl-2 inhibitors, such as oblimersen sodium pixantrone, farnesyltransferase 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 (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisone), and FOLFOX (an abbreviation for the treatment regimen using oxaliplatin combined with 5-FU and leucovorin). Chemotherapeutic agents also include poly ADP ribose polymerase (PARP) inhibitors: olaparib, rucaparib, niraparib, talazoparib.

[0203] Inhibitors that can be used together with the 5T4 binding agents (e.g., antibodies) 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, vemurafenib, vandetanib, luxolitinib, axitinib, bosutinib, regorafenib, tofacitinib, cabozantinib, ponatinib, trametinib, dabrafenib, afatinib, ibrutinib, ceritinib, idelalisib, nintedanib, palbociclib, lenvatinib, cobimetinib, abemaciclib, acalabrutinib, alectinib, binimetinib, brigatinib, encorafenib, erdafitinib, everolimus, fostamatinib, gilteritinib, larotrectinib, lorlatinib, netarsudil, osimertinib, pemigatinib, peqidaltinib, ribociclib, temsirolimus, XL-092, XL-147, XL-765, XL-499, and XL-880. In some embodiments, the compounds described herein can be used in combination with an HSP90 inhibitor (e.g., XL888), a liver X receptor (LXR) modulator, a retinoic acid-related orphan receptor gamma (RORy) modulator, a checkpoint inhibitor such as a CK1 inhibitor or a CK1α inhibitor, a Wnt pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor (e.g., esacerenone), or XL-888 for the treatment of diseases disclosed herein such as cancer.In some embodiments, the 5T4 binding agents (e.g., antibodies) disclosed herein can be combined with one or more inhibitors 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 / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYR, FRK, JAK (JAK1 and / or JAK2), ABL, ALK, CDK7, CDK12, KRAS and B-Raf.

[0204] Additional non-limiting examples of inhibitors that can be used, for example, in the treatment of cancer (e.g., combination therapy), together with the 5T4 binder (e.g., antibody or ADC) described herein, 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, olitinib, cetuximab, necitumumab, or panitumumab), VEGFR inhibitors or pathway blockers (e.g., bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, vandetanib, ramucirumab, lenvatinib, ziv-aflibercept), PARP inhibitors (e.g., olaparib, rucaparib, veliparib or niraparib), JAK inhibitors (e.g., ruxolitinib, baricitinib, itacitinib), IDO inhibitors (e.g., epacadostat, NLG919, or BMS-986205, MK7162), LSD1 inhibitors, TDO inhibitors, PI3K-delta inhibitors (e.g., parsaclisib), PI3K-gamma inhibitors such as PI3K-gamma selective inhibitors, pim inhibitors, CSF1R 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 inhibitors (HDAC), e.g., HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, or combinations thereof.

[0205] In some embodiments, the 5T4 binding agents (e.g., antibodies or ADCs) disclosed herein can be used in combination with an inhibitor of PD-1 or an inhibitor of 5T4, e.g., an anti-PD-1 monoclonal antibody or an anti-5T4 monoclonal antibody, e.g., nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, avelumab, semiprimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, sintilimab, AB122, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, and TSR-042, AMP-224, AMP-514, PDR001, durvalumab, pidilizumab (Imfinzi®, CT-011), CK-301, BMS 936559, 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.

[0206] In some embodiments, the multispecific binding agents disclosed herein can be used in combination with a CTLA-4 inhibitor, e.g., an anti-CTLA-4 antibody, e.g., ipilimumab (Yervoy), tremelimumab, and AGEN1884, or with a phosphatidylserine inhibitor, e.g., babitzuximab (PGN401), or with an antibody, or with a cytokine (IL-10, TGF-β, etc.), or with a bispecific antibody that binds to 5T4 and CTLA-4 (e.g., AK104) or PD-1 and CTLA-4, or in combination with other anti-cancer agents such as semiprimab.

[0207] The additional agent can be a pharmaceutically acceptable salt, ester, amide, hydrate, and / or prodrug of any of these therapeutic agents or other agents described above.

[0208] Modifications that do not substantially affect the activities of the various embodiments of the present invention are also provided within the definitions of the present invention provided herein. Accordingly, the following examples are intended to illustrate the present invention but not to limit it.

Example

[0209] Example 1. Antibody Generation To obtain binders against human 5T4, antibody discovery was performed by phage display.

[0210] A. Phage Display To obtain binders against human 5T4, antibody discovery was performed by phage display of a human Fab library 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 prepared. The antigens produced consisted of human 5T4, cynomolgus monkey 5T4, and mouse 5T4, all cloned into vectors containing a C-terminal 6-his-Avi tag and biotinylated in vitro using co-expressed biotin ligase enzyme using standard procedures if possible.

[0211] Phage clones were screened for their ability to bind to biotinylated human 5T4. Briefly, a phage library in Fab format was constructed using an expression vector capable of replication and expression in phage (also called phagemid). Both the heavy and light chains were encoded within the same expression vector where the heavy chain was fused to a truncated variant of the phage coat protein pIII. The light chain and heavy chain-pIII fusions were expressed as separate polypeptides and assembled in the bacterial periplasm where disulfide bond formation was enabled by the redox potential to form the antigen-binding domain (Fab) of the candidate antibody.

[0212] This library was generated using sequences derived from specific human heavy chain variable domains and specific human light chain variable domains. The light chain variable domains within the screened library were generated with diversity and introduced into VL CDR3 (L3), leaving the human germline sequences in light chain VL CDR1 (L1) and CDR2 (L2). For the screened library, all three CDRs of the VH domain were diversified to match the positional amino acid frequencies by 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, where "x" represents the CDR amino acids that were varied to create the library, and bold italics represent the CDR sequences that were constant. The sequence of SEQ ID NO: 72 is EVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKGLEWVAXXXXXXXXXXXXXXXXXRFTISADTSKNTAYLQMNSLRAEDTAVYYCARXXXXXXXXXXXXXXWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC. The sequence of SEQ ID NO: 73 is [Chemical formula] It was.

[0213] Diversity was created by mutagenesis as described in detail by Kunkel, TA (PNAS January 1, 1985. 82(2)488-492), which is hereby incorporated by reference in its entirety, 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 the natural antibody repertoire. 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. Chemically synthesized DNA was then electroporated into TG1 cells and subsequently recovered. The recovered cells were passaged and infected with M13K07 helper phage to generate a phage library.

[0214] Phage panning was performed using standard procedures. Briefly, the first round of phage panning was performed using a target immobilized on streptavidin magnetic beads, which was subjected to approximately 1×10 12 phages from a library prepared in a volume of 1 mL in PBST-2% BSA. After a 1-hour incubation, the bead-bound phages were separated from the supernatant using a magnetic stand. The beads were washed three times to remove non-specifically bound phages, and then OD 600 , approximately 0.6, was added to ER2738 cells (5 mL). After incubation at room temperature for 20 minutes, the infected cells were added to 25 mL of 2xYT + ampicillin and M13K07 helper phage (approximately 1x10 10It was passaged at a final concentration of pfu / ml and grown overnight at 37°C with vigorous shaking. The next day, phages were prepared using standard procedures by PEG precipitation. Pre-exclusion of phages specific to beads coated with SAV was performed prior to panning. The second panning was performed using a KingFisher magnetic bead handler with 50 nM or 100 nM of bead-immobilized 5T4 antigen using standard procedures (100 nM of 5T4 for the third time, 50 nM of 5T4 for the fourth time). In total, 3 to 4 rounds of phage panning were performed to enrich phage-displayed Fabs specific to the 5T4 antigen. Polyclonal ELISA was used to confirm 5T4-specific enrichment, individual clones were isolated, and further verified by performing monoclonal phage ELISA. The sequences of the CDRs of the isolated Fab clones containing the candidate antibodies were determined using DNA sequencing.

[0215] The genes encoding the heavy and light chain variable domains of the candidate antibodies were separately cloned into mammalian expression vectors for expression as full-length IgG in mammalian cells. For full-length IgG, the heavy chain constant regions (e.g., CH1 = normal text, hinge = italic text, CH2 = bold, and CH3 = underlined) contained the following amino acid sequences.

Chemical formula

[0216] IgG antibodies were purified from the culture supernatant using protein A resin.

[0217] Example 2. Screening assay Using a bivalent binding assay, data is provided regarding whether the antibodies having VH / VL obtained by phage display of Example 1 bind to immobilized 5T4 derived from all three clinically relevant species, human, cynomolgus (cyno), and mouse. For the bivalent binding and cross-reactivity assays, 5T4 human, cynomolgus, and mouse antigens were synthesized using sequences from the protein database, expressed as biotinylated proteins using a his-avi tag, and biotinylated in vivo with co-expressed BirA enzyme.

[0218] Using a standard Octet-based assay, it was qualitatively determined whether each antibody binds to each antigen. For these assays, 200 nM of biotinylated antigen was immobilized on streptavidin biosensors in 1X kinetic buffer (ForteBio). The loaded sensors were then immersed in wells containing the antibody of interest at 500 nM, and the association binding signal was observed. An increase in signal during the association step above the negative control was considered binding to the antigen.

[0219] In the qualitative binding affinity using Octet, all 27 antibodies tested showed binding to human 5T4, 13 of the antibodies showed binding to cynomolgus 5T4, and 13 of the antibodies showed binding to mouse 5T4. Data for exemplary antibodies are shown in Table 4 below.

Table 4

[0220] Using a monovalent binding assay, the relative affinity of each 5T4 binder obtained by phage display in Example 1 for human 5T4 was determined. Human 5T4 was purchased from Acro Biosystems (Acro catalog number TPGH52E) and also produced separately. The Octet (Pall ForteBio) instrument was used to monitor the monovalent interaction of the binders for human 5T4. In these assays, 36 nM antibody was immobilized on an anti-human Fc (AHC) sensor in 1X kinetic buffer (ForteBio). Next, the sensor was dipped into wells containing 3-fold serial dilutions of human 5T4 in the range of 1 μM to 1.37 nM. Association was allowed to reach equilibrium. Then, the sensor was transferred to wells containing only 1-fold kinetic buffer and dissociated to equilibrate. Association and dissociation rates were measured and from these, the on-board analysis software (ForteBio) was used to calculate the monovalent K D was calculated.

[0221] Of all 27 antibodies tested, most of them showed weak binding in the monovalent binding affinity - internal (lot 20 - 019 - 62) assay. mAbA4, mAbA15, and mAbA17, among others, showed strong or moderate binding in all three lots. The data are shown in Table 5 below.

Table 5

[0222] Example 3: Additional Screening and Selection Antibodies selected to bind 5T4, such as those described in Example 2, were evaluated for binding to cells expressing 5T4.

[0223] On the day of the assay, cells were harvested at 70 - 90% confluence. In a V-bottom 96-well plate (Costar 3897), 100,000 cells per well were incubated with 50 μL per well of an 8-point or 12-point antibody dilution series at +4°C for 30 minutes. After incubation, the cells were washed once with PBS by centrifugation at 200×g for 5 minutes. Next, the cells were incubated at +4°C for 30 minutes with 50 μL per well of a 1:100 dilution of Alexa Fluor 488 goat anti-human IgG Fab (Jackson Immuno Research 109-547-003). The cells were washed twice with PBS by centrifugation at 200×g for 5 minutes and acquired with an 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.

[0224] Of 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.

[0225] Antibodies selected to bind to all three of the HEK-5T4, MCF-7, and HEK-cyno-5T4 cell lines (including mAbA4, mAbA15, and mAbA17) were further evaluated in an internalization assay using HEK-5T4 and MCF-7 cells.

[0226] Next, the cells were harvested at 70 - 90% confluence and plated at 20,000 cells / well in 50 μL of medium in a 96-well black, clear / flattened bottom half-area plate (Corning 3882) overnight at 37°C. For floating cell lines, the 96-well plates were coated with poly-L-ornithine solution (Millipore-Sigma) at 37°C for 1 hour, and about 1 - 2 hours before the assay, the cells were seeded at a density of 40,000 cells / well in 50 μL of medium. The antibody was diluted in cell culture medium and mixed with a 3-fold molar concentration of the Incucyte human FabFluor-pH red antibody labeling reagent and incubated at room temperature for 15 minutes. A 2-fold final concentration of all dilutions of the antibody / FabFluor mix was prepared. Then, 50 μL of the 2X antibody / Fab-Fluor mix was added directly in duplicate to each well containing the pre-seeded cells, and the plate was quickly transferred to the Incucyte. Thereafter, the plate was scanned every 30 minutes for up to 24 hours. Care was taken to start the first scan (0 hours) within 2 - 3 minutes after adding the antibody / FabFluor mix to the cells. The plate was analyzed by reading the red signal generated over time and normalized to the % confluence of the cells in that well. Several antibodies were tested at different concentrations on the target cell line, and the best concentration was selected based on a good signal:noise ratio.

[0227] In the internalization assay, six antibodies (including mAbA4 and mAbA15) showed strong internalization in HEK-5T4 cells, one antibody showed moderate internalization in HEK-5T4 cells, and seven antibodies did not show internalization or non-specific internalization in HEK-5T4 cells. In further assays, two antibodies showed strong internalization in MCF 7 cells, four antibodies showed moderate internalization in HEK-5T4 cells, and eight antibodies did not show internalization or non-specificity in MCF-7 cells.

[0228] Example 4: Development potential assay Antibodies selected for binding to 5T4, such as those described in Examples 2 and 3, were tested in various development methods. For example, various chromatography methods including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and stand-up monolayer adsorption chromatography (SMAC) were used to evaluate development factors such as the percentage of monomer, solubility, and aggregation or precipitation of the antibody.

[0229] Size exclusion chromatography (SEC) analysis was performed using an Agilent 1100 HPLC with a 7.8 mm ID × 30 cm TSKgel G3000SWXL column (Tosoh Bioscience LLC, PN08541) and a 6 mm × 4 cm guard column (PN08543). The antibody was normalized to a concentration of 1 mg / mL in Dulbecco's PBS (pH 7.4, without Ca 2+ / Mg 2+ and clarified by microparticulation via centrifugation while retaining soluble aggregates. The mobile phase buffer was 2-fold concentrated (diluted from 10-fold stock concentration) Dulbecco's PBS (pH 7.4, without Ca 2+ / Mg 2+ . For each sample, 10 μL was loaded and eluted uniformly at 1.0 mL / min for 20 minutes at ambient temperature. Absorbance was monitored at 280 nm. Chromatographic peaks were integrated to determine the percentage of homogeneity and retention time. The stationary phase of the column and the selected mobile phase support hydrophobic and electrostatic interactions in addition to molecular sizing (secondary interactions are much milder compared to SMAC). Data analysis was performed using Agilent ChemStation B.04.03 SP1.

[0230] The results showed that mAbA4, mAbA15, and mAbA17 (among other antibodies tested) have high development potential based on SEC analysis.

[0231] Hydrophobic interaction chromatography (HIC) analysis was performed using an Agilent 1100 HPLC with a 4.6 mm ID × 3.5 cm TSKgel butyl-NPR column (Tosoh Bioscience LLC, PN14947). Antibodies were normalized to a concentration of 1 mg / mL in Dulbecco's PBS (pH 7.4, Ca 2+ / Mg 2+ -free). The column was equilibrated at ambient temperature with 100% mobile phase buffer A (2 M ammonium sulfate / 20 mM sodium phosphate, pH 7.0) at a flow rate of 1 mL / min. For each sample, 10 μL was loaded and eluted at 1.0 mL / min over 15 minutes using a gradient from 100% mobile phase buffer A to 100% mobile phase buffer B (20 mM sodium phosphate, pH 7.0), held at 100% B for 3 minutes to wash the column, and returned to 100% A for 2 minutes for equilibration. 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 hydrophobicity) and the presence of multiple species. Data analysis was performed using Agilent ChemStation B.04.03 SP1.

[0232] Results from hydrophobic interaction chromatography (HIC) analysis indicated that certain antibodies had strong development potential and other antibodies had moderate development potential. mAbA4, mAbA15, and mAbA17 were included among those with strong development potential based on HIC analysis. The hydrophobicity of antibodies can affect antibody aggregation, solubility, and viscosity. The results indicate a low tendency for aggregation and precipitation of these antibodies.

[0233] Stand-up monolayer adsorption chromatography (SMAC) analysis was performed using an Agilent 1100 HPLC with a 4.6 mm × 50 mm guard column (PN213300P-4605) and a 4.6 mm ID × 300 mm Zenix SEC 300 column (Sepax Technologies, PN213300P-4630). Antibodies were normalized to a concentration of 1 mg / mL in Dulbecco's PBS (pH 7.4, calcium / magnesium-free) and clarified by microfluidization via centrifugation while retaining soluble aggregates. The mobile phase buffer was 2-fold concentrated (diluted from a 10-fold stock concentration) Dulbecco's PBS (pH 7.4, calcium / magnesium-free). For each sample, 10 μL was loaded and eluted uniformly at 0.4 mL / min over 32 minutes at ambient temperature. Absorbance was monitored at 280 nm. Due to the selection of the column stationary phase and the mobile phase, secondary interactions are promoted in addition to molecular sizing, so the sample retention time was calculated and compared to a set of standard controls to identify antibodies with increased retention time (increased tendency to form aggregates and / or increased hydrophobic / electrostatic interactions). Data analysis was performed using Agilent ChemStation B.04.03 SP1.

[0234] The results of stand-up monolayer adsorption chromatography (SMAC) analysis indicated that certain antibodies have strong development potential while other antibodies have moderate development potential. mAbA4, mAbA15, and mAbA17 are included among those with strong production based on SMAC analysis. This measurement was based on good retention times indicating colloidal stability and low aggregation tendency.

[0235] Example 5: Epitope Binding Assay An epitope binding assay was used to determine which epitopes of 5T4 each antibody obtained by phage display in Example 1 binds to.

[0236] To enable measurement of epitope binding while maintaining the overall structure of the 5T4 protein, mouse-human chimeras were generated by cloning each of seven contiguous sequences (epitopes 1-7) of human 5T4 and replacing each corresponding mouse sequence. For chimera generation, full-length human and mouse 5T4 genes were synthesized by GenScript and cloned into a mammalian vector containing a 6-His-Avi tag. Each of the seven sequences (epitopes 1-7) described herein was then removed from the human gene and separately cloned into the mouse gene using appropriate primers. Epitope 1 of human 5T4 (H1) corresponds to SSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAART (SEQ ID NO: 76), epitope 2 of human 5T4 (H2) corresponds to VKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVR (SEQ ID NO: 77), epitope 3 of human 5T4 (H3) corresponds to AGAFEHLPSLRQLDLSHNPLADLSPFAFSG (SEQ ID NO: 78), epitope 4 of human 5T4 (H4) corresponds to SNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLE (SEQ ID NO: 79), epitope 5 of human 5T4 (H5) corresponds to LASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESL (SEQ ID NO: 80), epitope 6 of human 5T4 (H6) corresponds to HLEDNALKVLHNGTLAELQGLPHIRVFL (SEQ ID NO: 81), and epitope 7 of human 5T4 (H7) 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, the seven chimeric constructs of the generated human (H) and mouse (M) sequences were as follows: (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 of the chimeric constructs and to the full-length human protein. Any significant binding signal to the chimera indicates the region to which the antibody binds specifically.

Table 6-1

Table 6-2

Table 6-3

[0237] A standard Octet-based assay was used as an epitope binning assay to establish the epitope bins of each antibody. For these assays, 50 nM biotinylated antigen was immobilized on streptavidin biosensors in 1X kinetic buffer (ForteBio). The sensor was then dipped into wells containing the antibody of interest at 100 nM, and the association signal was monitored. The chimera(s) that effectively knocked out the binding of the antibody then corresponded to the specific epitope(s) to which the antibody binds. Full-length human 5T4 was included in the assay for reference.

[0238] The results showed that nine antibodies specifically bound to epitope 4 of 5T4, three antibodies specifically bound to epitope 7 of 5T4, three antibodies specifically bound to epitope 2 of 5T4, one antibody specifically bound to both epitope 4 and epitope 6 of 5T4, and eleven antibodies bound to full-length 5T4 (FL). The results of exemplary epitope binding (for mAbA4, mAbA15, and mAbA17) are summarized in Table 7.

Table 7

[0239] Example 6: Selection of Antibodies 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 into IgG were further tested as described in Examples 2 - 5. Three antibodies identified as mAbA4, mAbA15, and mAbA17 were selected based on a number of activities, including their specificity for human 5T4 binding, binding to cyno5T4, their cell surface binding signals, and strong developability evaluated by various methods such as SEC, HIC, and SMAC. The VH sequences, VL sequences, and CDR sequences of these antibodies are shown in Tables 1 - 3.

[0240] Example 7: Binding of Antibodies to Human 5T4 (ELISA) A. Normal Binding

[0241] The selected antibodies were tested for their ability to bind to hu5T4 using ELISA. On day 1, unconjugated human TPBG / 5T4 protein with a C-terminal polyhistidine and Avi (AVITAG™) tag (AcroBiosystems (Newark, DE, USA), catalog number TPG-H52E5) was diluted to 2 μg / mL in PBS, mixed thoroughly, and 100 μL per well was plated onto a 96-well plate (Nunc MAXISORP®) and incubated overnight at 4°C.

[0242] On the second day, the plates were washed four times with PBS containing 0.1% Tween®-20 (“PBS-T”). The wells were blocked with 200 μL of casein (Blocker™ Casein, available from ThermoFisher Scientific) in PBS and incubated for 2 hours at room temperature with shaking.

[0243] A 1:3 dilution series of IgG-ADC was prepared in PBS and added at a volume of 100 μL per well of a 96-well plate. The plate was incubated for 1 hour at room temperature with shaking.

[0244] Goat anti-human IgG Fc gamma specific HRP conjugate (Jackson Immunoresearch, catalog number 109-035-098) was diluted 1:15000 with PBS. The ELISA plate was washed 6 times with PBS-T using a plate washer (Bio Tek ELx405), and then 100 μL of goat anti-human IgG Fc gamma specific HRP conjugate was added to each well. The plate was incubated for 30 minutes at room temperature with shaking.

[0245] After the ELISA plate was washed 6 times with PBS-T using a plate washer (Bio Tek ELx405), 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB) was added to each well. The plate was developed until a dark blue color was visible. Then, H2SO4 (2N, 100 μL) was added to quench the reaction, and the absorbance of each well was read using a plate reader (SpetraMax M5 equipped with SoftMaxPro software available from Molecular Devices).

[0246] B. Stress Test

[0247] 1. High pH stress test. Antibodies were tested for their ability to bind to 5T4 using ELISA under basic (high pH) conditions. Antibodies at 1 mg / mL in 0.1% w / w H2O2 / 1xPBS were stored in the dark at room temperature for 24 - 48 hours, after which 50 mM methionine was added. Samples were frozen for storage prior to analysis and the analysis was performed by ELISA as described in Part A above. Table 8 below reports the EC 50 calculated for each antibody tested.

Table 8

[0248] 2. Low pH stress test. Antibodies were also tested for their ability to bind to 5T4 using ELISA under acidic (low pH) conditions. Antibody solutions at 1 mg / mL - 10 mg / mL in 20 mM Tris / 10 mM EDTA (pH 8.5) were prepared and stored at 37 °C for 1 - 2 weeks. Samples were frozen for storage prior to analysis and the analysis was performed by ELISA as described in Part A above. Table 9 below reports the EC 50 calculated for each antibody tested.

Table 9

[0249] 3. Oxidative stress. Antibodies were tested for their ability to bind to 5T4 using ELISA in an oxidative environment. Antibody solutions at 1 mg / mL - 10 mg / mL in 20 mM citrate / 50 mM NaCl were prepared at pH 5.5 and stored at 37 °C for 1 - 2 weeks. Samples were frozen for storage prior to analysis and the analysis was performed by ELISA as described in Part A above. Table 10 below reports the EC 50 calculated for each antibody tested.

Table 10

[0250] Example 8. Cross-reactivity of antibodies against cynomolgus monkey, rat, and mouse 5T4 (ELISA and Octet) Selected antibodies were tested for their ability to bind to non-human 5T4-T using ELISA and Octet. Instead of the human 5T4 antigen, recombinant cynomolgus monkey 5T4-Fc (supplied from either R&D Systems (Minneapolis, MN, USA), catalog number 2280-TG-100 or LSBio (Seattle, WA, USA), catalog number LS-G137131), recombinant mouse 5T4-Fc (R&D Systems, catalog number 5049-TG-100), 6His-SUMO, recombinant rat TPBG / 5T4 with an N-terminal tag (LSBio catalog number G56592), and recombinant mouse TPBG / 5T4 with a His, N-terminal tag (LSBio, catalog number G12168) antigen were substituted, and the ELISA protocol described in Example 7 was used. In the Octet analysis, Protein A biochips were coated with the antibody, washed with PBS, and then reacted with the antigen as specified above. All antibodies tested bound to human, cynomolgus monkey, and rat 5T4. No significant binding cross-reactivity to mouse 5T4 was observed.

[0251] Example 9. Cellular binding of antibodies to 5T4-transfected CHO and HEK293 cells Selected antibodies were tested for their ability to bind to HEK293 and CHO cells transfected to express human, cynomolgus monkey, rat, or mouse 5T4. Non-transfected cells were used as a control to observe non-specific binding. mAbA4, mAbA15, and mAbA17 were reactive with HEK293 and CHO cells transfected to express human and cynomolgus monkey 5T4, but not with cells transfected to express rat or mouse 5T4. * * * * *

[0252] Throughout this application, various publications, patents, patent applications, and other documents are referenced. The disclosures of these publications, patents, patent applications, and other documents are hereby incorporated by reference in their entirety for all purposes, including for the purpose of more fully describing the state of the art to which the present invention pertains. Although the invention has been described with reference to the embodiments provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Many variations of the invention will be apparent to those skilled in the art upon consideration of this specification.

Claims

1. An antibody or fragment that binds to 5T4, (i) VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 44, and VL CDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 45, (ii) VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 26, or (iii) The antibody or fragment thereof, comprising VH CDR1, VH CDR2, and VH CDR3 as described in VH, which contains the amino acid sequence of SEQ ID NO: 62, and VL CDR1, VL CDR2, and VL CDR3 as described in VL, which contains the amino acid sequence of SEQ ID NO:

63.

2. (a) (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35, and 39, (2) VH CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40, and 43, (3) A VH region including a VH CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 33, 37, and 41, and (b) (1) VL CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) VL CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 and 22, or an amino acid sequence called SAS, (3) The antibody or fragment thereof according to claim 1, comprising a VL region including a VL CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 38, and 42.

3. (i) to (vi): (i) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, (ii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, (iii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 34, VH CDR2 containing the amino acid sequence of SEQ ID NO: 28, VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, (iv) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 38, (v) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 39, VH CDR2 containing the amino acid sequence of SEQ ID NO: 40, VH CDR3 containing the amino acid sequence of SEQ ID NO: 41, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 42, and (vi) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 29, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, The antibody or fragment thereof according to claim 1, comprising one or more of the following.

4. (a) (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, (2) VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, (3) A VH region including VH CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20, and (b) (1) VL CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) VL CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 and 22, or an amino acid sequence called SAS, (3) The antibody or fragment thereof according to claim 1, comprising a VL region including a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

5. (i) to (vi): (i) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, (ii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 7, VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, VH CDR3 containing the amino acid sequence of SEQ ID NO: 9, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, (iii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, (iv) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 13, VH CDR2 containing the amino acid sequence of SEQ ID NO: 14, VH CDR3 containing the amino acid sequence of SEQ ID NO: 15, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17, (v) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 18, VH CDR2 containing the amino acid sequence of SEQ ID NO: 19, VH CDR3 containing the amino acid sequence of SEQ ID NO: 20, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 23, and (vi) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 24, VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

6. The antibody or fragment thereof according to claim 1, comprising one or more of the following.

6. (a) (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50, 53, 13, and 57, (2) VH CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 58, and 61, (3) A VH region including VH CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 55, and 59, and (b) (1) VL CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, (2) VL CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 and 22, or an amino acid sequence called SAS, (3) The antibody or fragment thereof according to claim 1, comprising a VL region including a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 56, and 60.

7. (i) to (vi): (i) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 49, (ii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, VH CDR2 containing the amino acid sequence of SEQ ID NO: 51, VH CDR3 containing the amino acid sequence of SEQ ID NO: 52, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 49, (iii) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 49, (iv) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 13, VH CDR2 containing the amino acid sequence of SEQ ID NO: 54, VH CDR3 containing the amino acid sequence of SEQ ID NO: 55, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence SAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56, (v) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 57, VH CDR2 containing the amino acid sequence of SEQ ID NO: 58, VH CDR3 containing the amino acid sequence of SEQ ID NO: 59, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 60, and (vi) A VH region including VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 61, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, and a VL region including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

49. The antibody or fragment thereof according to claim 1, comprising one or more of the following.

8. The antibody or fragment thereof according to claim 1, further comprising a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence, optionally described in any one of sequence numbers 44, 45, 25, 26, 62, and 63.

9. The antibody or fragment thereof according to claim 1, further comprising a human framework sequence.

10. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment comprises VH containing the amino acid sequence of SEQ ID NO: 44 and VL containing the amino acid sequence of SEQ ID NO:

45.

11. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO:

26.

12. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises VH having the amino acid sequence of SEQ ID NO: 62 and VL having the amino acid sequence of SEQ ID NO:

63.

13. The antibody or fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.

14. The antibody or fragment thereof according to claim 1, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

15. Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ The antibody or fragment thereof according to claim 1, which is a multispecific antibody formed from a single-chain antibody molecule, a bivariate region antibody, a single variable region antibody, a linear antibody, a V region, or an antibody fragment.

16. One or more polynucleotides encoding the antibody or a fragment thereof according to any one of claims 1 to 15.

17. One or more vectors comprising one or more polynucleotides according to claim 16.

18. A cell comprising one or more polynucleotides as described in Claim 16.

19. A pharmaceutical composition comprising an antibody or fragment thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

20. A method for treating cancer or tumors in a subject, for alleviating one or more symptoms associated with cancer or tumors in a subject, for reducing the size of a tumor in a subject, for enhancing the removal of tumor cells in a subject, or for treating a 5T4-related disease, disorder, or condition in a subject, comprising administering to the subject an antibody or fragment thereof according to any one of claims 1 to 15, wherein the subject is optionally administered one or more therapeutic agents in combination with the antibody or fragment thereof.

21. The method according to claim 20, wherein the subject is a human subject.

Images