Anti-5T4 antibodies and uses thereof

Abstract

The present disclosure provides anti-5T4 antibodies, variants and humanized versions thereof. The newly disclosed antibodies have shown high affinity to 5T4 protein and can be used to treat cancer. The present disclosure provides antibodies and antigen-binding fragments specific to human 5T4 protein in various embodiments. Experimental tests show that these newly identified antibodies can bind strongly and specifically to human 5T4 protein. Unlike naptumomab, a fusion protein containing a Fab fragment targeting 5T4, which has been evaluated in clinical trials, these newly identified antibodies can also bind to cynomolgus monkey 5T4 protein with comparable potency.

Description

[Technical field]

[0001] background 5T4 (also known as trophoblast glycoprotein, TPBG; 5T4 oncofetal trophoblast glycoprotein; and Wnt activation inhibitory factor 1, WAIF1) is a vertebrate-specific single-pass transmembrane protein first identified in human placental tissue. 5T4 contains a highly glycosylated rigid core that includes eight leucine-rich repeats (LRRs) in the extracellular domain, transmembrane helices, and cytoplasmic region. The cytoplasmic PDZ-binding motif Ser-Asp-Val of 5T4 is reported to interact with the PDZ domain of TIP-2 / GIPC, a cytoplasmic protein that associates with vesicles located near the plasma membrane. The further downstream mechanism of signal transduction remains unclear. 5T4 has also been found to inhibit the Wnt / β-catenin signaling pathway, a pathway important for embryonic development and a major target for anticancer therapeutics. [Background technology]

[0002] 5T4 is hardly expressed in normal adult tissues, but is present at high levels in the placenta and in more than 80% of the most common tumors, typically renal, breast, colon, prostate and ovarian cancers. 5T4 therefore has characteristics of an oncofetal antigen, which makes it stand out as a promising candidate for use as a diagnostic marker or target for cancer treatment. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides, in various embodiments, antibodies and antigen-binding fragments specific to human 5T4 protein. Experimental testing shows that these newly identified antibodies can bind strongly and specifically to human 5T4 protein. Unlike naptumomab, a fusion protein containing a Fab fragment targeting 5T4, which has been evaluated in clinical trials, these newly identified antibodies can also bind to cynomolgus monkey 5T4 protein with comparable potency.

[0004] According to one embodiment of the present disclosure, there is provided an antibody or antigen-binding fragment thereof having specificity for human 5T4 oncofetal trophoblast glycoprotein (5T4) protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0005] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 41, 42 (or any one of 47-53), 43, 44, 45 and 46 (or 264 or 265), respectively. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 264 or 265. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 197 and VL comprises the amino acid sequence of SEQ ID NO: 262 or 263.

[0006] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 54, 55 (or any one of 60-63, or 266 or 267), 56, 57, 58 and 59, respectively. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 64-69, respectively. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 70, 71 or 76, 72, 73, 74 or 75, respectively. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 77-82.

[0007] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 83-88. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 89-94. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 95, 96 (or any one of 101-104), 97, 98, 99 and 100. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 105-110. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO:111-116.

[0008] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 117-122. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 123, 124 or 129, 125, 126, 127 and 128. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 130-135. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 136-141. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 142-147.

[0009] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 148-153. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 154-159. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 160, 161 or 166, 162, 163, 164 and 165. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 167, 168 or 173, 169, 170, 171 and 172, respectively. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 174, 175 or 180, 176, 177, 178 and 179, respectively.

[0010] Also provided are antibody-drug conjugates comprising an antibody of the present disclosure or a fragment thereof conjugated to a drug moiety. In some embodiments, the drug moiety is a cytotoxic or cytostatic agent. In some embodiments, the drug moiety is a maytansinoid, an auristatin, or a macrocyclic ketone analog. In some embodiments, the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some embodiments, the drug moiety is attached to the antibody or a fragment thereof via a linker that is hydrolyzable under acidic conditions.

[0011] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 264 or 265. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 197 and the VL comprises the amino acid sequence of SEQ ID NO: 262 or 263.

[0012] Multispecific antibodies are also provided that comprise an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments that have binding specificity for a target antigen that is not 5T4.

[0013] In another embodiment, a chimeric antigen receptor (CAR) is also provided that comprises an antigen-binding fragment of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0014] Also provided is a polynucleotide encoding an antibody or antigen-binding fragment thereof or a CAR of the present disclosure. In some embodiments, the polynucleotide is an mRNA, optionally chemically modified.

[0015] Methods and uses for treating cancer and inflammatory conditions using the antibodies or antigen-binding fragments thereof of the present disclosure are also provided. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the ELISA binding activity of tested anti-5T4 chimeric monospecific antibodies against human 5T4 protein.

[0017] [Diagram 2] FIG. 2 shows the ELISA binding activity of tested anti-5T4 chimeric monospecific antibodies against cynomolgus 5T4 protein.

[0018] [Diagram 3] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay. [Figure 4] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay. [Diagram 5] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay. [Figure 6] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay. [Figure 7] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay. [Figure 8] Figures 3-8 show epitope binding of the tested anti-5T4 chimeric monospecific antibodies by competitive ELISA assay.

[0019] [Figure 9-1] FIG. 9 shows that the binding activity of the tested anti-5T4 chimeric mAbs to human 5T4 expressed on the surface of CHO-K1 cells. [Figure 9-2] FIG. 9 shows that the binding activity of the tested anti-5T4 chimeric mAbs to human 5T4 expressed on the surface of CHO-K1 cells.

[0020] [Figure 10] FIG. 10 shows that the majority of the humanized 14G12 antibodies have similar binding activity to the human 5T4 antigen compared to the chimeric 14G12 mAb.

[0021] [Figure 11-1] FIG. 11 shows that most of the humanized 14G12 antibodies tested have similar binding activity to CHOK1 overexpressing human 5T4 compared to the chimeric 14G12 antibody. [Figure 11-2]FIG. 11 shows that most of the humanized 14G12 antibodies tested have similar binding activity to CHOK1 overexpressing human 5T4 compared to the chimeric 14G12 antibody.

[0022] [Figure 12] FIG. 12 shows that the humanized 393E9 antibody and its chimeric mAbs tested have comparable binding activity to the human 5T4 antigen.

[0023] [Figure 13] FIG. 13 shows that some of the humanized 393E9 antibodies tested have similar or even stronger binding activity to CHOK1-hu5T4 cells than the chimeric antibody.

[0024] [Figure 14] FIG. 14 shows that the humanized 159D5 antibody and its chimeric mAbs tested have comparable binding activity to the human 5T4 antigen.

[0025] [Figure 15] FIG. 15 shows that the humanized 159D5 antibody and its chimeric antibodies tested have comparable binding activity to CHOK1 overexpressing human 5T4.

[0026] [Figure 16] FIG. 16 shows that some of the humanized 286B4 antibodies tested have similar binding activity to the human 5T4 antigen compared to the chimeric antibody.

[0027] [Figure 17] FIG. 17 shows that some of the humanized 286B4 antibodies tested have similar or even stronger binding activity to CHOK1-hu5T4 or MCF-7 cells than their chimeric antibodies.

[0028] [Figure 18]FIG. 18 shows that the PTM-depleted antibodies have enhanced binding ability to 5T4-expressing cells than their chimeric antibodies.

[0029] [Figure 19] FIG. 19 shows that the affinity matured antibodies have stronger binding activity for the human 5T4 antigen than their parent Hu14G12-28 antibody.

[0030] [Figure 20] FIG. 20 shows that the affinity matured antibodies have enhanced binding ability to 5T4-expressing cells than their parent Hu14G12-28 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0032] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment thereof or a single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0033] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0034] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively.

[0035] Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are expressly within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides of approximately 23,000 daltons molecular weight and two identical heavy chain polypeptides of 53,000-70,000 daltons molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains on either side, beginning at the mouth of the "Y" and continuing through the variable region.

[0036] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0037] As used herein, the term "chimeric antibody" should be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0038] The antibody disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibody is human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody.In some embodiments, the variable region can be of chondrichthyan origin (e.g., from shark).

[0039] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which may be made by combining polynucleotides that do not normally occur together.

[0040] Hybridoma techniques can be performed under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out at about 40°C in about 10xSSC, or a solution of equivalent ionic strength / temperature. Medium stringency hybridization is typically carried out at about 50°C in about 6xSSC, and high stringency hybridization reactions are generally carried out at about 60°C in about 1xSSC. Hybridization reactions can also be performed under "physiological conditions", which are well known to those of skill in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH and Mg normally found in cells. 2+ Concentration. Anti-5T4 antibody

[0041] As demonstrated in the attached experimental examples, the present inventors were able to generate anti-5T4 antibodies 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 and 95F10 (Table 1). Importantly, many of these antibodies showed greater biological activity than naptumomab (NeoTX), a benchmark fusion protein containing a Fab portion against 5T4. Furthermore, these antibodies showed cross-reactivity to cynomolgus 5T4, which allows for advancement of preclinical evaluation.

[0042] Competitive binding experiments showed that these newly identified antibodies, along with naptumomab, can be classified into four different bins based on where they bind to the 5T4 antigen. Interestingly, only 14G12, 393E9 and 113H5 compete with naptumomab in binding to 5T4 (referred to as "bin A", Table 4). Bin B includes 159D5, 24F10 and 493E10, bin C includes 257F1, 353H11, 367B8, 389G2 and 109H7, and bin D includes 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 and 95F10. Sequence examination shows that certain CDRs of the antibodies in each of these bins are highly homologous and therefore are assumed to be interchangeable.

[0043] According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or an antigen-binding fragment thereof has binding properties to human 5T4 protein. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0044] Sequence analysis revealed that some of the CDRs contain residues that can potentially be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify production, the present disclosure has designed and tested certain de-risked versions of the CDRs.

[0045] In some embodiments, an antibody or antigen-binding fragment thereof is provided that is derived from antibody 393E9. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:54; VH CDR2 comprises the amino acid sequence of SEQ ID NO:55; VH CDR3 comprises the amino acid sequence of SEQ ID NO:56; VL CDR1 comprises the amino acid sequence of SEQ ID NO:57; VL CDR2 comprises the amino acid sequence of SEQ ID NO:58; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO:59.

[0046] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 54; the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 55, or SEQ ID NOs: 60-63, or 266-267; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 58; and the VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 59.

[0047] In some embodiments, the VH CDR2 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 54; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 266; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 58; and the VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 59. In some embodiments, the VH CDR2 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 54; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 267; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 58; and the VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 59. Interestingly, as shown in Example 12, the PTM risk-avoiding versions, in particular Hu393E9-45-P2 and Hu393E9-62-P2 (both containing SEQ ID NO: 267 as VH CDR2), had enhanced affinity compared to the chimeric versions.

[0048] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 215-221 and 248-256. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 222-226.

[0049] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 3, 215-221 and 248-256, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 3, 215-221 and 248-256 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 4 and 222-226, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 4 and 222-226 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0050] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 393E9 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 393E9 for binding to 5T4 are also provided.

[0051] In some embodiments, an antibody or antigen-binding fragment thereof is provided that is derived from antibody 286B4. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 123; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 124; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 125; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 126; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 127; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128.

[0052] In some embodiments, VH CDR2 is PTM risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 123; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 124 or SEQ ID NO: 129; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 125; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 126; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 127; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 128, respectively. Interestingly, as shown in Example 12, the PTM risk avoided version has enhanced affinity compared to the chimeric version.

[0053] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 236-241 and 259-261. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 242-247.

[0054] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 23, 236-241, and 259-261, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 23, 236-241, and 259-261 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 24 and 242-247, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 24 and 242-247 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0055] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 286B4 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 286B4 for binding to 5T4 are also provided.

[0056] In some embodiments, an antibody or antigen-binding fragment thereof is provided that is derived from antibody 14G12. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0057] In some embodiments, the VH CDR2 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 42 or SEQ ID NO: 47 to 53; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and the VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0058] In some embodiments, the VL CDR3 is affinity matured. As shown in Example 20, the affinity matured antibodies Hu14G12-28-88# and Hu14G12-28-108# significantly increased the binding affinity to human 5T4 protein by 9.45-fold and 7.41-fold, respectively, compared to the parent 14G12-28 antibody.

[0059] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 264. In some embodiments, VH CDR2 is PTM risk averted. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 265.

[0060] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, or any one of SEQ ID NOs: 47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 264, and 265, respectively.

[0061] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 185-188, 193-194, 205-214, and 262-263. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263.

[0062] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204 while retaining the corresponding VH CDRs or PTM-avoided versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263 while retaining the corresponding VL CDRs or PTM de-risked versions thereof. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 197 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NO: 262, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 262 while retaining the corresponding VL CDRs or affinity matured versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NO: 263, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 263 while retaining the corresponding VL CDRs or affinity matured versions thereof.

[0063] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 14G12 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 14G12 for binding to 5T4 are also provided.

[0064] In some embodiments, an antibody or antigen-binding fragment thereof derived from antibody 159D5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 70; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 72; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 74; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 75.

[0065] In some embodiments, VH CDR2 is PTM risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 70; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 76; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 72; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 74; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 75. As shown in Example 12, the PTM risk avoided version (159D50-P1) has the same performance as the chimeric antibody.

[0066] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 227-229, and 257. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 230-235, and 258.

[0067] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 7, 227-229, and 257, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 7, 227-229, and 257 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 8, 230-235, and 258, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 8, 230-235, and 258 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0068] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 159D5 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 159D5 for binding to 5T4 are also provided.

[0069] In some embodiments, an antibody or antigen-binding fragment thereof derived from antibody 353H11 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 95; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 96; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100.

[0070] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 95; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 96 or any one of SEQ ID NOs: 101 to 104; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99; and the VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100.

[0071] An example VH sequence comprises the amino acid sequence of SEQ ID NO: 15, and an example VL sequence comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 15 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 16 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0072] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 353H11 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 353H11 for binding to 5T4 are also provided.

[0073] In some embodiments, an antibody or antigen-binding fragment thereof is provided that is derived from antibody 109H7. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 117; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 118; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 119; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 120; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 121; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 122.

[0074] An example VH sequence comprises the amino acid sequence of SEQ ID NO: 21, and an example VL sequence comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 21, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 21 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 22 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0075] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 109H7 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 109H7 for binding to 5T4 are also provided.

[0076] In some embodiments, an antibody or antigen-binding fragment thereof is provided that is derived from antibody 49C5. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 154; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 155; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 156; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 157; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 158; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 159.

[0077] An example VH sequence comprises the amino acid sequence of SEQ ID NO: 33, and an example VL sequence comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 33, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 33 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 34, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 34 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0078] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on 5T4 as 49C5 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 49C5 for binding to 5T4 are also provided.

[0079] Also provided are antibodies comprising any set of CDRs (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the antibodies disclosed herein, such as 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 and 95F10 (Table 1), or PTM risk-avoided versions thereof. The CDR sequences are listed in Tables 1-1 to 1-41.

[0080] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 41, 42 (or any one of 47-53), 43, 44, 45 and 46 (or 264 or 265). In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 54, 55 (or any one of 60-63, or 266 or 267), 56, 57, 58 and 59. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 64-69. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 70, 71 or 76, 72, 73, 74 or 75. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 77-82.

[0081] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 83-88. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 89-94. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 95, 96 (or any one of 101-104), 97, 98, 99 and 100. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 105-110. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO:111-116.

[0082] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 117-122. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 123, 124 or 129, 125, 126, 127 and 128. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 130-135. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise an amino acid sequence of SEQ ID NO: 136-141. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 142-147.

[0083] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 148-153. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 154-159. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 160, 161 or 166, 162, 163, 164 and 165. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 167, 168 or 173, 169, 170, 171 and 172, respectively. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 174, 175 or 180, 176, 177, 178 and 179, respectively.

[0084] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0085] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 9 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 10 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0086] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 11, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 11 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 12 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0087] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 13 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 14 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0088] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 17 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 18 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0089] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 19 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 20, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 20 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0090] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 25 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 26, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 26 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0091] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 27, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 27 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 28, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 28 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0092] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 29, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 29 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 30, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 30 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0093] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 31, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 31 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 32, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 32 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0094] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 35, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 35 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 36, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 36 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0095] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 37, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 37 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 38, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 38 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0096] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 39, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 39 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 40, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 40 while retaining the corresponding VL CDRs or PTM de-risked versions thereof.

[0097] In some embodiments, antibodies and antigen-binding fragments are also provided that comprise CDR sequences derived from those of the disclosure, with one, two or three amino acid substitutions, deletions and / or additions. Antibody-drug conjugates

[0098] In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0099] In one embodiment, the antibody or fragment of the present disclosure is covalently attached to a drug moiety. The drug moiety may be a reactive group that has a conjugation point on the antibody, or may be modified to include the group. For example, the drug moiety may be attached by alkylation (e.g., at the epsilon-amino lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification of hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation to thiols.

[0100] In some embodiments, the number of conjugated drug moieties per antibody molecule, p, ranges on average from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges on average from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p ranges on average from 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p ranges on average from about 1 to about 20, from about 1 to about 10, from about 2 to about 10, from about 2 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, or from about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, or from about 2 to about 3.

[0101] For example, if chemical activation of the protein results in the formation of free thiol groups, the protein can be conjugated with a sulfhydryl-reactive agent. In one embodiment, the agent is substantially specific for free thiol groups. Such agents include, for example, maleimides, haloacetamides (e.g., iodo, bromo, or chloro), haloesters (e.g., iodo, bromo, or chloro), halomethylketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodide, bromide, or chloride), vinylsulfones, and pyridylthios.

[0102] The drug may be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs through a maleimide group, which reacts with sulfhydryls (thiols, -SH), while the sulfo-NHS ester is reactive to primary amines (such as those found in lysine and protein or peptide N-termini). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a particular pH or pH range, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. Linkers, such as mc linkers, can be covalently attached to antibodies to such an extent that the antibody must be degraded intracellularly to release the drug.

[0103] The linker may contain a group for linking to an antibody. For example, the linker may contain an amino, hydroxyl, carboxyl, or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinylsulfone, and pyridylthio).

[0104] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, etc. The conjugate can be used to inhibit tumor or cancer cell multiplication, to cause apoptosis in tumor or cancer cells, or to treat cancer in patients. Thus, the conjugate can be used in a variety of situations for the treatment of cancer in animals. The conjugate can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in some embodiments, the conjugate can bind to or associate with cancer cells expressing CLDN6, and the conjugate and / or drug can be taken up into tumor or cancer cells by receptor-mediated endocytosis.

[0105] In some embodiments, the drug moiety is a maytansinoid or an auristatin. In some embodiments, the drug moiety is a macrocyclic ketone analog, such as eribulin. In some embodiments, the drug moiety is a topoisomerase inhibitor, such as exatecan and exatecan derivatives.

[0106] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolytically cleaved by one or more tumor or cancer cell-associated proteases, resulting in the release of the drug. The released drug is then free to migrate into the cell and induce cytotoxic or cytostatic or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor or cancer cell, and then the drug penetrates the cell or acts on the cell surface.

[0107] Examples of drug moieties or payloads are eribulin (2-(3-amino-2-hydroxypropyl)hexacosahydro-3-methoxy-26-methyl-20,27-bis(methylene)11,15-18,21-24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-furo(3,2-i)furo(2',3'-5,6)pyrano(4,3-b)(1,4)dioxacyclopentacosin-5-(4H)-one), DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxo-1,3-dioxacyclopentacosin-5-(4H)-one), isopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4- oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Va l-Cit-PABA-MMAE (6-Maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornityl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE and MMAF are derivatives of auristatins. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.

[0108] The antibody or fragment may be conjugated or fused to a therapeutic agent, which may include a detectable label, e.g., a radioactive label, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0109] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0110] Metals that emit fluorescence, e.g. 152Antibodies can also be detectably labeled using Eu, or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.(eds.), pp. 243-56 (Alan R. Liss, Inc. (1985);Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623- 53 (1987);Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985);"Analysis, Results, And Future Developments in Antibodies See, "Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).

[0111] It is understood that any antibody or antigen-binding fragment of the present disclosure is suitable for inclusion in an antibody-drug conjugate (ADC) as currently disclosed. In one embodiment, the antibody or fragment comprises the VH and VL CDRs of any of antibodies 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 or 95F10, or PTM-avoided or affinity-matured versions thereof.

[0112] In some embodiments, the antibody or fragment comprises the VH and VL CDRs of any of the antibodies in bin A (14G12, 393E9, and 113H5). In some embodiments, the antibody or fragment comprises the VH and VL CDRs of any of the antibodies in bin B (159D5, 24F10, and 493E10). In some embodiments, the antibody or fragment comprises the VH and VL CDRs of any of the antibodies in bin C (257F1, 353H11, 367B8, 389G2, and 109H7). In some embodiments, the antibody or fragment comprises the VH and VL CDRs of any of the antibodies in bin D (286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10).

[0113] In some embodiments, the antibody or antigen-binding fragment of the ADC comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 41; a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 42; a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 43; a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 44; a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0114] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, or any one of SEQ ID NOs: 47 to 53; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0115] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 264. In some embodiments, VH CDR2 is PTM risk averted. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 265.

[0116] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, or any one of SEQ ID NOs: 47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 264, and 265, respectively.

[0117] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 185-188, 193-194, 205-214, and 262-263. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263.

[0118] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204 while retaining the corresponding VH CDRs or PTM-avoided versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263 while retaining the corresponding VL CDRs or PTM de-risked or affinity matured versions thereof. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 197 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 262, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 262 while retaining the corresponding VL CDRs or affinity matured versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 263, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 263 while retaining the corresponding VL CDRs or affinity matured versions thereof. multifunctional molecules

[0119] Antibodies or antigen-binding fragments specific for 5T4, such as those disclosed herein and one or more antibodies or antigen-binding fragments having specificity for a second antigen.

[0120] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells.

[0121] In some embodiments, the second antigen is against CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0122] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-5T4 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0123] Bifunctional molecules are also provided that do not simply comprise antibodies or antigen-binding fragments.As tumor antigen targeting molecules, 5T4-specific antibodies or antigen-binding fragments, such as those described herein, can be combined with immunocytokines or ligands, optionally via peptide linkers.Linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL.Such bifunctional molecules can combine immune checkpoint blocking effects with tumor site local immune regulation.

[0124] In some embodiments, the anti-5T4 fragment comprises the VH and VL CDRs of any of the antibodies in bin A (14G12, 393E9, and 113H5). In some embodiments, the anti-5T4 fragment comprises the VH and VL CDRs of any of the antibodies in bin B (159D5, 24F10, and 493E10). In some embodiments, the anti-5T4 fragment comprises the VH and VL CDRs of any of the antibodies in bin C (257F1, 353H11, 367B8, 389G2, and 109H7). In some embodiments, the anti-5T4 fragment comprises the VH and VL CDRs of any of the antibodies in bin D (286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10).

[0125] In some embodiments, the anti-5T4 fragment comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 41; a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 42; a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 43; a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 44; a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0126] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, or any one of SEQ ID NOs: 47 to 53; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0127] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 264. In some embodiments, VH CDR2 is PTM risk averted. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; VL CDR3 each comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 265.

[0128] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, or any one of SEQ ID NOs: 47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 264, and 265, respectively.

[0129] An example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 185-188, 193-194, 205-214, and 262-263. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 181-184, 189-192, and 195-204. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 262. Yet another example VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197. An example VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 263.

[0130] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 1, 181-184, 189-192, and 195-204 while retaining the corresponding VH CDRs or PTM-avoided versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263 while retaining the corresponding VL CDRs or PTM de-risked versions or affinity matured versions thereof. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 197 while retaining the corresponding VH CDRs or PTM de-risked versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 262, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 262 while retaining the corresponding VL CDRs or affinity matured versions thereof. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 263, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 263 while retaining the corresponding VL CDRs or affinity matured versions thereof. Chimeric Antigen Receptor

[0131] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or fragment thereof of the present disclosure as a targeting unit. In some embodiments, the CAR comprises the antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0132] The transmembrane domain can be designed to be fused to the extracellular domain, including antibody or fragment, optionally via hinge domain.It can also be fused to an intracellular domain, such as a costimulatory domain.In some embodiments, the transmembrane domain can include the natural transmembrane region of the costimulatory domain (e.g., the TM region of CD28T or 4-IBB used as costimulatory domain) or the natural transmembrane domain of the hinge region (e.g., the TM region of CD8alpha or CD28T used as hinge domain).

[0133] In some embodiments, a transmembrane domain may comprise a sequence that spans a cell membrane, but extends into the cytoplasm of the cell and / or into the extracellular space. For example, a transmembrane may comprise a membrane-spanning sequence that itself may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of the cell and / or into the extracellular space. Thus, a transmembrane domain may comprise a region that spans the membrane, and may further comprise amino acids that extend beyond the inner or outer surface of the membrane itself, and still be considered to be a "transmembrane domain".

[0134] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS), a glycine-serine-glycine triplet (GSG), or an alanine-alanine-alanine triplet (AAA) would be a suitable linker.

[0135] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain is located between the transmembrane domain and the activation domain. Examples of costimulatory domains include CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A(B cell antigen receptor complex-associated alpha chain), CD79B(B cell antigen receptor complex-associated beta chain), CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(KG2D), CD319(SLAMF7), CD335(K-p46 ), CD336(K-p44), CD337(K-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF 18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD 11a / CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0136] In some embodiments, the cytoplasmic portion of the CAR also comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 zeta domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a CD3 zeta domain. Methods for expressing or preparing polynucleotides, mRNA, and antibodies

[0137] The present disclosure also provides a polynucleotide or nucleic acid molecule encoding the antibody, variant or derivative thereof, or CAR of the present disclosure. The polynucleotide of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotide of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. It may be encoded on the molecule.

[0138] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell for expression of the antibody or fragment thereof.

[0139] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using linear or circular template DNA, containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. Stringent conditions vary depending on specific applications.

[0140] In some embodiments, for the preparation of mRNA encoding an antibody, the template DNA is transcribed in vitro. A suitable template DNA typically has a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding a heavy or light chain), and a termination signal.

[0141] Using standard methods, the mRNA sequence encoding the desired antibody (e.g., encoding a heavy or light chain) can be determined and incorporated into the template DNA. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), a virtual back-translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select the appropriate codons. Typically, the G / C content can be optimized on the one hand to achieve as high a G / C content as possible, and on the other hand to take into account as much as possible the frequency of tRNA according to the codon usage frequency. The optimized RNA sequence can be established and displayed, for example with the aid of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or, respectively, regions of the RNA.

[0142] mRNA can be synthesized as unmodified or modified mRNA.Typically, mRNA is modified to improve stability.Modification of mRNA can include, for example, modification of nucleotide of RNA.Thus, modified mRNA can include, for example, backbone modification, sugar modification or base modification.In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil)), as well as modified nucleotides such as purines and pyrimidine analogs. Analogs or derivatives, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, These include, but are not limited to, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0143] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modification. Typically, backbone modification is a modification in which the backbone phosphate of the nucleotide contained in RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium, etc., which means that phosphodiester bond is replaced by other anionic, cationic or neutral groups.

[0144] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides they contain, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, 2'-deoxy-2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-amino ... and 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0145] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, and 6-azacytidine 5'-triphosphate. , 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.

[0146] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to the C-terminal (3') end. The presence of the cap is important in making the mRNA resistant to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0147] Thus, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0148] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(A) tail structure. The polyA tail at the 3' end of the mRNA typically comprises about 10-300 adenosine nucleotides (e.g., about 10-200 adenosine nucleotides, about 10-175 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-125 adenosine nucleotides, 10-100 adenosine nucleotides, about 10-75 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(C) tail structure. A suitable poly-C tail at the 3' end of an mRNA typically comprises about 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly-C tail can be added to or replace the poly-A tail.

[0149] In some embodiments, the mRNA (e.g., mRNAs encoding the heavy and light chains) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron response element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length (e.g., between about 50 and 400 nucleotides in length, between about 50 and 300 nucleotides in length, between about 50 and 200 nucleotides in length, or between about 50 and 100 nucleotides in length).

[0150] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is linked directly or indirectly to the sequence encoding the heavy or light chain at the N-terminus.

[0151] This technology can be used to deliver any antibody known in the art and that can be raised against a desired antigen using standard methods. The invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0152] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0153] As described herein, the antibodies, variants, or derivatives of the present disclosure can be used in certain treatment and diagnostic methods.

[0154] The present disclosure further relates to antibody-based therapy, including administering an antibody, or fragment, of the present disclosure to a patient, such as an animal, mammal, or human, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, an antibody of the present disclosure (including variants and derivatives thereof as described herein), and a nucleic acid or polynucleotide encoding an antibody of the present disclosure (including variants and derivatives thereof as described herein).

[0155] The antibodies of the present disclosure can also be used to treat or inhibit cancer. As mentioned above, 5T4 is hardly expressed in normal adult tissues, but is present at high levels in the placenta and in more than 80% of the most common tumors, typically kidney, breast, colon, prostate and ovarian cancers.

[0156] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method entails administering to the patient an effective amount of the antibody, or fragment, or antibody-drug conjugate of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses 5T4.

[0157] Cell therapy, for example, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are transduced with a vector that encodes or contacts a CAR that includes (or alternatively is engineered to express) the anti-5T4 antibody of the present disclosure. Thus, by such contact or manipulation, the cells can be introduced into a cancer patient that requires treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0158] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0159] Non-limiting examples of cancer include bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0160] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and Carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, These include, but are not limited to, alveolar carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0161] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the number of administrations, excretion rate, drug combinations, and the severity of the particular disease being treated.The judgment of such factors by medical practitioners is within the ordinary skill of the art.The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.The amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0162] Methods of administration of the antibody or fragment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as a powder, ointment, drops or transdermal patch), bucally, or as an oral spray or nasal drops.

[0163] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0164] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0165] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, nonporous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the proteins do not absorb.

[0166] The amount of the antibody or fragment of the present disclosure that will be effective in treating, inhibiting and preventing inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques.In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges.The exact dose to be used in the formulation will also depend on the route of administration and the severity of disease, disorder or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0167] As a general proposition, the dosage of the antibody or fragment of the present disclosure administered to a patient is typically between 0.001 mg and 100 mg per kg of the patient's body weight, between 0.01 mg and 20 mg per kg of the patient's body weight, or between 0.5 mg and 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other animal species due to the immune response to the foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are often possible. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0168] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0169] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0170] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody, or fragment, or antibody-drug conjugate, and acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., immune checkpoint inhibitor).

[0171] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0172] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0173] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0174] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0175] Example 1 Generation of mouse monoclonal antibodies against human 5T4. This example describes the generation of anti-human 5T4 mouse monoclonal antibodies using hybridoma technology.

[0176] Antigens: human 5T4-His protein and CHO-K1 expressed human 5T4.

[0177] Immunization: To generate mouse monoclonal antibodies targeting human 5T4, SJL mice, Balb / c mice and C57BL / 6 mice were first immunized with 5T4-His protein. The immunized mice were then boosted with 5T4-His protein or CHO-K1-expressed human 5T4. To select mice producing antibodies that bound to 5T4 protein, the sera of the immunized mice were subjected to antibody titer evaluation by ELISA and FACS. Briefly, microtiter plates were coated with 0.5 or 1 μg / mL human 5T4 protein or cyno 5T4 protein in ELISA coating buffer, 100 μL / well, overnight at 4°C, and then blocked with 150 μL / well 1% BSA. Dilutions of serum from immunized mice were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween® and then incubated with anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. The immune response was also tested by serum FACS against CHOK1-hu5T4 cell line, with CHOK1 parental cell line serving as a negative control. The resulting mice were used for fusion. Hybridoma supernatants were screened by ELISA.

[0178] Cell fusion: Fusion was performed by electrofusion. The fused cells were plated in 50 96-well plates per fusion.

[0179] Screening: Hybridoma supernatants were screened by ELISA against recombinant human (rh) 5T4-His protein and recombinant cyno 5T4-His protein. Positive supernatants from the primary screen were then subjected to confirmation screening by FACS binding to the CHOK1-hu5T4 cell line and protein binding by ELISA.

[0180] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parent cell. The subclones were screened using the same approach as the primary clones, and culture supernatants of positive clones were subjected to additional confirmatory screening by affinity ranking.

[0181] Hybridoma clones 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 and 95F10 were selected for further analysis. The amino acid sequences of the variable regions of these clones are listed in Table 1 below. Tables 1-1 to 1-20 include the original CDR sequences from this murine antibody, as well as versions in which potential post-translational modifications (PTMs) have been removed or which have been affinity matured. [Table 1] [Table 1A] [Table 1B] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

[0182] (Example 2) mAb binding affinity Binding of chimeric mAbs from these clones to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. mAbs were captured using a protein A chip. Serial dilutions of human 5T4-his tag protein were injected over the captured antibody for 2-3 min at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 360-1000 s. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software.

[0183] As shown by the results in Table 2 below, the majority of the antibodies tested exhibited nanomolar or subnanomolar binding affinity for recombinant human 5T4 protein. [Table 2]

[0184] ELISA tests were performed to evaluate the binding of the chimeric antibodies to humans and cynomolgus monkeys, respectively. The antibody portion of naptumomab (NeoTX) (abbreviated as naptumomab in this disclosure) targeting 5T4 was generated and used as a benchmark.

[0185] Briefly, microtiter plates were coated with 1 μg / ml human and cynomolgus 5T4 proteins in PBS, 100 μl / well, overnight at 4° C., then blocked with 150 μl / well 1% BSA. Serial dilutions of chimeric antibodies were added to each well and incubated for 1 h at RT. Plates were washed with PBS / Tween® and then incubated with mouse anti-human IgG Fc antibody conjugated with horseradish peroxidase (HRP) for 30 min at RT. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. All 5T4 chimeric mAbs bound to human and cynomolgus 5T4 (Figures 1-2 and Table 3). Naptumomab showed very weak cross-reactivity to cynomolgus 5T4. Example 3 Binding activity to 5T4 antigen 3.1 Cross-species activity [Table 3] 3.2 Epitope binning by competitive ELSA

[0186] A competitive ELISA was performed to classify the test 5T4 mAbs based on their binding epitopes on human 5T4.

[0187] Briefly, microtiter plates were coated with 100 μl / well of 0.5 μg / ml human 5T4 protein in PBS overnight at 4° C., then blocked with 150 μl / well of 1% BSA. Serial dilutions of chimeric antibodies and 0.2 ug / ml of biotin-conjugated reference mAb were added to each well and incubated for 1 hour at RT. Plates were washed with PBS / Tween® and then incubated with streptavidin-HRP for 15 minutes at RT. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. According to the competitive performance with reference mAb, 5T4 mAb was divided into four bins (bins A-D) as shown in Figures 3-8 and Table 4. [Table 4] 3.3 FACS Testing

[0188] Cell-based binding: FACS was used to assess the binding activity of all tested chimeric mAbs against CHO-K1 expressing human 5T4 (CHOK1-hu5T4).

[0189] Briefly, CHOK1-hu5T4 cells were washed with FACS buffer and distributed into each well with serially diluted 5T4 chimeric mAbs for 30 min at 4° C. After washing with FACS buffer, PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4° C. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was evaluated by MACSQuant Analyzer 16. As shown in FIG. 9, all tested 5T4 chimeric mAbs bound to CHOK1-hu5T4 cells, and antibodies in different bins showed different binding activities.

[0190] Example 4 Humanization of 14G12 The 14G12 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 14G12 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0191] The human germline sequences used for CDR grafting, and the resulting humanized sequences, are listed in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-5A] [Table 5-6]

[0192] Example 5 Binding activity of 14G12 humanized antibody to human 5T4 antigen This example examined the binding activity of the 14G12 humanized antibody to the human 5T4 protein. 5.1 ELISA binding of 14G12 humanized antibody to 5T4

[0193] To evaluate the binding activity of the 14G12 humanized antibody to human 5T4, the 14G12 chimeric antibody was subjected to an ELISA test together with the 14G12 humanized mAb.

[0194] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human 5T4-His protein in PBS overnight at 4° C., then blocked with 150 μl / well of 1% BSA. Serial dilutions of antibodies were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS / Tween® and then incubated with goat anti-human IgG-HRP for 30 minutes at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in FIG. 10 and Table 6, the majority of the humanized antibodies bound to human 5T4 with high activity. [Table 6-1] [Table 6-2] 5.2 Cell-based binding of 14G12 humanized antibody to 5T4

[0195] To evaluate the cell-based binding properties of the 14G12 humanized antibody to human 5T4, the tested antibodies were analyzed by FACS on CHOK1-hu5T4 cells. A total of 1×10 5CHOK1-hu5T4 cells were incubated with serially diluted antibodies in FACS buffer for 30 min at 4° C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4° C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As shown in FIG. 11, some of the listed 14G12 humanized antibodies showed binding ability comparable to that of the 14G12 chimeric antibody. 5.3 Protein affinity ranking of 14G12 humanized antibody against 5T4

[0196] Binding of 14G12 humanized antibody to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. mAb was captured using a Protein A chip. Serial dilutions of human 5T4-his tag protein were injected over the captured antibody for 120-180 seconds at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 360-1000 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 7 below. [Table 7]

[0197] Example 6 Humanization of the 393E9 antibody The 393E9 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 393E9 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0198] The human germline sequences used for CDR grafting, and the resulting humanized sequences, are listed in Table 8. [Table 8-1] [Table 8-2]

[0199] Example 7 Binding activity of 393E9 humanized antibody to human 5T4 antigen This example examined the binding activity of the 393E9 humanized antibody to human 5T4 protein. 7.1 ELISA binding of 393E9 humanized antibody to 5T4

[0200] To evaluate the binding activity of the 393E9 humanized antibody to human 5T4, the 393E9 chimeric antibody (393E9-C) was subjected to an ELISA test together with the 393E9 humanized mAb.

[0201] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human 5T4-His protein in PBS overnight at 4° C., then blocked with 150 μl / well of 1% BSA. Four-fold dilutions of antibodies starting at 20 nM were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS / Tween® and then incubated with goat anti-human IgG-HRP for 30 minutes at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in FIG. 12 and Table 9, the majority of the humanized antibodies bound to human 5T4 with high activity. [Table 9-1] [Table 9-2] 7.2 Cell-based binding of 393E9 humanized antibody to 5T4

[0202] To assess the cell-based binding properties of the 393E9 humanized antibody to human 5T4, the tested antibodies were analyzed by FACS on CHOK1-hu5T4 cells. A total of 1 x 10 5CHOK1-hu5T4 cells were incubated with 3-fold serial dilutions of antibodies starting from 50 nM in FACS buffer for 30 min at 4° C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4° C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As shown in FIG. 13, some of the listed 393E9 humanized antibodies showed binding abilities comparable to the 393E9 chimeric antibody. 7.3 Protein affinity ranking of 393E9 humanized antibody against 5T4

[0203] Binding of 393E9 humanized antibody to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. mAb was captured using a Protein A chip. Serial dilutions of human 5T4-his tag protein were injected over the captured antibody for 3 min at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 280-800 s. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 10 below. [Table 10]

[0204] Example 8 Humanization of 159D5 antibody The 159D5 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 159D5 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0205] The human germline sequences used for CDR grafting, and the resulting humanized sequences, are listed in Table 11. [Table 11-1] [Table 11-2]

[0206] Example 9 Binding activity of 159D5 humanized antibody to human 5T4 antigen This example examined the binding activity of the 159D5 humanized antibody to the human 5T4 protein. 9.1 ELISA binding of 159D5 humanized antibody to 5T4

[0207] To evaluate the binding activity of the 159D5 humanized antibody to human 5T4, the 159D5 chimeric antibody was subjected to an ELISA test together with the 159D5 humanized mAb.

[0208] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human 5T4-His protein in PBS overnight at 4° C., then blocked with 150 μl / well of 1% BSA. Serial dilutions of antibodies were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS / Tween® and then incubated with goat anti-human IgG-HRP for 30 minutes at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in FIG. 14 and Table 12, the majority of the humanized antibodies bound to human 5T4 with high activity. [Table 12] 9.2 Cell-based binding of 159D5 humanized antibody to 5T4

[0209] To assess the cell-based binding properties of the 159D5 humanized antibody to human 5T4, the tested antibodies were analyzed by FACS on CHOK1-hu5T4 cells. A total of 1×10 5CHOK1-hu5T4 cells were incubated with serially diluted antibodies in FACS buffer for 30 min at 4° C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4° C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As shown in FIG. 15, most of the listed 159D5 humanized antibodies showed binding ability comparable to that of the 159D5 chimeric antibody. 9.3 Protein affinity ranking of 159D5 humanized antibody against 5T4

[0210] Binding of 159D5 humanized antibody to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. mAb was captured using a Protein A chip. 50 nM of human 5T4-his tag protein was injected over the captured antibody for 3 minutes at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 600 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 13 below. [Table 13]

[0211] Example 10 Humanization of 286B4 antibody The 286B4 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 286B4 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0212] For the heavy chain of 286B4, VH4-28 / JH6 was selected as the humanized backbone. For the light chain of 286B4, VL-O18 / JK4 was the best-matched germline. Then, a humanized 286B4 CDR-grafted antibody was designed, with CDRL1, L2 and L3 grafted into the framework sequence of VL-O18-JK4, and CDRH1, H2 and H3 grafted into the framework sequence of VH4-28-JH6. Then, a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 286B4 CDR-grafted antibody sequence, four additional humanized heavy chains and five additional light chains were created.

[0213] The human germline sequences used for CDR grafting, and the resulting humanized sequences, are listed in Table 14. [Table 14-1] [Table 14-2]

[0214] Example 11 Binding activity of 286B4 humanized antibody to human 5T4 antigen This example examined the binding activity of the 286B4 humanized antibody to the human 5T4 protein. 11.1 ELISA binding to 5T4

[0215] To evaluate the binding activity of the clones, the 286B4 chimeric antibody was subjected to an ELISA test together with the 286B4 humanized mAb.

[0216] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human 5T4-His protein in PBS overnight at 4° C., then blocked with 150 μl / well of 1% BSA. Four-fold dilutions of test antibodies starting at 20 nM were added to each well and incubated for 1 hour at 37° C. Plates were washed with PBS / Tween® and then incubated with goat anti-human IgG-HRP for 30 minutes at 37° C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in FIG. 16 and Table 15, the majority of humanized clones bound to human 5T4 with high potency. [Table 15-1] [Table 15-2] 11.2 Cell-based binding to 5T4

[0217] To evaluate the cell-based binding properties of the 286B4 humanized antibody to human 5T4, the tested antibodies were analyzed by FACS for their binding to CHOK1-hu5T4 (a human 5T4 high expressing cell line) or MCF-7 cells (a human 5T4 low expressing cell line). A total of 1 x 10 5 CHOK1-hu5T4 or MCF7 cells were incubated with 4-fold or 3-fold serially diluted antibodies starting from 50 nM in FACS buffer for 30 min at 4° C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4° C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As shown in FIG. 17A (CHO-hu5T4 cells) and FIG. 17B (MCF-7 cells), some of the listed 286B4-3 / 8 / 13 / 18 / 5 / 10 / 15 humanized antibodies showed binding capacities comparable to the 286B4 chimeric antibody. 11.3 Protein affinity ranking for human 5T4

[0218] Binding of 286B4 humanized antibody to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. mAb was captured using a Protein A chip. Serial dilutions of human 5T4-his tag protein were injected over the captured antibody for 3 minutes at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 280-800 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 16 below. [Table 16]

[0219] Example 12 Confirmation of PTM removal of 393E9, 159D5 and 286B4 12.1 Confirmation of PTM removal in 393E9 393E9 VH CDR2 contains NG and NS residues (Kabat numbering), which are at risk of post-translational modification (PTM) and are a challenge for future manufacturing. Therefore, this example mutated NG to NA and NS to YS on VH to prevent PTM. The sequences of potential PTM removal sites are listed in Table 17. [Table 17-1] [Table 17-2]

[0220] To evaluate the binding properties of the PTM-depleted antibodies to surface 5T4, Hu393E9-45-P2, Hu393E9-53-P2, Hu393E9-62-P2 or trispecific antibodies carrying the anti-5T4 portion of chimeric 393E9 were analyzed by FACS for their binding to CHOK1-hu5T4 cells. A total of 1 × 10 cells were collected in each well. 5The cells were incubated with 4-fold serial dilutions of antibodies starting from 100 nM for 30 min at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4°C. The MFI of PE was evaluated by a MACSQuant Analyzer 16. As shown in Figure 18A, the trispecific antibodies with anti-5T4 moieties of Hu393E9-45-P2 or Hu393E9-62-P2 showed enhanced binding ability to 5T4-expressing cells than those with chimeric 393E9. The only difference between the tested trispecific antibodies was the anti-5T4 moiety. 12.2 Confirmation of PTM removal in 159D5

[0221] 159D5 VH CDR2 contains DS residue (Kabat numbering), which is at risk of post-translational modification (PTM) and is a challenge for future manufacturing. Therefore, this example mutated DS to DA on VH to prevent PTM. The sequences of potential PTM removal sites are listed in Table 18. As shown in Figure 14B, Figure 15 and Table 13, the PTM-removed antibody of 159D5-P1 showed binding ability equivalent to that of the 159D5 chimeric antibody of 159D5-C. [Table 18-1] [Table 18-2] [Table 18-3] 12.3 Check for PTM removal on 286B4

[0222] 286B4 VH CDR2 contains DG residues (Kabat numbering), which are at risk of post-translational modification (PTM) and are a challenge for future manufacturing. Therefore, this example mutated the DG to DA on VH to prevent PTM. The sequences of potential PTM removal sites are listed in Table 19. [Table 19-1] [Table 19-2]

[0223] To evaluate the binding properties of the PTM-depleted antibodies to surface 5T4, Hu286B4-3-P1, Hu286B4-5-P1, Hu286B4-8-P1, Hu286B4-15-P1 or trispecific antibodies carrying the anti-5T4 portion of chimeric 286B4 were analyzed by FACS for their binding to CHOK1-hu5T4 cells. A total of 1 × 10 cells were collected in each well. 5 The cells were incubated with 4-fold serial dilutions of antibodies starting from 100 nM for 30 min at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4°C. The MFI of PE was evaluated by a MACSQuant Analyzer 16. As shown in Figure 18B, the trispecific antibodies with PTM-depleted 286B4 fragments showed enhanced binding ability to 5T4-expressing cells than those with chimeric 286B4 fragments. The only difference between the tested trispecific antibodies was the anti-5T4 moiety.

[0224] (Example 13) Affinity maturation of humanized antibody Hu14G12-28 To enhance the affinity of the humanized antibody of Hu14G12-28, affinity maturation was carried out. Briefly, four phage libraries containing single or two saturation mutations in the CDR region of Hu14G12-28 were constructed. Two candidates with unique mutations in the CDR were obtained by one round of screening using solid or liquid panning. The CDRs of these candidates were grafted into the Hu14G12-28 framework to generate antibodies for further binding and affinity validation.

[0225] The amino acid sequences of the variable regions of the affinity maturation candidates grafted into the framework are listed in Table 20 below. [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] 13.1 ELISA binding to 5T4

[0226] To evaluate the binding activity of the affinity matured antibodies against human 5T4, Hu14G12-28 was subjected to ELISA testing together with Hu14G12-28-88# and Hu14G12-28-108#.

[0227] Briefly, microtiter plates were coated with 2 μg / ml human 5T4-His protein in PBS, 30 μl / well, overnight at 4° C., then blocked with 5% PBS / milk. Serial dilutions of antibody were added to each well and incubated for 1 hour at room temperature. Plates were washed with PBS / Tween® and then incubated with goat anti-human IgG-HRP for 50 minutes at room temperature. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in FIG. 19, affinity matured antibodies bound to human 5T4 with higher potency than parental Hu14G12-28 antibody. 13.2 Cell-based binding to 5T4 To assess the binding properties of affinity matured antibodies to surface 5T4, the tested antibodies were analyzed by FACS for their binding to CHOK1-hu5T4, HEK293-hu5T4 or MCF-7 cells. A total of 1×10 5The cells were incubated with 4- or 5-fold serial dilutions of antibodies starting at 133 nM or 100 nM for 60 min at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4°C. The MFI of PE was assessed by a MACSQuant Analyzer 16. As shown in Figures 20A-B (CHOK1-hu5T4 cells), 20C-D (HEK293-hu5T4 cells) and 20E (MCF-7 cells), the affinity matured antibodies showed enhanced binding ability to 5T4-expressing cells compared to the parental Hu14G12-28 antibody. 13.3 Binding affinity to 5T4

[0228] The binding affinity of the tested antibodies to recombinant 5T4 protein (human 5T4-his tag) was tested on Biacore using the capture method. Antibodies were captured using a protein A chip. Serial dilutions of human 5T4-his tag protein were injected over the captured antibody for 3 min at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 300 s. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software.

[0229] As shown by the results in Table 21 below, the affinity matured antibodies Hu14G12-28-88# and Hu14G12-28-108# significantly increased the binding affinity to human 5T4 protein by 9.45-fold and 7.41-fold, respectively, compared to the parent Hu14G12-28 antibody, making these affinity matured antibodies ideal anti-5T4 antibodies for antibody-drug conjugates (ADCs) and for bispecific antibodies. [Table 21] * * *

[0230] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0231] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. An antibody or antigen-binding fragment thereof having specificity for human 5T4 oncofetal trophoblast glycoprotein (5T4) protein, comprising a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are, respectively: SEQ ID NOs: 41, 42 (or any one of 47-53), 43, 44, 45 and 46 (or 264 or 265); SEQ ID NOs: 54, 55 (or any one of 60-63, or 266 or 267), 56, 57, 58 and 59; SEQ ID NOs: 64-69; SEQ ID NOs: 70, 71 (or 76), 72, 73, 74 and 75; SEQ ID NOs:77-82; SEQ ID NOs: 83-88; SEQ ID NOs: 89-94; SEQ ID NOs: 95, 96 (or any one of 101-104), 97, 98, 99 and 100; SEQ ID NOs: 105-110; SEQ ID NOs: 111-116; SEQ ID NOs: 117-122; SEQ ID NOs: 123, 124 or 129, 125, 126, 127 and 128; SEQ ID NOs: 130-135; SEQ ID NOs: 136-141; SEQ ID NOs: 142-147; SEQ ID NOs: 148-153; SEQ ID NOs: 154-159; SEQ ID NOs: 160, 161 or 166, 162, 163, 164 and 165; SEQ ID NOs: 167, 168 or 173, 169, 170, 171 and 172; or SEQ ID NOs: 174, 175 or 180, 176, 177, 178 and 179 An antibody or antigen-binding fragment thereof comprising the amino acid sequence of:

2. the VH CDR1 comprises the amino acid sequence of SEQ ID NO:54; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 60-63 and 266-267; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:57; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:58; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 59; The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 215-221 and 248-256, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 222-226.

4. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 123; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124 and 129; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 125; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 126; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 127; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 128; The antibody or antigen-binding fragment thereof according to claim 1.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 236-241, and 259-261, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, and 242-247.

6. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 and 47-53; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 46, 264 or 265; The antibody or antigen-binding fragment thereof according to claim 1.

7. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 264 or 265; The antibody or antigen-binding fragment thereof described in claim 6.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 181-184, 189-192, and 195-204, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 185-188, 193-194, 205-214, and 262-263.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the VH comprises the amino acid sequence of SEQ ID NO: 197, and the VL comprises the amino acid sequence of SEQ ID NO: 262 or 263.

10. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 70; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 and 76; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 72; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 74; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 75; The antibody or antigen-binding fragment thereof according to claim 1.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 227-229, and 257, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 230-235, and 258.

12. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 95; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 and 101-104; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:98; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:99; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 100; The antibody or antigen-binding fragment thereof according to claim 1.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15 and the VL comprises the amino acid sequence of SEQ ID NO:

16.

14. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 117; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 118; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 119; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 120; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 121; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 122; The antibody or antigen-binding fragment thereof according to claim 1.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the VH comprises the amino acid sequence of SEQ ID NO: 21 and the VL comprises the amino acid sequence of SEQ ID NO:

22.

16. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 154; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 155; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 156; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 157; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 158; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 159; The antibody or antigen-binding fragment thereof according to claim 1.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the VH comprises the amino acid sequence of SEQ ID NO: 33 and the VL comprises the amino acid sequence of SEQ ID NO:

34.

18. 18. The antibody or fragment thereof according to any one of claims 1 to 17, which is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

19. 10. An antibody-drug conjugate comprising the antibody of claim 1 or a fragment thereof conjugated to a drug moiety.

20. 20. The antibody-drug conjugate of claim 19, wherein the drug moiety is a cytotoxic or cytostatic agent.

21. 21. The antibody-drug conjugate of claim 20, wherein the drug moiety is a maytansinoid, an auristatin, or a macrocyclic ketone analog.

22. 22. The antibody-drug conjugate of claim 21, wherein the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

23. 20. The antibody-drug conjugate of claim 19, wherein the drug moiety is attached to the antibody or fragment thereof via a linker that is hydrolyzable under acidic conditions.

24. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 264 or 265; The antibody-drug conjugate of claim 19.

25. A multispecific antibody comprising the antigen-binding fragment of claim 1 and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than 5T4.

26. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment of claim 1, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

27. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof of claim 1.

28. 28. The polynucleotide of claim 27, which is one or more mRNAs.

29. 29. The polynucleotide of claim 28, wherein the mRNA is chemically modified.

30. A cell comprising the polynucleotide of claim 27.

31. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the antibody-drug conjugate according to claim 19, the multispecific antibody according to claim 25, the CAR according to claim 26, the polynucleotide according to claim 27, or the cell according to claim 30, and a pharmaceutically acceptable carrier.

32. A composition for use in treating cancer in a patient in need thereof, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody-drug conjugate according to claim 19, a multispecific antibody according to claim 25, a CAR according to claim 26, a polynucleotide according to claim 27, or a cell according to claim 30.

33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the antibody-drug conjugate according to claim 19, the multispecific antibody according to claim 25, the CAR according to claim 26, the polynucleotide according to claim 27, or the cell according to claim 30, for the preparation of a medicament for treating cancer.

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